Form Approved. OMJ3. No. 2070-00J2.·Approval Expires IO-J 1-96 AGENCY USE ONLY U.S. ENVIRONMENTAL PROTECTION AGENCY Unte ofReceIpt

### _~_I::DA 

## PREMANUFACTURE**~C--I-\** 

## NOTICE
FORNEW CHEMICAL SUBSTANCES 

### CompanySanitized

1*

___ ...**~._** .....uuv""'. VI**paeC~** n the Premanufacture Notice

• You must provide all intill'lnation requested in this form to the extent that it is known to or reasonable ascertainable by vou. Make reasonahle estimates if}ou do not have actual data. • Bef(lrc you complete this form. you should rcad the "Instructions Manual for Premanulacture Notification" (the Instructions Manual is available from theToxic Substances Control Act (TSCA) Information Service by calling 202-554-1404. or faxing 20:'-554-5603) • Ifa user fee has been remitted for this notice (40 CFR 70045). indicate in the boxes abo\t' the TS-user fee identification number Y(lU havegenerated. Remember. your user fee If) number must also appear on vour corresponding fee remittance. which is sent [() EPA. HQ ,\cwunting Operalions Branch (PM-266), P.O Box 360399M. Pittsburgh, PA 15251·6399. Alln" TSCi\ User Fcc. Part I - CE:\ER,\l. lNf'ORMAT10N You must**rro\ld~ th~** **curr('lltl~** correct Chemical**AbstraCl~** ICA) Name "rth" ilL''' chemical**'ub'lal1c~,** C\'c'11 ifYOU d,lim the ltknuty as confidential You rna\' authori?c another pns{)l1 In slIbmll ehemkal identIty mi()rmatlon for VOIL but your slIbnllSS10n \\ II llll\ be wmr;ct" and the [(?Ikw will nm begin until EPA r,-,(elves tillS inf(lrmJlinn A kacr 111 support ofyour submission should rckrcl1cc vour TS user fee idcl1l1i1cauoll numhcr 'iou must submit un origlnul and t\vO cop;'::, "fthi,nOllce lIlc!udmg "lileS, \Jain Ifvou claimed anv ll1tormatlOn as confidenllaL a slllgie sunitizeJ COP' must "Iso he suhmltted Pan 1l·111'MAN EXPOSllRF AND fNVIRO"lMfNTAl. RELEASE I fthere' arc several manufacturc, processmg. or usc operations to he**dcscflb~d** In Pan 11. sectIOn !\ and Bol'this notice, rcproduce the seclions as needed Panlll- LIST OF ATTAClIMEJ'.:TS Attach additional sheets ifthere IS mIL enough space to :lnswer HqL.cslion till'y Lahd each COllllnU31IO" sheet \\lIh the corresponding scelion headlllg In Part Ill. ht these atlJchnWIllS. :m" lest <.lat:l orother data and any optIOnal ll1fnrmallon lI1c1uUt'lt In the nOlie;; OPTIONAL INFORMAlf()N You l11a\ Il1Clcldc any 1l11{lfln<lllon that YOU want FP1\ to c:onslder III c'valuatlng the new suh,tiU1CC. On page II 01' Ihis form. sp:lec Ims been pf()\'ldcd I,l[Hnl 10**(kscrih~** pollution rn:\clliHHl Ul1J**1\\.:~cll11g** lnionnarinn you**m;j~** lla\.: regardmg lhl'**n~~w** SUbSl:1I1l.'t.' So-called "bmdlllg" box"s arc 111dud"d througlltlUI tillS form 1,)[ you to Indlcale your**\\'l!lJngn~ss** lo**h~** bound to ccrtl:in**statcln~111S** you make. m thiS puoll( notice. slich as usc, produc[lon \'olul1w. rlOl"ctll<c cqui[lmcnt Th,s OpllOll IS Illlcnded to**r~ducc'** ddavs Iltilt rOlltil1ch**3~C"'npan\** the development 01 consent ord"rs Llr SlgmfteJnt NeVI Rules L\ccpt mtlt" C'ase or**cx~mrllon** J[lpilcaU1ll1' (such us 'I'M!',\, LVle. LORFX)**wlt~r.c** cert:lln lllrOrl11JIIOn rl'mtded ill tim notl1lcJtml1 " binding on tilt' submitter**wh~n** the**Ag~nc\'** J[lrrovcs the exemption J[l[lilc
..tlon, dlccKlI1g a bmding box 1I1 thIS nollce docs nlll hI IIselfprohibit the**'lIhlmtt~r** rrom later deVl:ltll1g I[om the 1Il1l1l'malion (exceptchcmical,"kntllyl re[loned In the I<,rm. CONFlDENTI\LlTY CLA1MS You mas clalln am' inlornwtion llltilis nOllCc as conf'dential. To assen a ckllm on the lonn. mark (X) the confidential hox next to the m:bnrmtron that you**d~Hm** as conlidcntial. To assert a cilllm 111 an attachment, Circle or hracket the ll1t(ll1natiol1 vou claim as conlldcl1tlal. lrvou dUlm ml()rmallon In the nollce as confidential. vou must also provide a sanitized versIOn ofthe notice fincludtne attachments) for <ldditional ll1struc!tons on claiming inlormation as conlldenuaL read the Instructions Manual**~** Mark (x)**ir~ny** mhmnalion in thIS notice IS clalmed a, contidential TEST DATA AND OTHER DATA You arc**feljUlr~d** to suornll al1t
....sl daw 111 your posseSSion ,Ifconlrul <md to pro\,ld-: a descnpuoll ofall other data known to orreasonanle as,cnamahle to you, irthese dala arc rdatcd to Ihe health and environmental e:kcls ofthe manuta<:turc, [lrocessmg. dlSmhllll()1l III C(lml1lcrcc'. usc. or dlsposal ofthe**ne\~** chemJcal substallce Standurd Ineralurc cllUllOllS may he suhmllled for dma mille oren sClcnl1t1c literature. Complete tes! datu IwrutC11111 Em,ilsh), not summafle5 {Ifdata, must he suhmlltcd lfth"v do no! appear m the open Illcraturc You should dearl" ldcnllf'y 'Whether test data IS on the sut>s:ance or on an analog Also. tlw chemKul Ulmp()Sil ion oftiw lested mulerlill should be ,har;]cterized. Followmg are e,ampic's oftes! data and other dut;]. Data should b" suhmllled accllrdlOg to the rCljuiremel1ts of §72050ofthe Premanufactllfe Notlficallon Ruk 140 CrR 1':111720) Tesi Data IClwck Bdml un\' mcluded in this nOllee)

Em lronmenlillinte daw Other datu DYes

• Ikalth l'lli:c!> Data Y<.:, Risk assessment;,

I:nvironmental En;:"s Data Structure/aclivlt'· rclatiOllSlllps PhYSical Chemica! i'ro[lenies

## D y", reSI dala nol In Ihe posSeSSIOl1 orcontrol ofIhe Submlllcr '\ pl1\SlCal and ('hellllc'al prnpcr1les**\~(\r)..sheet** IS iocated on the last page nl'thls 1(lfIn TYPf- ()I' \'1 lTKT j( hcdlmlvOllcl 1'\1".; tl'rcmallulaJ.'lurc "olice.1 

## D**~** <­ I"TLR'I,lIJ)I!\TI; I'M\, isubmlltcu ill sequellce "'llh**Iinal~duct:r.~INV** 

## D 

# o

# 47~f TMIA (Test Marketing EXclllpuon /\ppl!<:<1110n)

-....... •.. ' - J 

# D 1.V!;.lLo\\' Volume FxcmptlOI1) a 4(1 erR 7::3.50(c)( 1)';9 

## D LOREX ILow Rcleasdl.ow Exposurc LxelTIrtion) 7is:jofc)(2) 

# o l.VI- Modillcalloll 

## D LOREX Mod i11catlOn lS 1II1S·\ CO!\SOLIDATED PMN') 

# 0 Ycs

# ofchemIcals (l'rcnotlcc COmlllUl1lCation # required. enter # on page 3)

EPA FORM 7710-25 (Rev 5-(5) Replaces previous editions ofEPA Form 7710-25 

## IPublic reporting burden for this collection ofinformation is estimated to avcrage 110 hours perresponse, including time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send commentsregarding the burden estimate orany other aspect ofthis collection ofinformation, including suggestions for reducing this burden. to Chief, Information Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M St.. S.W., Washington, D.C. 20460; and to the Office ofManagement and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. CERTIFICATION Icertify that to the best ofmy knowledge and belief: 1. The company named in Part I, Section A, subsection Iaofthis notice form intends to manufacture or import for a commercial purpose, otherthan in small quantities solely for research and development, the substance identified in Part L Section B. 2. All information provided in this notice is complete and truthful as ofthe date ofsubmission. 3. 1am submitting with this notice all test data in my possession or control and a description ofall other data known to or reasonably ascertainable by me as required by §720.50 ofthe Premanufacture Notification Rule.
Additional Certification Statements: Ifyou are submitting a PMN, Intermediate PMN, Consolidated PMN. or SNUN. check the following user fee certification statementthat applies: 

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

# o The Company named in Part I, Section A has remitted the fee of$1 000 for an Intermediate PM;\! (defined @ CFR 700.43) in accordance with 40CFR 700.45 (b), or 

# o The Company named in Part I, Section A is a small business concern under40 CFR 700.43 and has remitted a fee of$100 in accordance with 40 CFR 700.45 (b). Ifyou are submitting a low volume exemption (LVE) application in accordance with 40 CFR 723.50 (c (I)ora Low release and low exposureexemption (LoREX) application in accordance with 40 CFR 723.50 (c (2), check the following certification statements: 

# o The manufacturer submittingthis notice intends to manufacture or import the new chemical substancefor commercial purposes, otherthan in small quantities solely for research and development, undertheterms of40 CFR 723.50. 

# o The manufacturer is familiar with the terms ofthis section and will comply with those terms; and 

# o The 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 (cl(I), the manufacturer intends tocommence manufacture of the exempted substance for commercial purposes within I year ofthe date ofexpiration ofthe 30 day review period.

Theaccuracy ofthe statementsyou makein this notice should renect your best prediction ofthe anticipated facts regarding the chemical substancedescribed herein. Any knowing lind willful misinterpretation is subject to criminal penalty pllnllantto 18esc 100I. Confidential Signatureand Title ofAuthorized Official (Original Signature Required) Date
Signatureofagent
- (ifapplicable) Date

FORM EPA 7710-25 (Rev. 5-95) Page 2 

### xPartI

-- GENERAL INFORMATION Section A - SUBMITTERIDENTIFICATION Mark (X) the "Confidential" box next to any subsection you claim as confidential la. Person Name ofauthorized ofticial Position Submitting Notice

(in U.S.) Company Confidential
X
Mailing address (number and street) City, State, ZIP Code
b. Agent (if applicable) Name ofauthorized official Company Position

Mailing address (number and street) City, State, ZIP Code Telephone

C. lfyou are submitting this notice as part ofajoint submission, mark (X) this box. I I I I Area Code I I I I Number 

# D Joint Name ofauthorized official Submitter (if applicable) Company Mailing address (numberand street) City, State, ZIP Code
2. Technical Name Contact (in U.S.) Company Mailing address (numberand street) City, State, ZIP Code
3. Ifyou have had aprenotice communication (PC) concerning this notice and EPA assigned aPC Number to this notice, enterthe number.
4. Ifyou have submitted an exemption application for the chemical substance covered by this notice, enter the exemption number assigned by EPA. Ifyou previously submitted a PMN for this substance, enterthe PMN number assigned by EPA (i.e. withdrawn or incomplete). 5. Ifyou have submitted a notice of Bona fide intent to manufacture or import for the chemical substance covered by this notice, enterthe notice number assigned by EPA.

6. Type ofNotice

- Mark (Xl I. 

# D Manufacture Only 

# D Binding Option Mark (X)

- Position

Telephone I Area Code I I Position I Number I I X

Telephone : Area Code I 

## : :. Number I I Mark (X) ifnone 

# D

Mark (X) ifnone 

# D X

Mark (X) ifnone 

# D X

2. 

# 12I Irrp:rtOJIy 

# D BindingOption Mark (X) 3. 

# D Both X

FORM EPA**7710-2~** (Rev. 5-95) Page 3Part I - GENERAL INFORMATION

- Continued You must provide a currently correct Chemical Abstracts (CAl name ofthe substance based Section B - CHEMICAL IDENTITY INFORMATION: on the ninth Collective (9Cll ofCA nomenclature rules and conventions. Mark (X) the "Confidential" box next to any section you claim as confidential Complete either item I (Class I or 2 substances) or 2 (Polymers) as appropriate. Complete all other items.
Ifanother person will submit chemical identity information for you (for either item I or 2) mark (Xl the box at the right. 

# D IdentitY the name. company, and address ofthat person in a continuation sheet. I. Class I or 2 chemical substances (for definitions ofclass I and class 2 substances, see the Instructions Manual) a. Class ofsubstance

- Mark (X) I.

# D Class I or 2. 

# D Class 2 b. Chemical name (Currently correct Chemical Abstracts (CA) Name that is consistent with TSCA Inventory listings for similar substances. For Class I substances a CA Index name must be provided. For Class 2 substances either a C A Index name or C A Preferred Name must be provided, whichever is appropriate based on C A 9CI nomenclature rules and conventions.)

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



### x

X

d. Method I (CAS Inventory Expert Service - a copy ofthe Identification report obtained from the CAS Inventory Expert Service must be submitted as an attachment to this notice) Molecular formula and CAS Registry Number (ifa number already exists for the substance) 

# D Method 2 (Other Source)

X

e. CAS#

For a class I substance, provide a complete and correct structural diagram. For a class 2 substance

- (I) List the immediate precursor substances with their respective CAS Registry Numbers. (2) Describe the nature ofthe reaction or process. (3) Indicate the range ofcomposition and the typical composition (where appropriate). (4) Provide a correct representative or partial structural diagram, as complete as can be known, ifone can be reasonably ascertained. X

X

Mark (X) this box ifyou attach a continuation sheet FORM EPA 7710-25 (Rev. 5-95) Page 4Continuation Sheet Contains TSCA ConfidentialBusiness Information Section B - CHEMICAL IDENTITY INFORMATION l.d



### I.e

Page4(A) 

## 000005Part I - GENERAL INFORMATION

- Continued Section B - CHEMICAL IDENTITY INFORMATION

- Continued Not Applicable 2. Polymers (For a definition ofpolymer, see the Instructions Manual.) Confidential a. Indicate the number-average weight ofthe lowest molecular weight composition ofthe polymer you intend to manufacture.

Indicate maximum weight percent oflow molecular weight species (not including residual monomers, reactants or solvents) below 500 and below 1000 absolute molecular weight ofthat composition. GPCD D (Specify) Describe the methods ofmeasurement or the bases for your estimates: Other i) lowest numberaverage molecular weight: ii) maximum weight % below 500 molecular weight:
D iii) maximum weight % below 1000 molecular weight: Mark (X) this box ifyou attach a continuation sheet See attachment, page 14. b. You must make separate confidentiality claims for monomerorotherreactant identity, composition infomlation and residual information.

Mark (X) the "Contidential" box next to any item you claim as confidential. (I)
- Provide the specific chemical name and CAS Registry Number (ifa number exists) ofeach monomer or other reactant used in the manufacture ofthe polymer. (2)

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

- Indicate the Iypical weight percent ofeach monomer or otherreactant in the polymer. (4)

- Mark (X) the identity column ifyou want a monomerorother reactant used at two weight percent or less to be listed as part ofthe polymer description on the TSCA Chemical Substance Inventory. (5) Mark (Xl this column ifentries in columns (3) and (4) are confidential. (6)

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

- Mark (X) this column ifentry in column (6) is confidential. Monomerorotherreactant and CAS Registry Number Contid- Typical Identity Confid- Maximum Confidential Composition Mark (X) ential residual ential (I) (2) (3) (4) (5) (6) (7)

D Mark (Xl this box ifyou attach a continuation sheet.

c. Please idenOWhiCh method you used to develop orobtain the specitied chemical identity information reported in this notice: (check one) Method I (CAS Inventory Expert Service a copy ofthe Identification D Method 2 (Other Source) report obtained from the CAS Inventory Expert Service must be submitted as an attachment to this notice) d. The currently correct Chemical Abstracts (CA) name for the polymer that is consistent with TSCA Inventory listings tor similarpolymers.

e. Provide a correct representative orpartial chemical structure diagram, as complete as can be known, ifone can be reasonably ascertained. D

D Mark (X) this box ifyou attach a continuation sheet. r>.r..",,...,,. FORM EPA 7710-25 (Rev. 5-95) Page 5 VVVUUUPart I - GENERAL INFORMATION

- Continued Section B - CHEMICAL IDENTITY INFORMATION

- Continued 3. Impurities (a)

- Identify each impurity that may be reasonably anticipated to be present in the chemical substance as manufactured for commercial purposes. Provide the CAS RegistryNumber ifavailable. Ifthere are unidentitied impurities, enter "unidentified". (b)

- Estimate the maximum weight % ofeach impurity. Ifthere are unidentified impurities. estimate their total weight%. Impurity and CAS Registry :'IJumber Maximum Confid­ (a) percent (b) ential % X % X % X °10 X % X 0/0 % 

# D Mark (X) this box ifyou attach a continuation sheet. 4. Synonyms

- Enterany chemical synonyms for the new chemieal substance identified in subsection I or2. Confidential

X 

# D Mark (X) this box ifyou attach a continuation sheet.
5. Trade identification

- List trade names for the new chemical substance identified in subsection I or2. 

# D Mark IX) this box ifyou attach a continuation sheet. 6. Generic chemical name - Ifyou claim chemical identity as confidential, you must provide a generic chemical name for your substance that reveals the specific chemical identity ofthe new chemical substance to the maximum extent possible. Refer to the TSCA Chemical Substances Inventory, 1985 Edition, Appendix B for guidanceon developing generic names. Substituted aryl sulfonium polynuorophosphate salts 

# D Mark (X) this box ifyou attach a continuation sheet. 7. Byproducts

- Describe any byproducts resulting from the manufacture, processing, use, ordisposal ofthe new chemical substance at sites you controL Provide i the CAS Registry Number ifavailable. Byproduct CAS Registry Number Confid­ (2) (I) ential X


# D Mark (X) this box ifyou attach a continuation sheet. FORM EPA 7710-25 (Rev. 5-95) Page 6


## 000007 XPart I - GENERAL INFORMATION

- Continued Section C - PRODUCTION, IMPORT AND USE INFORMATION Mark (X) the "Confidential" box next to any item you claim as confidential. I. Production volume

- Estimate the maximum production volume during the first 12 months ofproduction. Also estimate the maximum production volume for any

consecutive 12-month period during the first three years ofproduction. Estimates should be on 100%new chemical substance basis. Fora Low Volume Exemption applicalion, ((you choose 10haveyournolice reviewedala IOlrerproduclion volume Ihan/a.nnn kg
..yr. specifyIhe volume andmark (x) in/he binding box. If gran/ed, you are bound /0 this volume. Maximum first 12-month production (kg/yr) Maximum 12-month production (kg/yr) Confid- Binding Option (100% new chemical substance basis) (100%new chemical substance basis) ential Mark (XJ
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 orthenew 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 ofthe new chemical substance by function and application.

(2) Mark (X) this column ifentry in column (I) is confidential business information (CBI). (3) Indicate your willingness to have the information provided in column ( I ) binding. (4) Estimate the percent oftotal production for the first three years devoted to each category ofuse. (5) Mark (X) this column ifentry in column (4) is confidential business information (CSI). (6) Estimate the percent ofthe new substance as formulated in mixtures, suspensions, emulsions. solutions or gels as manufactured for commercial purposes at sites under your control associated with each category ofuse. (7) Mark (X) this column ifentryin 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 information provided in (8) binding. (9) Mark (X) this column ifentry(ies) in column (8) is (are) confidential business information (CBI). Category ofUse (I) CBI Binding Produc- CSI %in CBI % ofsubstance expected peruse CBI (by function and application, i.e. a dispersive dye for finishing Option tion% Form­ (8) polyester fibers) Mark (X) ulation Site­ 'Con­ lndus- Com- Binding (2) (3) (4) (5) (6) (7) limited sumer trial mercial Option (9) X 

# . X X
oj, ';'

0/ OJ.
o/, 0/ 

### I 0' 0,

0" I**~I**
0,**~/** '; 0; *Ifyou have identified a "consumer" use, please provide on a continuation sheet a detailed description ofthe use(s) ofthis chemical substance in consumer products. In addition include estimates ofthe concentration ofthe new chemical substance as expected in consumer products and describe the chemical reactions by which this substance loses its identity in the consumer product. 

# D Mark (Xl this box ifyou attach acontinuation sheet. b. Generic Ifyou claim any category ofuse description in subsection 2aas confidential, enter a generic description ofthat category. Read the Instructions

use Manual for examples ofgeneric use descriptions. description Catalyst 

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

- Include in the notice a copy ofreasonable facsimile ofany hazard warning statement, label, material safety data sheet, or other Binding Option

information which will be provided to any person who is reasonably likely to be exposed to this substance regarding protective equipment or Mark (X) Gtices for the safe handling. transport, use, or disposal ofthe new substance. List in part III hazard information you include.

X Mark (X) this box ifyou attach hazard information. See Material Safety Data Sheet



### - FORM EPA 7710-25 (Rev. 5-9::» Page 7 

## 000008Part II

- HUMAN EXPOSUREAND ENVIRONMENTAL RELEASE Mark (X) the "Confidential" box next to any item you claim as Section A - INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER confidential. Complete Seetion A for each type ofmanufacture, processing or use operation involving the new chemical substance at industrial sites you control. Importers do not have to complete this section for operations outside the U.S.; however, you may still have reporting requirements ifthere are further industrial processing or use operations after import. You must describe these operations. I. Operation Description

a. Identity

- Enter the identity ofthe site at which the oper Name See instructions manual.
ation will occur. Confidential

X
Site address (number and street) City, County, State, ZIP Code

I Ifthe same operation will occur at more than one site. enter the number ofsites. Identify the additional sites on a continuation I # ofsites I sheet, and ifany ofthe sites have significantly different production rates oroperations. include all the infonnation requested in I I this section for those sites as attachments. 

### l 

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

# D Manufacturing Processing Use 

# D D

c. Amount and Duration

- Complete I or 2 as appropriate.
I Batch Maximum kglbatch (100% new chemical substancel I I I I Hours/batch I I I I Batches/year I I I I I I 2. Continuous Maximum kg/day (100% new chemical I I Hours/day I I Days/year substance) I I I I I I d. Process Description 

# D Mark (Xl your willingness to have our process description binding. X

(I) Diagram the major unit operation steps and chemical conversions. Include interim storage and transport containers (specify

- ego 5 gallon pails, 55 gallon drum, rail car, tank truck, etc.1 (2) Provide the identity, the approximate weight (by kg/day orkglbatch on a 100% new chemical substance basis), and entry point ofall starting materials and feedstocks (including reactants, solvents, and catalysts, ete.), and ofall products. recycle streams, and wa,tes. Include cleaning ehemicals (note frequency ifnot used dailyorper batch). (3) Identifyby number the points ofrelease, including small orintennittent releases, to the environment ofthe new chemical substance.



# D Mark (Xl this box ifyou attaeh a continuation sheet.**FOR~** EPA 7710-25 (Rev. 5-95) Page 8 

## 000009Part II HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

- Continued Section A - INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER

- Continued
2. Occupational Exposure

- You must make separate confidentiality claims for the description ofworker activity, physical form oflhe new chemical substance, number

ofworkers exposed, and duration ofactivity. Mark (Xl the "Confidential" box next to any item you claim as confidential. (I)-- Describe the activities in which workers may be exposed to the new chemical substance. (2)-- Mark (Xl this column ifentry in column (I) is confidential business information (CBI). 0)-- Describe any protective equipment and engineering controls used to protect workers. (4) and (6)--lndicate your willingness to have 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. or dust) and % new chemical substance (ifpart ofamixture) at the time ofexposure. (7)-- Mark (X) this column ifentry in column (5) is confidential business information (CBI). (8)-- Estimate the maximum number ofworkers involved in each activity. (9)-- Mark (X) this column ifentry in column (8) is confidential business information (CBI). (10) and (II)-- Estimate the Maximum duration ofthe activity for any worker in hours/day and days/year. (12)--Mark (X)this column ifentries in column (10) and (I I)are confidential business information (CBI). Worker Activity CBI Protective Equipment! Binding Physical form(s) Binding CBI # of CBI I Maximum Duration CHI (eg., bagdumping, filling drums) Enginecring Controls Option (eg. solid, powder) Option Workers Mark (X) and % new Mark (X) Exposed substance Hrs/day Days/yr (I) (2) (3) (4) (5) (6) (7) (8) (9) (10) (II) ( 12) N/A

I 

# D Mark (X) this box ifyou attach a continuation sheet. 3. Environmental Release and Disposal

- You must make separate confidentiality claims for the release number and the amount ofthe new chemical substance released

and other release and disposal information. Mark (X) the "Confidential" box next to each item you claim as confidential. (l)-- Enter the number ofeach release point identified in the process description, part II, section A subsection Id(3) (2)-- Estimatethe amount ofthe new substance released (a) directly to the environment or (b) into control technology (in kg/day or kg/batch). (3)-- Mark (X) this column ifentries in columns (I) and (2) are confidential business information (CBI). (4)-- Identify the media i.e. stack air, fugitive air (optional-see Instruction Manual), surface water. on-site or off-site land orincineration, POTW, orother (please specify) to which the new substance will be released from that release pom!. (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. characterize the disposal method and state whether it is approved for disposal ofRCRA hazardous waste. On a continuation sheet for each site describe any additional disposal methods that will be used and whether the waste is subJect to secondary or tertiary on-site treatment. b. Estimate the amount released to theenvironment aftercontrol technology (in kg/day). (6)-- Mark (Xl this column ifentries in columns (4) and (5) are confidential business information (CBI). (7)-- Identify the destination(s) ofreleases to water. Please supply NPDES (!'Jational Pollutant Discharge Elimination System) #s for direct dischargers orNPDES numbers ofthe POTW (Publiciy Owned Treatment Works). Mark (X) ifthe POTW name or NPDES # is confidential business information (CBr). Release Amount ofnew substance CBI Media of Control technology and efficiency (you may with to optionally attach efficiency data) CBII Number released Release (eg. stack air) (2a) (2b) I ( I ) (3) (5a) I Binding (5b) (6) (4) I Mark (X) 

### i I I I I I I I I I I I I I I I (7) Mark (X) the 

# D POTW provide name(s) below: CBI 

# D Navigable 

# D Other

- Specify Provide NPDES # eBr destination(5) of f-­ releases to water. Waterway 

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

## 00001.0 FORM EPA 7710-25 (Rev. 5-95) Page 9PartII

- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

- Continued Section B

- INDUSTRIAL SITES CONTROLLED BY OTHERS Complete section B for typical processing or use operations involving the new chemical substance at sites you do not control. Importers do not have to complete this section for operations outside the U$.; however, you must report any processingoruse activities after import. See the Instructions Manual. Complete a separate section B/oreach type o/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 a continuation sheet. I. Operation Description To claim information in this section as confidential, circle or bracket the specitic information that you claim as confidential. (I) Diagram the major unit operation steps and chemical conversions, including interim storage and transport containers (specify

- e.g. 5 gallon pails, 55 gallon drums, rail cars, tank trucks, etc.). On the diagram, identify by letter and briefly describe each worker activity. (2) Provide the identity, the approximate weight (by kg/day or kg/batch. on a 100% new chemical substance basis) and entry point ofall feedstocks (including reactants, solvents, and catalysts, etc.) and ofall products, recycle streams, and wastes. Include cleaning chemicals (note frequency ifnotused daily or per batch). (3) Identify by number the points ofrelease, including small or intermittent releases, to the environment ofthe new chemical substance. (4). Please enterthe number ofsites (rememberto identify the locations ofthese sites on a continuation sheet): User Sites [ I # ofsites See continuation page IO(A)



## [3 Mark (X) this box ifyou attach a continuation sheet. 2. Worker ExposurelEnvironmental Release (I) From the diagram above, provide the letter for each worker activity, Complete 2-8 for each worker activity described. (2) Estimate the numberofworkers exposed for all sites combined. (4) Estimate the typical duration ofexposure per worker in (a) hours per day and (b) days per year. (6) Describe physical form ofexposure and % new chemical substance (ifin mixture), and any protective equipment and engineering controls used to protect workers. (7) Estimate the percent ofthe new substance as formulated when packaged orused as a final product. (9) From the process diagram above, enterthe numberofeach release point. Complete 9-13 for each relea.'ic point identified. (10) Estimate the amount ofthe new substance released (a) directly to the environment or (b) into control technology to the environment (in kg/day orkg/batch). (12) Describe mediaofrelease i.c. stack air, fugitive air (optional see Instructions Manual), surface water, on-site oroff-sitc land or incineration, POTW, or other (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), (11), (13), and (15)

- Mark (X) this column ifany orthe preceding entries are confidential business information (CBI), Letterof Activity

(I) # of Workers Exposed

(2) CBI

(3) Duration of Exposure CBI
(5) Protective EquipJ Engineering Controls! Physical Form and % new substance (6) %inFormulation

(7) CHI
(8) Releasc Number

(9) Amount ofNew Substance Released CBI
(11 ) Control Technology (12) CBI
(13) (4a) (4b) (lOa) kg/batch total all

sites (lOb) kg/batch total all

sites X X X X X X X X X X X X X 

### xi X X X X X (14)

- Byproducts ( 15) 

## [3 Mark (X) this box ifyou attach a continuation sheet. See continuation pages IO(A) FORM EPA 7710-25 (Rev. 5-95) Page 10 

## 0000:11

### Continuation Sheet ContainsTSCAConfidential Business Information PartII

- HumanExposure and EnvironmentalRelease Section B.1 & 

### B.2



## 00001-2 10 (A)

### Continuation Sheet Contains TSCA Confidential Business Information
OPTIONAL POLLUTION PREVENTION INFORMATION To claim information in this section as confidential circle orbracket the specific information that you claim as confidential. 

### Inthis sectionyou may provide infonnation not reported elsewhere in this fonn regarding your efforts to reduce orminimize potential risks associatedwith activities surrounding manufacturing, processing, use and disposal ofthe PMN substance. Please include new infonnationpertinentto pollution prevention, including source reduction, recycling activities and saferprocesses orproducts available due tothe new chemical substance. Source reduction includesthe reduction in the amount oftoxicity ofchemical wastes by technological modification, process andprocedure modification, product refonnulation, raw materials substitution, and/or inventory control. Recycling refers to the reclamation ofuseful chemical components from wastes that would otherwise be treated orreleased as air emissions orwater discharges, orland disposal. Descriptions ofpollution prevention, source reduction and recycling shouldemphasize potential risk reduction subsequentto compliance with existing regulatoryrequirements and canbe either quantitative orqualitative. TheEPA is interested in this infonnationto assess overall net reductions in toxicity ofenvironmental releases and exposures, not the shifting ofrisks to otherenvironmental media ornon-environmental areas (e.g., occupational orconsumerexposure). In addition, infonnation onthe relative cost orperfonnance characteristics ofthe PMN substance to potential alternatives may beprovided. All information provided in this section will be taken into consideration during the review ofthis substance. See the revised Instructions Manual that includes a Pollution Prevention manual for guidance and examples. Describe the expected net benefits. such as (1) an overall reduction in risk to human health orthe environment; (2) a reduction in the volume manufactured; (3) a reduction in the generation ofwaste materials through recycling. source reduction or othermeans; (4) a reduction in potential toxicity ofhuman exposure and/or environmental release: (5) an increase in product performance. a decease in the cost ofproduction and/or improved operation efficiency ofthe new chemical substance in comparison to existing chemical substances used in similar applications; or (6) the extent to which the ncw chemical substance may be a substitute toran existing substance that poses a greater overall risk to human health orthe environment. [

J 

# 0 Mark (X) this box ifyou attach a continuation sheet. FORM EPA 7710-25 (Rev. 5.95) Page 11 VUUU..l.uContinuation Sheet Contains TSCA ConfidentialBusiness Information


### Part III -- LIST OFATTACHMENTS

Attach continuation sheets for sectionsofthe fonn and test data and other data (including physical/chemical properties and structure/activity intonnation), and optional infonnation after this page. Clearly identify the attachment and the section ofthe form to which it relates. if appropriate. Numberconsecutively the pages ofthe attachments. In the column below. enterthe inclusive page numbers ofeach attachment. Mark (X) in the "Confidential" box next to any attachment name you claim as confidential. Read the Instructions Manual for guidance on how to claim any infonnation in an attachment as confidential. You must inelude with the sanitized copy ofthe notice fonn a sanitized version ofany attachment in which you claim infonnation as contidential Attachment name Attachment page 

### I Conlinumbcrs(s) dential X Physical and Chemical PropertiesWorksheet 13 14

- 16 X Appendix 1 - HPLC 17

- 23 X Appendix 2 - IES Reportwith attachments Appendix 3 - Material Safety Data Sheet(MSDS) 24

- 27 X 28-41 X Appeudix 4 - Acute Dermal Irritation/Corrosion

- OECD 42-62 X Appendix 5 - Acute Eye Irritation/Corrosion

- OECD Appendix 6·Acute Oral Toxicity Test (I;p-aud-Down Procedure·OECD) 63·74 X Appendix 7 •Acute Dl!rmal Toxicity

- OECD 75-88 X 89-106 ApPl!ndix 8 - BUl!hler Sl!usitization Tl!st OECD X ApPl!ndix 9 . Salmonella Typhimurium and Escherichia Coli Reverse Mutation Assay· OECD 107-125 X 

### I



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

## 00001.4Continuation Sheet Contains TSCA Confidential Business Information PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET To assist EPA's review ofphysical and chemical properties data, please complete the following worksheet for data you provide and include it in the notice. Identify the property measured, the page ofthe notice on which the property appears, the value 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 or formulations should be so noted (% PMN substance in**~).** You are not required to submit this worksheet; however, EPA strongly recommends that you do so, as it will simplify review and ensure that confidential information is properly protected. You should submitthis worksheet as a supplement to your submission oftest data. This worksheet is not a substitute for submission oftest data. Property

(a) Mark (X) ifprovided Page number

(b) Value (c) Measured or Extimate (M orE) Physical state ofneat substance Vapor pressure @ Temperature "C __ (s)

__ (I)

__ (g) Torr
Density Irelative density Solubility @ Temperature Solvent "C giL
Solubility in water @ Temperature Melting temperature "C gllOOL "C
Boiling I sublimation temperature @ torr pressure Spectra X
Dissociation constant
Particle size distribution
Octanollwater partition coefficient Henry's Law constant
Volatilization from water
Volatilization from soil pH @ concentration -1% (of[]%in [] M Flammability Explodability Adsorption I coefficient
Other

- Specify Confidential Mark(X)

(d)

Page 13 

## 000015 X

X

XContinuation Sheet Contains TSCA**~usiness** Information**~"h~c** IJ

Appendix 1 HPLC Chromatograph



## 000016 Page 14

# ANALYTICAL METHOD ANALYSIS BY HPLC HPLC Model Column

Eluent Detector Injection Volume Flow rate Sample Concentration Time for analysis HP 

### 1100 ZORBAX ODS 4.6 mm 

### x25 cm MeOH I Water UV255nm 

### 10 j.tI 

### 1.0 mllmin 

### 10 mg in 5 ml MeOH ca. 

### 40min



## 00001.7 

# 15Injection Date Sample Name Acq. Operator Acq. Method Last changed Analysis Method Last changed 11/14/03 3:00:57 PM Vial 1 C:\HPCHEM\2\METHODS\1176N.M 11/14/03 3:41:14 PM (modified after loading) C:\HPCHEM\2\METHODS\1176N.M 5/20/04, 4:30:22 PM (modified after

l'1li 1 NomI, 3CIOO
3OQO
2SOO
:IGOIl
1500
100G
500

0
...... , .u .._, 

## '.-, 

# . , 

### ., , i I 7 I I I 5 10 UI 30 IIIIIi 

### , I 31**~** Area Percent Report Sorted By Multiplier Dilution Signal
__ 1.0000 1&0000 Signal 1: VWDl A, Wavelength=255 nm Peak**RetTi~eType** -Width t [min] [min] Area mAU *5 .. Height [mAU 1 Area

,**----I-------I-----------I-------~-I----------i------1** 

# . 1 2 ­ 3 4 5 6 7 8 

# lB· 11 Totals

Results obtained with enhanced Integracor! *** End of Report ***



## 00001.8 

# 16Continuation Sheet Contains TSCA**~siness** Information 

### SelM

### crl-z..eJ

Appendix 2 lES Report



## 000019 Page 11

# InventorY.. 

# Exeert Service

A division of theAmerican Chemical Society Phone: 800-631-1884. 614·447·3870 Fax: 614-447-3747 E-mail: answers@cas.org Internee http://www.cas.org/Supportldient.html

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 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.



## 000020 

# 18

### · 00002:1 

# 19

### l

0

0**~** CI:) • 0.

-.j U1

....., c.

en U'I

en

(Jt (Jt

U1



### .... UI
"CA."CXO**~**

W U1

W 0

# ~1 0 

### 1


## .. 

# :j

o _

U'I

0 0 PPM



# i 000022 

# 20

### [ Mass Spectrum ] Date 08-0ct-73 16:40 

### Data : FAb-81B9**.~** Sample: C\t 

### Note : 

# * Inlet: DIrect 

### Ion Mode FAB+ 

### Spectrum Type : 

### Regu Iar (MF-Li

### near] RT : .2. 10 min Scan. : 

### 19 Temp : 13. 1 del). C C"'j 

### BP : m/z 

### 427.0000 Int.: 

### 700.48 N 

### Output m/z range: 

### 45.0000 to 1200.0000 Cut 

### Level: 0.00 % 

# o o 7345024 

# o -, 100 

# -

90 

### 83· 

### 70
60 

### 50 

### 40 3 

### 30 

### 20
10 1

,...... L "•••,.. .. T' t I ','

## .*1, "e. 

### 0-, I I 1 I r I II 300 400 500 600 700 800 900 IOOVl 1100 1200 100 200 m/z

## 000024 

# 22To. CASInventoryExpert Service Pax: ++1-614-447-3747 JenniferMoore Da
..: 2003111114

.. CASNo. registration 1

I:::J u......t



### BestRegards,



## 000025 

# 23Continuation Sheet Contains TSCA**C~iness** Information Scvvt

### ltr2
..

## <.J

Appendix 3 Material Safety Data Sheet

Page 24 

## 000026MATERIAL SAFETY DATA SHEET

1. Substance/preparation and company name Trade name: Company:

TEL: FAX: ; 2. Composition/information on ingredients Component CAS. Amount r/.WIW)
:0:::40% Propylene carbonate 108-32-7 :2:60% 3. Possible hazards May causeseye irritation May cause allergic skin reaction 4. First aid measures IfInlHlled: Remove to fmsh airand summon medical help if respiratory irritation develops or if breathing becomes difficult. On skIn contact: Wipe away excess material with drytowel and then wash affected areaswith plenty ofwater for several minutes. Getmedical attention immediately ifirritation occurs. On contactwlfh eyes: Immediatelywash affected eyesfor several minutes underrunning waterwith eyelids held open. Get medical attention immediately if irritation occurs. Ifswallowed: Wash out mouth with water and then drink plenty ofwater, and summon physician. Do notgive anything by mouth In an unconsciousorconvulsing person. 5. Firefighting and measures Flash point : >140 °C (284 OF) (Closed Cup) Flammable limits : Notapplicable Decomposition temp. : >100 °C. Extinguishing media : Carbon dioxide. foam, dry chemical. waterspray. Fire fighting procedures-speciat : Use self-contained breathing apparatus.

REVISED DATE: JANUARY 1.2004 1/3

### ~.Q:;121 

## 0000276.

7.

8.

9.

10.

11.

12. 13. MATERIAL SAFETY DATA SHEET

Accidental release measures Wear appropriate personal protective equipment, including appropriate respiratory protection to ventilate contaminated area. Contain spill if possible. Wipe up or absorb on suitable material and shovel up, Prevent entry into sewers and waterways. Avoid contactwith skin, eyes orclothing, Handling and storage Handling precautions: In accordance with good industrial practice, handle with care and avoid unnecessary personal contact. Avoid contactwith eyes and prolonged or repeated skin contact. isfor industrial use only. Shipping and storing precautions: Keep container tightly closed in cool condition INhen not in use and during transport Personal hygiene: Washthoroughly after handling. Exposure controls and personal protection Protective equipment: Eye protection : Safety goggle isrecommended. Skin protection : Glove and lab coat is recommended. Respiratory protection : Suitable mask is recommended INhere vapor arises from use. Physical and chemical properties Appearance Specific gravity Boiling point Vapor pressure pH value Viscosity Soluble in water Stabilityand reactivity Stability: Stable. Incompatibilities: Mineral acid. Avoid contactwith excessive heat, open flame, sparks, or ignition source. Avoid exposure to sunlight, ultraviolet lightorfluorescent light. Hazardous decomposition: Toxic fumes ofcarbon dioxide, carbon monoxide, sulfur oxides, and hydrogen fluoride gas. Toxicological information Acute effects May be harmful by inhalation, ingestion, orskin absorption. May cause eye and skin irritation. May be irritating to mucous membranes and upper respiratory tract. Ecological information Data notyet available. Disposal considerations Waste disposal method: Incinerate in a chemical incineratorequipped with an afterbumerand scrubber. Followall federal, state and local regulations,

2/3 

## 000028**WI-MS1~6** REVISED DATE: JANUARY 1 , 2004MATERIAL SAFETY DATA SHEET

14. Transport information DOT proper shipping name: Not regulated as a hazardous material by the U.S. dept oftransportation (DOT) 49 CFR 172.101 HAZADOUS MATERIAL TABLE. DOT class: None. DOT number: None. HMIS Rating Health: 2 Flammability: 1 Reactivity : 1 Protective equipment: B NFPA Rating Health: 2 Flammability: 1 Reactivity : 1 Special:
­ EmergencyTEL: 15. Regulatory information 15.1 FEDERALI NATIONAL RCRA status: Nota hazardous waste under RCRA (40 CFR 261). CERCLA status: Not listed. SARAITITLE III - TOXIC CHEMICALS LIST: This productdoes notcontain a toxic chemical for routine annual toxic chemical release reporting under sec. 313 (40 CFR 372). 15.2 STATE I LOCAL CALIFORNIA SCAQMD RULE 443.1(SOUTH COAST AIR QUALITY MANAGEMENT DISTRICT RULE 443.1, LABELING OF MATERIALS CONTAINING ORGANIC SOLVENTS) VOC: Vapor pressure <0.03 mmHg(l20 °C 699 gllVOC 16. Other information Shelf life: One-year minimum in sealed containers protected from light and heat Suggested uses: A cationic photoinitiator for**UV~urable** systems.
The information and recommendations contained herein are based upon data believedto be correct However. no guaranty or warranty ofany klnd expressed or implied is made with respectto the information contained here.

REVISED DATE JANUARY 1.2004 3/3 

## 000029 W'-MS1227Continuation Sheet Contains TSCA

### Co~usiness Information SaM

### \.1.

### '"l.-e.J

Appendix 4 Acute Dennal Irritation/Corrosion

- OECD

Page 28 

## 000030TOXIKON FINALREPORT:
ACUTE DERMAL IRRITATION/CORROSION

- OEeD
Autbor Shirley Lister. B.S.• LATG
Final 

### Report Date



### March 02, 2004

MANAGEMENT OF THE STUDY Performing Laboratory

Toxikon Corporation

15 Wiggins Avenue Bedford. MA 01730
Sponsor



## 000031 

# 29Toxikon Corporation Acute Dermal Irritation/Corrosion

- OBeD Project Number: 

### TABLE OF CONTENTS Title Page Table ofContents Study Summary Quality Assurance Statement Study Director Signature and Verification Dates 1.0 Purpose 2.0 References 3.0 Compliance 4.0 Identification ofTest and Control Substance 5.0 Identification ofTest System 6.0 Justification ofTest System and Route ofAdministration 7.0 Experimental Design 8.0 Dosage 9.0 Evaluation Criteria 10.0 Results 11.0 Conclusion 12.0 Records 13.0 Confidentiality 14.0 Policy on Pain and Suffering in Animals 15.0 Animal Usage Table I: Body Weights/Clinical Observations Table II: Skin Irritation Scores Appendix I: Grading for Skin Reactions



### 000032 

## 30 Page 2 of 13TomonCorporation Acute Dermal Irritation/Corrosion

- OECD Project Number: 

### STUDY SUMMARY
The test substance, •was evaluated for its potential to produce primary dennal irritation or corrosion after a single topical 4 hour application to the skin ofNew Zealand White rabbits. The test substance is considered a negligible irritant.



### 000033 

# 31Toxikon Corporation Acute DermallrritationiCorrosion

- OECD Project Number: 

### QUALITY ASSURANCE STATEMENT
This study was conducted in compliance with U.S. Food and Drug Administration regulations set forth 

### in21 CFR. Part 58 and OECD GLPs. 1982. The sections ofthe regulations not performed by orunder the direction ofToxikon Corporation, exempt from this Good laboratory Practice statement, include characterization and stability ofthe test and its mixture with carriers. 21 CFR. Part 58.105 and 58.113. The Quality Assurance Unit conducted inspections on the following dates. The findings were reported to the Study Director and to Toxikon's management.
DATE OF DATE REPORTED DATE REPORTED INSPECTIONS INSPECTION STUDY DIRECTOR MANAGEMENT ANIMAL 01128/04 01128/04 01128/04 

### WEIGHTS RAW DATA 03/02/04 03/02/04 03/02/04 

### FINAL REPORT 03/02/04 03/02/04 03/02/04

# '-~k~ C8f6?/1Lf Felice Randi LaMadeleine, B.S. Date Quality 

### Assurance



## 000034 

# 32TomonCor,'oration Acute Dermal Irritation/Corrosion

- OECD Project Number: STUDY DIRECTOR SIGNATURE AND VERIFICATION DATES
This study meets the technical requirements ofthe protocol. The study also meets with the requirements ofthe Good Laboratory Practice Regulations, 21 CFR, Part 58, with the exemptions as stated 

### in the Quality Assurance Statement.
Protocol Number: CHT/VIVO/OOI-03/000 Study Director: Shirley Lister, B.S., LATG Company: Toxikon Corporation
Signature: Date: Study Supervisor: William Entwistle, A.S., LATG
VERIFICATION DATES: The Toxikon Protocol Effective Date is the GLP Study Initiation Date. The study dates were as follows: Protocol pffective Date: 12/24/03 Test Substance Receipt: 12/24/03 Project Log Date: 01/08/04 Technical Initiation: 01128104 Technical Completion: 

### Ofl31104



## 000035 

# 33 Pa.e;e 5 of 13Toxikon Corporation Acute Dermal Irritation/Corrosion

- OBCD Project Number: 1.0PURPOSE Thepurpose ofthe test is to evaluate the potential ofthe test substance to produce primary dermal irritation or corrosion after a single topical 4 hour application to the skin ofalbino rabbits. 

### 2.0 REFERENCES The test was conducted based upon the following references: 2.1 Organization for Economic Co-operation and Development (OECD), Guidelines for Testing ofChemicals, "Acute Dermal Irritation/Corrosion". Section404, 2002. 

### 3.0 COMPLIANCE The study conformed to all applicable laws and regulations. Specific regulatory requirements included the current OECD GLPs; AAALAC, International; "Guide for the Care and Use of Laboratory Animals," National Research Council. 1996. (NIH) (OLAW). "Public Health Service Policy onHumane Care and Use ofLaboratory Animals." Health Research Extension Act of 1985 (Public Law 99-158 November 

### 20. 1985), Reprinted 1996; USDA, Departmentof Agriculture, Animal and Plant Health Inspection Service, 9 CFR Ch. 1 (1-1-95 edition), Subchapter A-Animal Welfare. 

### 4.0 IDENTIFICATION OFTHE TEST AND CONTROL ARTICLES The following information was supplied by the Sponsor wherever applicable; itdoes not apply to confidential information. The Sponsor was responsible for all test substance characterization data as specified in the GLP regulations. Test and control substances (exclusive ofextracts) that are mixed with carriers require verification ofconcentration. homogeneity, and stability. Samples oftest and control substance mixtures shall be returned to the Sponsor for characterization. 

### 4.1 Test Substance Name: CAS/Code #: Not Suppliedby Sponsor (N/S) Lot/Batch

### #: Physical State: Color: Expiration Date: 12/23/2005 Density: Stability: stable in 

### dark and cool closed containers Solubility: pH: N/S Storage Conditions: Room Temperature Safety Precautions: StandardToxikon Laboratory Safety Precautions


## 000036 

# 34 Page 6 of 13Toman Corporation Acute Dermal Irritation/Corrosion

- OECD Project Number: 

### 4.2 A control article was not utilized. Untreated skin ofeach animal served as it's own control. 5.0IDENTIFICATION OF THE TEST SYSTEM 5.1 Animals Used in the Test: Number and species: 3 New Zealand White rabbits (Oryctolagus cuniculus) 

### Sex: 1male and 2 females Weight/Age range: 2.11

- 2.37 kilograms / at least 10 weeks old (adult) Weighed to the nearest 10 gm Animal identification: ear marker Health status: healthy, unused in other experimental procedures Animal purchase: Millbrook Breeding Labs, Amherst, MA Acclimatization: minimum of5 days under the same conditions as for the actual test. Animal selection: selected from a larger pool ofanimals and observed to ensure lack of adverse clinical signs. 5.2 Animal Care and Maintenance Housing: individually housed Cages: suspended stainless steel Bedding: hardwood chips, P.W.I. Industries, St-Hyacinthe, Quebec, Canada (non-contact) Animal room temperature: 68 

### ±50P Animal room relative humidity: 30

- 70% Air exchanges per hour: 10 to 15 Lights: 12-hour light/dark cycle, full spectrum fluorescent lights The laboratory and animal rooms were maintained as limited-access facilities. Animal rations: TEK 8630 Rabbit Diet, Harlan Teklad, Madison, WI, adlibitum 

## 000037 Page 7 of 

### n 

# 35TomonCorporation Acute Dermal Irritation/Corrosion

- OECD Project Number: 

### Water: tap water, 

### adlibitum There were no known contaminants present in the feed, water, or bedding expected to interfere with the test data. 6.0JUSTIFICATION OF TEST SYSTEM AND ROUTE OF ADMINISTRATION 6.1 In vivo irritation testing was not undertaken until all available data relevant to the potential dennal corrosivity/irritation ofthe substance had been evaluated in a weight-of-the-evidence analysis. This data includes evidence from existing studies in humans and/or laboratory animals, evidence ofcorrosivity/irritation ofone ormore structurally related substances or mixtures ofsuch substances, strong acidity or alkalinity ofthe substance and results from validated and accepted 

### in vitro or 

### ex vivotests. It was the Sponsor's responsibility that these data, ifavailable be provided to Toxikon prior to testing. 6.2 Rabbits was used in this study because they have historically been used indennal safety evaluation studies. Dennal application corresponds to the likely route ofhuman exposure. The species and number ofanimals used was recommended by the OECD guidelines. 7.0EXPERIMENTAL DESIGN 7. 1 Pretreatment Screening Procedure and Selection ofAnimals Animals selected for the study were examined to ensure that their skin is free from irritation, trauma, and disease. 7.2 Preparation ofTest Animals The application sites were prepared by clipping the skin ofthe trunk free ofhair approximately 24hours before application ofthe test substance. The site ofapplication was not abraded intentionally or accidentally during preparation. Not less than 10% ofthe body surface was clear for application ofthe test substance. 7.3 Test Substance Application 7.3.1 The test substance was applied to a small area (approximately 6 cm2) 

### ofskin. Each application area was covered with a gauze patch, which was held inplace with non-irritating tape. Thepatch was loosely held in 

### contact with the skin for the duration ofthe exposure period. Care was taken to prevent access by the animal to the patch. which may result in 

### accidental ingestion/inhalation ofthe test substance. 7.3.2 Initial test Based on the suspicion that the test substance might produce severe irritancy or corrosion. a single animal 

### test was employed. Three test patches were applied simultaneously to the animaL The first patch was removed after three minutes. Since no serious skin reaction was 

### 0000.18**P~(JpR('\fl1** 

# 36TomonCorporation Acute DermallrritationlCorrosion

- OECD Project Number: 

### observed, the second patch was removed after one hour. If

### a corrosion effect was observed after either the three-minute or one-hour exposure, the test would be immediately terminated by removal ofthe remaining patches. The observation at this stage indicated that exposure could humanely be allowed to extend to four hours so the third patch was removed after four hours and the responses graded. <0 

### 7.3.3 Confirmatory Test As a corrosive effect was not observed in the initial test, the response was confirmed using two additional animals, each with one patch only, for an exposure period of4 hours. 7.4 After the 4 hour exposure period, animals were observed for signs oferythema and edema at 

### 60 minutes, and then at 

### 24, 48, and 

### 72 hours after patch removal. Observations were scored according to the "Grading ofSkin Reaction" (Appendix I). 

### Daily clinical observations include all toxicologic and pharmacologic signs. 

### 7.5 No therapeutic agents were used in any phase ofthe study. 

### 7.6 Animals were weighed at the end ofthe observationperiod and were sacrificed with an injectable barbiturate. Information was recorded in the raw data and was provided in the fmal report. 

### 8.0 DOSAGE The test substance was applied directly to the skin ofthe rabbits as supplied by the Sponsor. A dose of

### 0.5 

### mL was applied to each application site. 

### 9.0 EVALUATION CRITERIA 9.1 Evaluation ofAnimal Data Erythema and edema scores (see Appendix I) were determined for each of3 individual animals at 

### 60 minutes, 

### 24, 48, and 

### 72 hours after patch removal (at a minimum). 

### 9.2 Evaluation ofTest Results Dermal irritants are those test substances that produce reversible changes in the derma. Those test substances that destroy the structure ofthe intact skin orchange it irreversibly are considered corrosive. The dermal irritation scores were evaluated in conjunction with the nature and reversibility or otherwise ofthe responses observed. 

### 10.0 RESULTS 10.1 Body Weights (Table I) All ofthe test animals exhibited a gain in body weight during the study. 

# 37 000039Tomon Corporation Acute DermallrritationlCorrosion

- OECD Project Number: 

### 10.2 Clinical Observations (Table I) No overt signs oftoxicity were evident in any ofthe animals during the course ofthe study. 

### 10.3 Dermal Irritation (Table 

### II) In both the initial test and the confirmatory test there were slight signs oferythema at one hour after the four hour exposure period. No signs oferythema oredema were present at the 

### 24,48 or the 72 hour observation points in any ofthe animals. None ofthe control sites ofany animal at any ofthe observationperiods showed signs oferythema or edema. 11.0 CONCLUSION The test substance, , was evaluated for its potential to produceprimary dermal irritationor corrosion after a single topical 

### 4 hour application to the skin ofNew Zealand White rabbits. The test substance is considered a negligible irritant. 12.0RECORDS 

### 12.1 Original raw data 

### is archived at Toxikon Corporation. 

### 12.2 A copy ofthe final report and any report amendments is archived at Toxikon Corporation. 

### 12.3 The original final report and a copy ofany protocol amendments ordeviations is forwarded to the Sponsor. 

### 12.4 All 

### unused test substance shall be disposed ofby Toxikon, per Sponsor's specifications. 

### 12.5 Final reports shall not be reproduced except in 

### full, without the written authorization/approval from Toxikon 13.0 CONFIDENTIALITY AGREEMENT Statements ofconfidentiality may be agreed upon prior to study initiation. 14.0POLICY ON PAIN AND SUFFERING IN ANIMALS No evidence ofpain and suffering was observed in the animals. 15.0 ANIMAL USAGE The Sponsor assures that to the best oftheir knowledge this study does not unnecessarily duplicate previous testing.



## 000040 

# 38 Page 10 

### of 13Toxikon Corporation Acute DermalIrritation/Corrosion

- OECD Project Number:
TABLE I Animal Weights and Clinical Observations
Test Substance: Lot/Batch#:
Body Weight (Kg) Day 0 Day 3 Weight Signs of Animal 

### # Sex 01127/04 01/30104 Change Toxicity

40072 Female 2.37 2.42 0.05 none

Body Weight (Kg) Day 0 Day 3 Animal # Sex 01/27/04 01130104

40109 Male 2.14 2.18 0.04 none 40110 Female 2.11 2.16 0.05 none 

## * Excluding erythema and edema observations at 60 minutes after the 4 hour exposure, 24, 48 and 72 h.



## 000041 

## 39 P':In

.. 11 "f**1~**• VA.t!\.UH l._UJjJ\.ll ulhJU Acute Dermal Irritation/Corrosion

- OECD Project Number: TABLE II SKIN IRRITATION SCORES Test Substance: LotlBatch 

### #:


# --_. .._.....

.

# _------

_ .....

# _­ 01127/04 60 Minutes* ANIMAL 

### # Erythema Edema 40072 

### 1/0 0/0 01128/04 01129/04 24 Hours 48 Hours Erythema Edema Erythema Edema 

### 0/0 0/0 0/0 0/0 01/30/04 72 Hours Erythema Edema 

### 0/0 0/0

Test Sites I Control Sites
ANIMAL# 

### 01128/04 60 Minutes* Erythema Edema 

### 01129/04 24 Hours Erythema Edema 

### 01130/04 48 Hours Erythema Edema 01131104 72 Hours Erythema Edema 40109 110 

### 0/0 0/0 0/0 0/0 0/0 0/0 0/0 40110 

### 1/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 

# g *Observations made 60 minutes after four hour exposure 

# o o**~** 

### N Page 12 of 13



## o**~**
•Toxik.on Corporation Acute Dermal Irritation/Corrosion- OECD Project Number:
APPENDIX 1: Grading ofSkin Reactions
Reaction Erythema and Eschar Formation No erythema
........................................................ Very slight erythema (barely perceptible)
..................... Well defined erythema
............................................ Moderate erythema
................................................ Severe erythema (beet redness) to slight eschar formation

(injuries in depth)
................................................ Total possible erythema score 

## = 4 Edema Formation 

### 0 1 2 

### 3 4

No edema............................................................ 0 Very slight edema (barely perceptible)
. . . . . . . . . . . .... . ........ 

### 1 Slightedema (edges are well defined by definite raising)
.. 

### 2 Moderate edema (raised approximately 

### 1 mm)

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

### 3 Severe edema (raised more than 1 mm and extending beyond

area ofexposure)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Total possible edema score = 4

Page 13 of 13 

## 000043 

# 41

### Continuation Sheet Contains TSCA

### C~usiness Information**~ltl7.-d**

Appendix 5 Acute Eye Irritation/Corrosion- OECD



## 000044 Page 42

### TOXIKON FINALREPORT:

ACUTE EYE IRRITATION/CORROSION- OECD

Author Kathleen Krenzer, O.D., Ph.D.

Final Report Date February 25, 2004

MANAGEMENT OF THE STUDY Performing Laboratory

Toxikon Corporation

15 Wiggins Avenue Bedford, MA 01730 Sponsor



## 000045 

# 43Toxikon Corporation Acute Eye Irritation/Corrosion

- OECD Project Number:



### Title Page Table ofContents Study Summary Quality Assurance Statement TABLE OF CONTENTS

Study Director Signature and Verification Dates 1.0 Purpose 2.0 References 3.0 Compliance 4.0 Identification ofthe Test and Control Substances 5.0 Identification ofthe Test System 6.0 Justification ofTest System and Route ofAdministration 7.0 Experimental Design 8.0 Dosage 9.0 Evaluation Criteria 10.0 Results 11.0 Conclusion 12.0 Records 13.0 Confidentiality Agreement 14.0 Policy on Pain and Suffering 

### in Animals 15.0 Animal Usage Table 1: Animal Weights and Clinical Observation Table II: Ocular Lesion Scores and Fluorescein Examination Appendix 1: Testing and Evaluation Strategy for Eye Irritation/Corrosion Appendix II: Grading for Ocular Lesions Appendix III: Fluorescein Staining Grading Scale

Page 2 of20 

## 000046 

# 44Toxikon Corporation Acute Eye Irritation/Corrosion

- OECD Project Number: 

### STUDY SUMMARY
The test substance,
. was evaluated for its potential to produce an irritating effect on the ocular tissue of New Zealand White rabbits, when administered according to the procedures detailed in the study protocol. Slight signs ofirritation (conjunctival redness) were noted in the treated eyes oftwo ofthe animals at the 1 hour time point. All signs resolved in all animals by the 48 hour observation point. No signs ofirritation were present in any ofthe control eyes. Based on the methods employed, the test substance is considered to be a slight irritant to the ocular tissue ofNew Zealand White rabbits.



## 000047 

# 45 Page 3 of20Toxikon Corporation Acute Eye IrritatioDlCorrosion

- OECD ProjectNumber: QUALITY ASSURANCE STATEMENT
This study was conducted. in compliance with U.S. Environmental Protection Agency regulations set forth in 40 CFR, Part 792. The sections ofthe regulations not perfonned by or under the direction ofToxikon Corporation, exempt from this 

### Good Laboratory Practice Statement, included characterization and stability of the test substance and its mixture with carriers, 40 CFR, Parts 792.105 and 792.113. The Quality Assurance Unit conducted inspections on the following dates. The findings were reported to the Study Director and to Toxikon1s Management.

INSPECTIONS DATE OF DATE REPORTED DATE REPORTED INSPECTION STUDY DIRECTOR MANAGEMENT CLINICAL OBSERVATIONS 02/06/04 02/06/04 02/06/04 RAW DATA 02/25104 02/25104 02/25/04 FINAL REPORT 02125104 02125104 02/25104

# ~eCVNJ,L**'M~** Felice Randi LaMadeleine, B.S. Quality Assurance



## 000048 Page 4 of20 

# 46Toxikon Corporation Acute Eye Irritation/Corrosion

- OECD ProjectNumber: STUDY DIRECTOR SIGNATURE AND VERIFICATION DATES
This study meets the technical requirements of the protocol. The study also meets with the requirements of the Good Laboratory Practice Regulations, 40 CFR, and Part 7CJ2 with the
... exceptions as stated in the Quality Assurance Statement.
Protocol Name: CHT

### NIVO/OO2-03/000 Study Director: Kathleen Krenzer, 0.0., Ph.D. Company: Toxikon Corporation
Signature: Study Completion Date: Study Supervisor: William Entwistle, A.S., LATG
VERIFICATION DATES: The Toxikon Protocol Effective Date 

### is the GLP Study Initiation Date. The study dates were as follows: Protocol Effective Date: 12/29/03 Test Article Receipt: 12/24/03 Project Log Date: 01108104 Technical Initiation: 02/03/04 Technical Completion: 02/06/04



## 000049 Palle 5 of20Toxikon Corporation Acute Eye Irritation/Corrosion

- OECD Project Number: 1.0 PURPOSE The purpose ofthis test was to evaluate the potential ofthe test substance to produce an irritating and/or corrosive effect on the ocular tissue ofalbino rabbits. 2.0 REFERENCES The test was conducted based upon the following reference: 

### 2.1 Organization for Economic Co-operation and Development (OECD), Guideline for Testing ofChemicals, "Acute Eye Irritation/Corrosion" Section 405, Adopted 24 April, 2002. 

### 3.0 COMPLIANCE The study conforms to all applicable laws and regulations. Specific regulatory requirements include the current OECD GLPs; AAALAC, International; the "Guide for the Care and Use of Laboratory Animals," National Research Council, 1996; (NIH) (OLAW), "Public Health Service Policy on Humane Care and Use ofLaboratory Animals," Health Research Extension Act of 1985 (Public Law 99-158 November 20, 1985), Reprinted 1996; USDA, Department of Agriculture, Animal and Plant HealthInspection Service, 9 CFR Ch. 1 (1/1195 edition), Subchapter A-Animal Welfare. 4.0 IDENTIFICA

### nONOF THE TEST AND CONTROL SUBSTANCES The following information was suppliedby the Sponsor on a test requisition form or other correspondence wherever applicable; it does not apply to confidential information. The Sponsor will be responsible for all test substance characterizationdata as specified in the GLP regulations. Test and control substances (exclusive ofextracts) that are mixed with carriers require verification.ofconcentration, homogeneity and stability. Samples oftest and control substance mixtures will be returned to the Sponsor for characterization and verification, wherever applicable. 4.1 Test Substance 4.1.1 Test Substance Name: CAS/Code #: Not Supplied by Sponsor (N/S) Lot/Batch

### #: Physical State: Color: Expiration Date: 

### 12/23/2005 Density: Stability: stable in dark and cool closed containers Solubility: pH: N/A 

## 000050 

# 48 Pa2"e 

### 6 of20TomonCorporation Acute Eye Irritation/Corrosion

- OECD Project Number: 

### Storage Conditions: Room Temperature in dark and cool closed containers Safety Precautions: Standard Toxikon Laboratory Safety Precautions 4.2 Control 4.2.1 The untreated eye served as the negative control. 5.0 IDENTIFICATION OFTHETEST SYSTEM 5.1 Animals Used in the Test: Number and species: 3 New Zealand White rabbits (Oryctolagus cuniculus) 

### Sex: 1male and 2 females Weight range: 3.11

- 3.98 kg \ at least 10 weeks old (adult) Weighed to nearest 10 gm. 

### Health Status: healthy, not previously used in other experimental procedures Housing: individually housed Cages: suspended stainless steel Animal Identification: ear marker 5.2 Animal Care and Maintenance: Acclimatization: minimum 5 days under the same conditions as for the actual test. Animal Purchase: Millbrook Breeding Labs, Amherst, MA Bedding: hardwood chips P.W.I Industries, St-Hyacinthe, Quebec, Canada (non-contact) Animal room temperature: 68 

### ± 5 

### OF Animal room relative humidity: 30-70% Air exchanges per hour: 10 to 15 Lights: 12-hour light/dark cycle, full spectrum fluorescent lights The laboratory and animal rooms are maintained as limited-access facilities. 

## 000051. 

# 49 Page 70f20Toxikon Corporation Acute Eye Irritation/Corrosion

- OECD Project Number: Animal rations: TEK 8630 Rabbit Food, Harlan Teldad, Madison, WI Water: tap water, adlibitum Animal Selection: chosen from larger pool and examined for lack ofadverse clinical
... signs There was no known contaminants present in the feed, water, or bedding expected to interfere with the test data. 6.0 JUSTIFICATION OF TEST SYSTEM AND ROUTE OF ADMINISTRATION 

### 6.1 Test systemjustification Albino rabbits were used in this study because they have historically been used in ocular safety evaluations and the guidelines have no alternative (non-animal) methods. The number of animals used in this study was recommended by the OECD guidelines. Ocular exposure corresponds to a likely route ofhuman exposure. 

### 6.2 Definition ofirritation and corrosion Eye irritation is the production ofreversible changes in the eye following the application ofa test substance to the anterior surface ofthe eye. Eye corrosion is the production ofirreversible tissue damage in the eye following application ofa test substance to the anterior surface ofthe eye. 6.3 Testing and Evaluation Strategy for Eye IrritationlCorrosion The OECD guideline recommends weight-of-the-evidence analysis ofall available relevant data as to the potential eye corrosivity/irritationofthe test substance prior to vivo testing. A summary ofthe weight-of-the-evidence analysis and the preferred sequential testing strategy is provided in Appendix 

### I. 7.0 EXPERIMENTAL DESIGN 

### 7. 1 Pretreatment Screening Procedure and Selection ofAnimals Animals selected for the test were not subjected to any previous experimental procedures. Animals selected for the study were chosen from a larger pool ofanimals and were examined within the 

### 24hours before administration ofthe test substance to insure that the eyes were free ofabnormality, damage, and disease.



## 000052 

# 50 Page 8of20Toxikon Corporation Acute Eye Irritation/Corrosion

- OECD


### Both eyes ofeach rabbit were examined for macroscopic findings and were scored according to the Grades for Ocular Lesions, as described in Appendix II. The rabbits' eyes were scored by Fluorescein Staining as described in Appendix III. Only rabbits showing no eye irritation. ocular defects, or pre-existing corneal injury were used in the study. 7.2 Weight and Identification The animals were weighed to the nearest 10 grams and individually identified by ear marker. 7.3 Designation ofEye Test Parameters The left eye ofeach animal were treated with the test substance. The right eye remained untreated, and thus served as a control. 7.4 Severely Irritating Materials The test article was not anticipated to be a severe irritant. 7.5 Dosage The test substance was dosed as described in Section 8.0. 7.6 Post-Treatment Procedures Eyes were examined at 1, 24, 48, and 72 hours after treatment using the scale supplied (Appendix II). After recording the observations at 24hours, the eyes ofall rabbits were further examined with the aid offluorescein to further characterize corneal opacity (Appendix Ill). Reading ofreactions was facilitated by the use ofa slit lamp. The eyes were rinsed with 20 rnL 

### of0.9% USP Sodium Chloride for Injection (NaCl) after the 24 hour observation point. 7.7 Clinical Observations**Anirnal~** 

### were observed daily for clinical manifestations. 7.8 Gross and Microscopic Examinations No animals died during the course ofthe study. 7.9 Therapeutic Agents No therapeutic agents were used in any phase ofthe study. 7.10 Study Termination At the end ofthe study, all animals were weighed and then: sacrificed by injectable barbituate. 

### 000053 

# 51 Page 9 of20Toxikon Corporation Acute Eye Irritation/Corrosion

- OECD ProjectNumber: 

### 8.0 DOSAGE The test substance was dosed neat. Each animal received adose of0.1 

### mL ofthe test substance. The test substance was placed in the conjunctival sac ofone eye ofeach animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for one second. The other eye remained untreated and served as the control. The eyes were rinsed with 0.9% 

### USP Sodium Chloride for Injection following the 24 

### hour observations. 9.0 

### EVALUATION CRITERIA The grading system for ocular irritation is presented in Appendix II

### and Fluorescein Scoring in Appendix III. Eye corrosion is defmed as irreversible ocular damage. The ocular irritation scores were evaluated in conjunction with the nature and reversibility ofthe scores observed. Atest substance was rated satisfactory ifthe ocular changes ofthe test are equivalent to or less than those for the untreated (control) eyes. The determination ofthe irritation and/or corrosive potential ofthe test substance was based on the interpretation ofthe results by the Study Director. 10.0 

### RESULTS 10.1 

### Animal Weights (Table I) All ofthe animals exhibited an increase in body weight during the course ofthe study. 10.2 

### Clinical Observations (Table I) No overt signs oftoxicity other than the ocular effects were evident during the course ofthe study in any ofthe animals. 10.3 Eye Scores (Table II) Corneal Opacity: No corneal opacity was seen in any ofthe rabbits at any observationpoint. Iris: No iris 

### involvement was seen in any ofthe rabbits at any observation point. Conjuctiva: Slight conjunctival redness was present atthe 1 

### hour observation in two ofthe treated eyes. These findings resolved by the 48 hour observationpoint. 10.4 

### Fluorescein Staining (Table II) No Fluorescein Staining was seen in any ofthe rabbits at any observation point.



## 000054 Palle 10 

### of20 

# 52TomonCorporation Acute Bye Irritation/Corrosion

- aBCD Project Number: 

### 11.0 CONCLUSION The test substance, , was evaluated for its potential to produce an irritating effect on the ocular tissue ofNew Zealand White rabbits, when administered according to the procedures detailed in the study protocol. Slight signs ofirritation (conjunctival redness) were noted in the treated eyes oftwo ofthe animals at the 1hour time point. All signs resolved in all animals by the 

### 48 hour observation point. No signs ofirritation were present in any ofthe control eyes. Based on the methods employed, the test substance is considered to be a slight irritant to the ocular tissue ofNew Zealand White rabbits. 12.0 RECORDS 12.1 Original raw data is archived at Toxikon Corporation. 12.2 A 

### copy ofthe final report and any amendments is archived at Toxikon Corporation. 12.3 The original final report and a copy ofany protocol amendments or deviations is forwarded to the Sponsor. 12.4 All unused test article shall be returned. 13.0 CONFIDENTIALITY AGREEMENT Statements ofconfidentiality were as agreed upon prior to study initiation. 14.0 

### POUCYON PAIN AND SUFFERINGIN ANIMALS No evidence ofpain or suffering was observed inthe animals during the study. 15.0 ANIMAL USAGE The Sponsor assures that to the best oftheir knowledge this study does not unnecessarily duplicate previous testing.



### 000055 

# 53 Page 11 of20Toxikon Corporation Acute Eye Irritation/Corrosion

- OECD Project Number: 

### TABLE I ANIMALWEIGHTS AND CLINICAL OBSERVATIONS Test Substance: LotIBatch#:


### Body Weight (Kg) 

### Animal Number Sex 

### Day 0 02/03/04 

### Day 3 02/06104 Weight Change Signs of Toxicity* 40030 Female 3.51 3.56 0.05 None 40032 Female 3.98 4.03 0.05 None 40033 Male 3.11 3.15 0.04 None

. . 

### * Summary ofcluucal observations on Day 0through Day 3excluding Eye Scores
.



### 000056 

# 54 Page 12 of20Toxikon Corporation Acute Eye Irritation/Corrosion

- OECD Project Number:


### Table IT OCULAR LESION SCORES AND FLUORESCEIN EXAMINATION Test Substance: Lot/Batch

### #: Animal #: 40030 Date Time from Dose 02/02/04 02/03/04 02/04/04 02/05/04 02/06/04 -24 hr 1hr 24hr 48 hr 72hr CORNEA OCULARLESION SCORES OPACITY L/R 0/0 0/0 0/0 0/0 0/0 AREA INVOLVED L/R 0/0 0/0 0/0 0/0 0/0 IRIS 

### VALUES LIR 0/0 0/0 0/0 0/0 0/0 CONJUNCTIVA REDNESS L/R 0/0 1/0 1/0 0/0 0/0 CHEMOSIS L/R 0/0 0/0 0/0 0/0 0/0 FLUORESCEIN EXAM INTENSITY OF STAINING LIR 0/0 N/A 0/0 0/0 0/0 AREA INVOLVED L/R 0/0 N/A 0/0 0/0 0/0 Test: Left Eye Control: Right Eye (untreated) N/A: Not Applicable LlR: Left Eye Score/Right Eye Score



## 000057 

# 55 13 of20Tomon Corporation Acute Eye Irritation/Corrosion

- OECD Project Number:
Table II OCULARLESION SCORES AND FLUORESCEIN EXAMINATION (Cont.) Test Substance: LotlBatch

### #: Animal #: 40032 Date Time from Dose 02/02/04 02/03/04 02/04/04 02/05/04 02/06/04 -24 hr Ihr 24hr 48 hr 72 hr OCULARLESION SCORES CORNEA OPACITY LIR 0/0 

### 010 0/0 0/0 0/0 AREA INVOLVED LIR 0/0 

### 010 0/0 

### 010 010 IRIS VALUES LIR 

### 010 0/0 0/0 

### 010 010 CONJUNCTIVA REDNESS L/R 

### 010 0/0 

### 010 010 010 CHEMOSIS LIR 

### 010 0/0 0/0 

### 010 010 FLUORESCEIN EXAM INTENSITY OF STAINING LIR 

### 010 N/A 

### 010 010 010 AREA INVOLVED L/R 

### 010 N/A 

### 010 010 010 Test: Left Eye Control: Right Eye (untreated) NIA: Not Applicable LlR: Left Eye ScorelRight Eye Score



## 000058 

# 56 14 of20Toxikon Corporation Acute Eye Irritation/Corrosion· OECD Project Number: __ _ .
Table IT OCULARLESION SCORESAND FLUORESCEINEXAMINATION (Cont.) Test Substance: Lot/Batch

### #: Animal 

### #: 40033 Date Time from Dose 

### 02102/04 02103/04 02/04104 02105/04 02/06104 

### -24 hr 1hr 24hr 48 hr 12hr CORNEA OCULARLESION SCORES OPACITY 

### L/R 0/0 0/0 0/0 0/0 0/0 AREA INVOLVED 

### L/R 0/0 0/0 0/0 0/0 0/0 IRIS VALUES L/R 

### 0/0 0/0 0/0 0/0 0/0 CONJUNCTIVA REDNESS LIR 

### 0/0 110 110 

### 0/0 0/0 CHEMOSIS 

### L/R 0/0 0/0 0/0 0/0 0/0 FLUORESCEINEXAM INTENSITY OF STAINING L/R 

### 0/0 N/A 

### 0/0 0/0 0/0 AREA INVOLVED L/R 

### 0/0 N/A 

### 0/0 0/0 0/0 Test: Left Eye Control: Right Eye (untreated) N/A: Not Applicable 

### LlR: Left Eye ScorelRight Eye Score



## 000059 

# 57 15 of 20TomonCorporation Acute Eye Irritation/Corrosion

- OECD Project Number: 

### APPENDIX I TESTINGAND EVALUAnON STRATEGY FOR EYE IRRITATION/CORROSION OECD/OCDE405 Supplement Summary Step 1: Evaluation ofexisting human and animal data Pre-existing data demonstrating direct ocular effects (e.g clinical studies, occupational studies, case reports, ocular studies) or indirect ocular effects (e.g. dermal corrosionlirritation studies) should be evaluated first. Substances with known corrosivity or severe irritancy to the eye should not be instilled into the eye ofanimals, nor should substances showing corrosive or severe irritant effects to the skin; such substances should be considered to be corrosive and/or irritating to the eyes as welL Substances with sufficient evidence ofnon-corrosivity and nonirritancy from previously performed ocular studies should also not be tested in 

### in 

### vivoeye studies. Step 2: Analysis ofstructure activity relationships (SAR) When available, the results oftest ofstructurally related substances should be considered. Human and/or animal data onthe potential eye irritationlcorrosivity ofstructurally related substances or mixtures ofsuch substances should be evaluated; ifthese data indicate irritationlcorrosivity it can be presumed that the test substance will produce the same responses and the test substance may not need to be tested. Negative data from studies ofstructurally related substances or mixtures ofsuch substances do not constitute sufficient evidence to noncorrosivity/non-irritancy ofa substance under the sequential testing strategy. Step 3: Physiochemical properties and chemical reactivity Substances exhibiting pH extremes such as**~** 

### 2.0 or**~** 

### 11.5 may have strong local effects. If 

### extreme pH is the basis for identifying a substance as a corrosive or irritant to the eye, then its acid/alkaline reserve (buffering capacity) may also be taken into consideration. If

### the buffering capacity suggest that a substance may NOT be corrosive to the eye, then further testing should be undertaken to confirm this, preferably by the use ofa validated and accepted 

### in vitroor 

### in vivotest (see Steps 5 and 6). Step 4: Consideration ofother existing information All data relating to systemic toxicity via the dermal route should be evaluated. Acute dermal toxicity ofthe test substance should also be considered. If

### the test substance has been demonstrated to be highly toxic by the dermal route ofadministration, it may not need to be tested in the eye. Itcan be presumed that ifa test substance is highly toxic via the dermal route, it will also exhibit high toxicity when instilled into the eye.



## 000060 

# 58 Page 160f20Toxikon Corporation Acute Eye Irritation/Corrosion

- OECD Project Number: APPENDIX I (Cont.) TESTINGANDEVALUAnON STRATEGY FOREYEIRRITATION/CORROSION OECD/OCDE

### 40S Supplement Summary Steps 5 and 6: Results from 

### in vitro and 

### ex vivotests Test substances that have demonstrated corrosive or severe irritant properties in an 

### in vitro or 

### ex vivotest that has been validated and is accepted for the assessment specifically ofeye or skin corrosion/irritation need not be tested in animals. It can be presumed that the substance(s) will produce similar effects 

### in vivo. If validated and accepted 

### in 

### vitro/ex vivo tests are not available, one can bypass Steps 

### 5 and 

### 6 and proceed directly to Step 

### 7. Step 

### 7: Assessment of

### in vivo dermal irritancy orcorrosivity ofthe substance When insufficient evidence exists with which to perform a conclusive weight-of-the-evidence analysis ofthe potential eye irritation/corrosivity ofa substancebased upon data from the studies list above, the 

### in vivo skin irritation/corrosion potential should be first evaluated using OECD Guideline 404 and the accompanying Supplement. Ifthe substance is shown to produce corrosion or severe skin irritation, it should be considered to be a corrosive eye irritant unless other information supports an alternative conclusion. Thus, an 

### in vivo eye test would not need to be performed. Ifthe substance is not corrosive or severely irritating to the skin, an 

### in 

### vivo eye test should be performed. Steps 

### 8 and 

### 9: 

### In vivo test in rabbits 

### In vivo ocular testing should begin with an initial test using one animal. Ifthe results ofthis test indicate the substance to be a severe irritant or corrosive to the eyes, further testing should notbe performed. Ifthat test does not reveal any corrosive or severe irritant effects, a confirmatory test should conducted with two additional animals. Depending on the results of the confirmatory test, further tests may be needed.



## 000061 

# 59 Page 

### 17 of20Toxikon Corporation Acute Eye Irritation/Corrosion

- OECD Project Number: APPENDIX II GRADES FOROCULARLESIONS CORNEA* OPACITY-degree ofdensity (areas most dense taken for reading) No ulceration or opacity 

# o Scattered or diffuse areas ofopacity (other than slight dulling ofnormal luster), details ofiris clearly visible 

### 1 Easily discernible translucent area, details ofiris slightly obscured 

### 2 Nacrous area, no details ofiris visible, size ofpupil barely discernible 

### 3 Opaque cornea, iris not discernible through the opacity 4
IRIS Normal 

## o Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any ofthese or combination ofany thereof, iris reactive to light (a sluggish reaction is positive score) 1 No reaction to light. hemorrhage, gross destruction (any or all ofthese) 

### 2 

## * The area ofcorneal opacity should be noted.



## 000062 

# 60 Page 18 of20TomonCorporation Acute EyeIrritation/Corrosion

- OECD Project Number: 

### APPENDIX II (Cont.) 

### GRADES FOR OCULAR LESIONS CONJUNCTIVAE A. REDNESS**(re~ers** to palpebral and bulbar conjunctivae excluding cornea and iris) Blood vessels normal 

# o Some blood vessels defmitely hyperemic (injected) 

### 1 Diffuse, crimson color, individual vessels not easily discernible 2 Diffuse beefy red 

### 3
B. CHEMOSIS:lids and/or nictating membranes No swelling; nonnal 

## o Any swelling above normal (includes nictitating membranes) 

### 1 Obvious swelling with partial eversion oflids 

### 2 Swelling with lids about halfclosed 

### 3 Swelling with lids more than halfclosed 

### 4



### O(J0063 

# 61 Paee 19 of20TomonCorporation Acute EyeIrritation/Corrosion

- OECD Project Number: 

### APPENDIXll FLUORESCEIN STAINING GRADING SCALE
A. INTENSITY OF STAINING Absence offluorescein staining 

# o Slight fluorescein staining confined to a small focus. With diffuse illumination the underlying structures are easily visible. The outline ofthe pupillary margin is as ifthere were no fluorescein staining. 1 Moderate fluorescein staining confmed to a small focus. With diffuse illumination the underlying structures are clearly visible, although there 

### is some loss ofdetail. 2 Marked fluorescein staining. Staining may involve a larger portion of the cornea. With diffuse illumination underlying structures are barely visible but not completely obliterated. 

### 3 Extreme fluorescein staining. With diffuse illumination the underlying structures cannot be observed. 

### 4 B. AREA INVOLVEO No area offluorescein 

# o One-quarter ofless. but not 0 1 Greater than one-quarter. but less than one-half 2 Greater than one-half, but less than three-quarters 

### 3 Greater than three-quarters, up to whole area 

### 4



## 000064 

## 62 Pae:e 20 of20Continuation Sheet Contains TSCA

### C~Business Information**~(VA.C·\n~J**

Appendix 6 Acute Oral Toxicity Test Acute Toxic Class Method

- OECD



## 000065 Page 63

### TOXIKON FINAL REPORT:

ACUTE ORAL TOXICITY TEST UP-AND-DOWN PROCEDURE

- OECD

Author Chin H. Tay, Ph.D.

Final Report Date February 26, 2004

MANAGEMENT OF THE STUDY Performing Laboratory

Toxikon Corporation

15 Wiggins Avenue Bedford, MA 01730 Sponsor



### V{J0066 

# 64Toxikon Corporation Acute Oral Toxicity Test: Up and Down Procedure

- OECD Number:


### TABLE OF CONTENTS Title Page Table ofContents Study Summary Quality Assurance Statement Study Director Signature and Verification Dates 1.0 Purpose 2.0 References 3.0 Compliance 4.0 Identification ofthe Test Substance 5.0 Identification ofthe Test System 6.0 Justification ofTest System and Route ofAdministration 7.0 Experimental Design 8.0 Dosage 9.0 Evaluation Criteria 10.0 Results 11.0 Conclusion 12.0 Records 13.0 Confidentiality Agreement 14.0 Policy on Pain and Suffering in Animals 15.0 Animal Usage Table I: Dosing, Animal Weights, Necropsy Data and Clinical Observations



## 000067 Page 2 of 11 

# 65TomonCorporation AcUte Oral Toxicity 1cst: Up and Down Procedure

- OECD Project Number: 

### STUDY SUMMARY
The test substance, , was evaluated for its potential to produce death following oral administration. No deaths were observed in this study. No clinical signs of toxicity were observed in the study. Using OECD 425, the Up-And-Down Procedure, the test substance was dermed to have an estimated LDso ofgreater than 2000 mg/kgbody weight.



## 000068 

# 66 Paae 3 of 11ToxikonCorporation Acute OralToxicity Test: Up and Down Procedure

- OECD Project Number: 

### QUALITY ASSURANCE STATEMENT

This study was conducted in compliance with the current Organization for Economic Co-operation and Development (OECD) GLP. The sections ofthe regulations not performed by or under the direction ofToxikon Corporation, exempt from this Good laboratory Practice statement, include characterization and stability ofthe test substance and its mixture with carriers, 40 CFR. Part 792.105 and 792.113. The Quality Assurance Unit conducted inspections onthe following dates. The findings were reported to the Study Director and to Toxikon's management.
INSPECTIONS DATE OF DATE REPORTED DATEREPORTED INSPECTION STUDY DIRECTOR MANAGEMENT
ANIMAL WEIGHTS 02/06/04 02/06/04 02/06/04 

### RAW DATA 02/26/04 02/26/04 02/26/04 

### FINAL REPORT 02/26/04 02/26/04 02126104

# ~h~-~~ Fe Ice Randi LaMadeleine, B.S. Quality 

### Assurance Officer Date



## 000069 

# 67 Page 4 of 11Toxikon Corporation Acute Oral Toxicity Test: Up and Down Procedure

- OECD Project Number: 

### STUDY DIRECTOR SIGNATURE AND VERIFICATION DATES
This study meets the technical requirements ofthe protocol. The study also meets with the requirements ofthe Good Laboratory Practice Regulations, 40 CFR, Part 792, and the current OECD GLPs, with the exemptions as stated in the Quality Assurance Statement.
Protocol Number: CHT/VIVOIOO4-03/000 

### Study Director: Chin H. Tay, Ph.D. Company: Toxikon Corporation
Signature:

# ~-

Study Supervisor: William Entwistle, AS, LATG
VERIFICATION DATES: The Toxikon Protocol Effective Date is the GLP Study Initiation Date. The study dates were as follows: Protocol Date: 12124/03 

### Test Substance Receipt: 12/24/03 

### Project Log Date: 01108104 

### Technical Initiation: 01120/04 

### Technical Completion: 02/17/04



## 000070 

# 68 Page 5 of 11Toxikon Corporation Acute OralToxicit} Test: Up and Down hocedure

- OECD ProjectNumber: 

### 1.0 PURPOSE The test was designed to assess the acute oral toxicity ofa test substance via oral administration. Theup and down test procedure as described in OECD 425 was used. This procedure minimized the number ofanimals used, allowed the observation ofsigns oftoxicity and provided information both for hazard assessment and for hazard classificationpurposes. 

### 2.0 REFERENCES Thetest was basedon the following reference: 2.1 Basedupon the standards ofthe Organization for Economic Co-operation and Development (OECD), Guideline for Testing ofChemicals, Acute Oral Toxicity, Upand-Down Procedure, Section425,2001. 

### 3.0 COMPLIANCE The study conformed to all applicable laws andregulations. Specific regulatoryrequirements include the current OECD GLPs; AAALAC, International; the "Guide for the Care andUse of LaboratoryAnimals," National Research Council, 1996; (Nlli) (OLAW), "Public Health Service Policyon Humane Care and Use ofLaboratoryAnimals," Health Research ExtensionAct of 1985 (Public Law 99-158 November 20, 1985), Reprinted 1996; USDA, Department of Agriculture, Animal and Plant Health Inspection Service, 9 CFRCh. 1(1/1/95 edition), SubchapterA-Animal Welfare. 

### 4.0 IDENTIFICATION OFTHETEST SUBSTANCE The following information was suppliedbythe Sponsoron a testrequisition form or other correspondence wherever applicable; it does not apply to confidential information. The Sponsor was responsible for all test substance characterizationdata as specified in the GLP regulations. 4.1 Test Substance Test SubstanceName: CAS/Code#: Not SuppliedbySponsor (N/S) LotlBatch #: Physical State: Color: ExpirationDate: 12/23/2005 Density: Stability: stable indark and cool closed containers Solubility: pH: Not Applicable Storage Conditions: RoomTemperature SafetyPrecautions: Standard Toxikon Laboratory SafetyPrecautions


## 000071. 

# 69TomonCorporation Acute Oral Toxicity Test: Up and Down Procedure

- OECD Project Number: 

### 5.0IDENTIFICATION OFTHETESTSYSTEM 5.1 Animals Used in the Test: Number and Species: 5 Outbred Albino Rats (Rattus norvegicus) 

### Sex: Female; females were nulliparous and non-pregnant WeightlAge range: 207.4

- 239.5 grams/at least 49 days old (adult) Weighed to nearest 0.1 gm. The weight variation of animals used in the test did not exceed 

### ±20% ofthe mean weight for each sex. Health Status: Healthy, not previously used in other experimental procedures Animal Identification: ear punch Animals were purchased from Harlan, Indianapolis, IN. 5.2 Animal Care and Maintenance: Housing: Single housing The laboratory and animal rooms are maintained as limited-access facilities. Cages: Polycarbonate Bedding: Hardwood chips, (contact), P.W.I. Industries, St-Hyacinthe, Quebec, Canada Animal room temperature: 68 

### ±SOF Animal room relative humidity: 30-70% Air exchanges per hour: 10 to 15 Lights: 12-Hour light/dark cycle, full spectrum fluorescent lights Animal rations: TEK 7012 Rodent Diet, Harlan Tekiad, Madison, WI adlibitum 

### Water: Tap water, adlibitum 

## 000072 

# 70 Page 7 of 11TomonCorporation Acute Oral Toxicity Test: Up and Down Procedure

- OECD Project**Number~** 

### There were no known contaminants present in the feed, water, or bedding that interfered with the test data. Acclimatization: Minimum 5 days under the same conditions as for the actual test.

... 

### Selection ofAnimals: Randomly selected from a larger pool and examined for a lack ofadverse clinical signs 6.0 JUSTIFICATION OFTEST SYSTEM AND ROUTE OF ADMINISTRAnON Albino rats were used in this study because they have historically been used for acute oral toxicity studies and the guidelines have no alternative (non-animal) methods. The number ofanimals used was recommended by the 0ECD guidelines. Oral administration corresponded to a likely route ofhuman exposure. The test was based on a up and down procedure with the use ofa minimum number ofanimals for the entire study, yet enabling sufficient information to be obtained on the acute toxicity ofthe test substance. 7.0 EXPERIMENTAL DESIGN 7.1 Test Substance Application Food was withheld from the animals the night prior to dosing. Animals were administered a single dose ofthe test substance by intragastric intubation by means ofa ball tip gavaging needle and a syringe. After dosing, the animals were returned to their cages and supplied with feed and water adlibitum. 

### 7.2 Test Substance Preparation The test substance was a liquid and no preparation is necessary. The test substance was administered as provided by the Sponsor. Reference is made to Section 

### 8.0 for dosing information. The maximum volume ofa liquid that can be administered at one time depends on the size ofthe test animal. The volume did not exceed 1 ml/100 g body weight. 7.3 Limit Test (Per OECD 425) Limit Test (2000 mg/kg) The first animal was dosed at 2000 mg/kg. As the animal did not die from this dose, four additional animals were dosed sequentially at the same dose, so that a total of5 animals were tested. Reference is made to OECD 425 and the statistical computer program (AOT425StatPgm) will be used to determine the termination point ofthe study. 7.4 Post Treatment Procedures Careful clinical observations were made at least twice on the day ofdosing. Special attention was given during the first 4 hours. The frequency was determined by the 

# 71 Page 8 of 11 

## 000073Toxikon Corporation Acute Oral Toxicity Test: Up and Down Procedure

- OECD ProjectNumber: response ofthe test animals to the treatment. Animals were observed daily for 14 days for clinical manifestations. Cageside observations included changes inthe skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous system, and somatomotor activity and behavior pattern. Particular attention was directed to observations oftremors, convulsions, salivation, diarrhea, lethargy, sleep and coma. The time ofdeath was recorded as precisely as possible. 7.5 Moribund and Dead Animals Any animal found dead was necropsied as soon as possible, but in no case later than 12 hours. A gross necropsy was performed on all animals whether found dead 

### in extremis or sacrificed at the end ofthe study and all gross pathological changes was recorded. 7.6 No therapeutic agents were used in any phase ofthe study. 7.7 Animals were weighed at Day 0 prior to dose administration, Day 7, Day 14, and at death. Changes in weights were calculated and recorded when survival exceeded one day. At the end ofthe study, surviving animals were sacrificed by C02 inhalation. 7.8 Control Group Untreated control groups were not required. 

### 8.0 DOSAGE 

### 8.1 Test Substance Preparation The test substance was a liquid and was dosed as provided by the Sponsor. The animals were dosed at 2000 mg/kg using a Sponsor-provided density of 1.3 g/ml. Reference is made to Table 

### 1 for actual dose and volume. 9.0 EVALUATION CRITERIA An evaluation ofacute toxicity data included the relationship, ifany, between the animals exposed to the test substance and the incidence and severity ofall abnormalities, including behavioral and clinical abnormalities, gross lesions, bodyweight changes, effects onmortality, and any other toxic effects. Calculation of the LD50 and the confidence intervals ofthe test substance were detennined by using the 

### maximum likelihood method. A statistical computerprogram (AOT425StatPgm) developed by the United States Environmental Protection Agency was used for the calculation. 

### 10.0 RESULTS 10.1 Body weights (Table I)

- All the animals survived the duration ofthe study gained weight. 

# 72 Page 9 of 11TomonCorporation Acute Oral Toxicity Test: Up and DownProcedure

- OECD Project Number:


### 10.2 Necropsy (Table I)

- No unusual findings were found during necropsy in all the dosed animals. 10.3 Clinical Observations (Table I)

- 

### No clinical manifestations oftoxicity were observed in any ofthe animals for the duration ofthe study. lOA 

### Input ofthe results into the statistical computer program (AOT425StatPgm) developed by the United States Enviromnental Protection Agency, the test substance is defined to have a LDso greater than 2000 mg/kg. 11.0 

### CONCLUSION The test substance, , was evaluated for its potential to produce death following oral administration. No deaths were observed in this study. No clinical signs of toxicity were observed in the study. Using OECD 425, the Up-And-Down Procedure, the test substance was defined to have an estimated LDso ofgreater than 2000 mg/kg body weight. 12.0

### RECORDS 12.1 Original raw data will be archived at Toxikon Corporation. 12.2 A copy ofthe Final Report and any amendments will be archived at Toxikon Corporation. 12.3 All unused test substance shall be disposed ofby Toxikon, per Sponsor's specifications. 13.0 

### CONFIDENTIAUTY AGREEMENT Statements ofconfidentiality were 

### as agreed upon prior to study initiation. 14.0 

### POLICY ON PAIN AND SUFFERING IN ANIMALS There was no evidence ofpain and suffering during the course ofthe study. 15.0 

### ANIMAL USAGE The Sponsor assures that to the best oftheir knowledge this study did not unnecessarily duplicate previous testing.



## 000075 Page 10 of 11 

# 733

4

5 Toxikon Corporation Acc,e Oral Toxicity Test· Up-Alld-Down Procedure

- OECD Project Number:

TABLE I Dosing. Animal Weights. Necropsy Data. and Clinical Observations

Test Anicle: LotIBatch #: Dose: Animal # 2000 mgtts Sex Body Weight (grams) Dose Day 0 Day 7 Day 14 Weight Clinical Necropsy gIn mL 01120/04 01127104 02103/04 Change (grams) ObservatioN Observations Female 0.41 0.32 207.4 234.5 244.7 37.3 Normal Normal

Dose: 2000mg/tg Animal I Sex sm Dose mL Body Weight (grams) Day 0 Day 7 01123/04 01130104 Day 14 02/06104 Weight Cba!!&e**igrams~** Clinical Observations Necropsy ObservatiollS 2 Female 0.43 0.33 214.5 230.6 240.4 25.9 Nonnal Nonnal

Dose: 2000 mg/kg Body Weight (grams) Animal I Sex Dose Day 0 Day 7 Day 14 Weight Clinical Necropsy gm mL 01127104 02103104 02/10104 Change**{&rams~** Observations Observations Female 0.44 0.34 219.6 238.9 255.6 36.0 Normal Normal

Dose: 2OOOmr/y Body Weight (grams) Animal # Sex Dose Day 0 Day 7 Day 14 Weight Clinical Necropsy gIn mL 01/30/04 02106104 02113104**Cge!Srams~** Observations Observations Female 0.48 0.37 239.5 253.7 270.3 30.8 Normal Normal

Dose: 2000 mg/k& Body Weight (grams) Animal I Sex Dose Day 0 Day 7 Day 14 Weight Clinical Necropsy sm mL 02/03/04 02/10104 02/17104 Change**!srams~** Observations Observations Female 0.44 0.34 218.6 237.1 240.5 21.9 Nonnal Nonnal

Page II of II 

## 000076 

# 74Continuation Sheet Contains TSCA**Co~usiness** Information**~",1(** 

### ......

## -eJ

Appendix 7 Acute Dermal Toxicity

- OECD



## 000077 Page 75

### TOXIKON FINAL REPORT:

ACUTE DERMAL TOXICITY

- OECD

Author Chin H. Tay, Ph.D.

Final Report Date

March 22, 2004

MANAGEMENT OF THE STUDY Performing Laboratory

Toxikon Corporation

15 Wiggins Avenue Bedford, MA 01730 Sponsor



## 000078 

# 76Toxikon Corporation Acute Dermal Toxicity

- OECD ProjectNumber: 

### TABLE OF CONTENTS
Title Page Table ofContents Study Summary Quality Assurance Statement Study Director Signature and Verification Dates 1.0 Purpose 2.0 References 3.0 Compliance 4.0 Identification ofTest and Control Articles 5.0 Identification ofTest System 6.0 Justification ofTest System and Route ofAdministration 7.0 Experimental Design 8.0 Dosage 9.0 Evaluation Criteria 10.0
. Results 11.0 Conclusion 12.0 Records 13.0 Confidentiality 14.0 Policy on Pain and Suffering in Animals 15.0 Animal Usage Table I: Dosing, Body Weights, Necropsy Data and Clinical Observations Table II: Draize Scale Skin Reactions Appendix I: Draize Scale for Scoring Skin Reactions 

## ..

000079 

# 77 Page 2 of 13TomonCorporation Acute DermalToxicity

- OECD Project Number: 

### STUDY SUMMARY
The test substance, , was evaluated for its potential to produce systemic toxicity or death following a single topical 24 hour application to the skin of New Zealand White rabbits. The test substance when administered at 2000 mg/Kg body weight is considered non-toxic based on the absence of mortality and according to the criteria of the study protocol.



## 000080 

# 78 Page 3of 13Toxikon COIporation Acute Dermal Toxicity

- OECD Project Number: 

### QUALITY ASSURANCE STATEMENT
This study was conducted 

### in compliance with U.S. Food and Drug Administration regulations set forth in 

### 21 CFR, Part 58 and OECD GLPs, 1982. The sections ofthe regulations not perfonned by or under the direction ofToxikon Corporation, exempt from this Good laboratory Practice statement, include characterization and stability ofthe test and its mixture with carriers. 21 CFR, Part 58.105 and 58.113. The Quality Assurance Unitconducted inspections onthe following dates. The fmdings were reported to the Study Director and to Toxikon 1

### s management.

INSPECTIONS DATE OF DATE REPORTED DATE REPORTED INSPECTION STUDY DIRECTOR MANAGEMENT
ANIMAL WEIGHTS 02/18/04 02/18/04 02118104 

### RAW DATA 03/22/04 03/22/04 03122104 

### FINAL REPORT 03/22/04 03/22/04 03122104

# ~~0JNJ1~**03l~d-l6\f** Felice Randi LaMadeleine, B.S. Date Quality Assurance



## 000081. 

# 79 Page 4 of 13TomonCorporation Acute Dermal**To~jty** - OECD ProjectNumber: STUDYDIRECTOR SIGNATUREAND VERIFICATION DATES
This study meets the technical requirements ofthe protocol. The study also meets with the requirements ofthe Good Laboratory Practice Regulations, 21 CFR, Part 58, with the exemptions as stated in the Quality Assurance Statement.
Protocol Number: CHTNIVO/OO3-03/000 Study Director: Chin H. Tay, Ph.D. Company: Toxikon Corporation
Signature: Date: Study Supervisor: WilliamEntwistle, AS, LATG
VERlFICATION DATES: The Toxikon Protocol Effective Date is the GLP Study Initiation Date. The study dates were as follows: Protocol Effective Date: 

### 12/29/03 Test Article Receipt: 

### 12/24/03 ProjectLog Date: 01108/04 Technical Initiation: 

### 02/18/04 Technical Completion: 03/03/04



## 000082 Pae:e 5 of 13 

# 80Toxikon Corporation Acute Dermal Toxicity· OEeD Project Number: 1.0 PURPOSE The purpose ofthe test is to evaluate the potential ofthe test substance to produce systemic
. toxicity ordeath after a single topical 24 hour application to the skin ofalbino rabbits. 2.0 REFERENCES The study was performed based on the following references: 2.1 Organization for Economic Co-operation and Development (OECD), Guidelines for Testing ofChemicals, Section402, 2002. 2.2 Draize, J.H., "Dermal Toxicity", Appraisal ofthe Safety ofChemicals in Food, Drugs and Cosmetics-Dermal Toxicity,

## pp. 49-52. Association ofFood and Drug Officials ofthe U.S., Topeka Kansas, 1965. 3.0 COMPLIANCE The study conformed to all applicable laws and regulations. Specific regulatory requirements included the current OECD GLPs; AAALAC, International; "Guide for the Care and Use of Laboratory Animals," National Research Council, 1996. (NIH) (OLAW), "Public Health Service Policy onHumane Care and Use ofLaboratory Animals," Health Research Extension Act of 1985 (Public Law 99-158 November 20, 1985), Reprinted 1996; USDA, Department of Agriculture, Animal and Plant Health Inspection Service, 9 CFR, Ch.1 (111195 edition), Subchapter A-Animal Welfare. 4.0 IDENTIFICATION OFTHETEST AND CONTROL SUBSTANCES The following information was suppliedby the Sponsor wherever applicable; it does not apply to confidential information. The Sponsor was responsible for all test substance characterization data as specified in the GLPregulations. Test and control substances (exclusive ofextracts) that are mixed with carriers require verification ofconcentration, homogeneity, and stability. Samples oftest and control substance mixtures shall be returned to the Sponsor for characterization. 4.1 Test Substances Name: CAS/Code #: Not Suppliedby Sponsor (N/S) Lot/Batch 

### #: Physical State: Color: pH: N/S Density: Stability: Stable in dark and cool closed containers Solubility: Expiration Date: 12/23/2005 Storage Conditions: Room Temperature Safety Precautions: Standard Toxikon Laboratory Safety Precautions 

## 000083 

# 81Toxikon Corporalicn Acute DermalToxicity

- OECD Project Number: 

### 4.2 A control substance was not utilized. Untreated skin ofeach animal served as it's own control. 5.0 IDENTIFICATION OF THETEST SYSTEM 5.1 Animals Used in the Test: Number and species: 10 New Zealand White rabbits 

### (Oryctolagus cuniculus) Sex: 5 males and 5 females Weight/Age range: 2.03

- 2.46 kilograms / at least 10 weeks old (adult) Weighed to the nearest 10 

### gm Animal identification: earmarker Health status: healthy, previously unused in other experimental procedures Animal purchase: Millbrook Breeding Labs, Amherst, MA Acclimatization: minimum of5 days under the same conditions as for the actual test. Animal selection: selected from a larger pool ofanimals and observed to ensure lack of adverse clinical signs. 5.2 Animal Care and Maintenance Housing: individually housed Cages: suspended stainless steel Bedding: hardwood chips, P.W.I. Industries, St-Hyacinthe, Quebec, Canada (non-contact) Animal room temperature: 68 

## + 5°F Animal room relative humidity: 30-70% Air exchanges per hour: 10 to 15 Lights: 12-hour light/dark cycle, full spectrum fluorescent lights The laboratory and animal rooms were maintained as limited-access facilities. 

## 000084 

# 82 Page 7 of 13ToxikonCorporation Acute Dermal Toxicity

- OECD Project Number: 

### Animal rations: TEK 8630 Rabbit Diet. HarlanTeklad. Madison. WI. adlibitum Water: tap water. adlibitum There were no known contaminants present in the feed, water. orbedding expected to interfere with the test data. 6.0 JUSTIFICATION OFTEST SYSTEM AND ROUTE OFADMINISTRATION 6.1 Albino rabbits were used in this study because they have historically been used in dermal safety evaluation studies and the guidelines have no alternative (non-animal) methods. The species and number ofanimals used is recommended by the OECD guidelines. Dermal application corresponds to the likely route ofhuman exposure. 6.2 Acute dermal toxicity is the adverse effects occurring within a short time ofdermal application ofa single dose ofa substance ormultiple doses given within 24 hours. The albino rabbit is preferred because ofits size. skinpermeability. and 

### extensive database. 7.0 EXPERIMENTALDESIGN 7.1 Pretreatment Screening Procedure and Selection ofAnimals Animals selected for the study were examined to ensure that their skin was free from irritation. trauma and disease. 7.2 Preparation ofTest Animals The application sites were prepared by clipping the skin ofthe trunk 

### free ofhair approximately 24 hours before application ofthe test substance. The site ofapplication was not abraded intentionally nor accidentally during preparation. 7.3 Test Substance Application The test substance was introduced under gauze patches two single layers thick and applied directly to the skinofthe body surface (approximately 10%) ofeach often animals. Animals were immobilized and the patches were secured inplace by wrapping the entire trunk 

### ofthe animal with an impervious bandaging. Thetest sites were secured to prevent the animals from ingesting the test substance. 7.4 Post-Treatment Procedures At the completion ofthe exposureperiod, the wrapping was removed and the skin was gently wiped and rinsed with USP Water for Injection to remove any test substance still remaining. The animals were observed for signs oferythema and edema after the 24 hour exposure period (see Appendix I). Animals were observed daily for 14 days for clinical manifestations. 

## 000085 

# 83 Page 8 of 13Toxikon Corporation Acute DermalToxicity

- aBeD ProjectNumber: 7.5 Moribund and Dead Animals No animals died during the course ofthe study. 

### 7.6 No therapeutic agents were used in any phase ofthe study. 

### 7.7 Animals were weighed on Day 

### 0, Day 

### 7, and Day 

### 14; at the end ofthe study animals were sacrificed by an injectablebarbiturate. 8.0 DOSAGE 

### 8.1 Test Substance Preparation A weighed amount ofthe test substance was first mixed with USP Water for Injection into a paste prior to dosing. Thetest substance was dosed directly onto the skin ofthe test animals. 

### 8.2 Acute Toxicity A limit test was performed on 10 rabbits, 

### 5 males and 

### 5 females, at a dose level of 

### 2000 mg/Kg. 

### 8.3 LDso Determination A complete**LD~o** was not required. 8.4 Control Groups An untreated control group was not required. 9.0 EVALUATION CRITERIA 

### Ifthere is no toxicity at the 2000 

### mg/Kgdose level, the test substance is considered non-toxic. 10.0 RESULTS 10.1 Body Weights All animals gained weight during the post-treatment period (Table I). 10.2 Clinical Observations No overt signs ofsystemic toxicity were evident during the course ofthe study (Table

# n. 10.3 Skin Reactions No signs oferythema/edema or necrosis were noted at the application sites ofany of the animals (Table II).



## 000086 

# 84 Page 9 of 13Toxikon Corporation Acute Dermal Toxicity

- OECD Project Number: 

### lOA Necropsy There were no abnormalities or lesions noted during necropsy. 10.5 Mortality No animals died during the course ofthe study. 

### 11.0 CONCLUSION The test substance, • was evaluated for its potential to produce systemic toxicity or death following a single topical 24 hour application to the skin of New Zealand White rabbits. The test substance when administered at 2000 mg/Kg body weight is considered non-toxic based on the absence of mortality and according to the criteria of the study protocoL 12.0 

### RECORDS 12.1 Original raw data is archived at Toxikon Corporation. 12.2 A copy ofthe fmal report and any report amendments is archived at Toxikon Corporation. 12.3 The original final report and a copy ofany protocol amendments ordeviations is forwarded to the Sponsor. 12.4 All unused test article shall be disposed ofby Toxikon, per Sponsor's specifications. 12.5 Final reports shall not be reproduced except in full, without the written authorization/approval from Toxikon. 13.0 

### CONFIDENTIALITY AGREEMENT Statements ofconfidentiality may be agreed upon prior to study initiation. 14.0 

### POLICY ON PAIN AND SUFFERING IN ANIMALS No evidence ofpain and suffering was observed in the animals. 15.0 

### ANIMAL USAGE The Sponsor assures that to the best oftheir knowledge this study does not unnecessarily duplicate previous testing.



## 000087 

# 85 Page 10 of 13ToxikonCorporation Acute Dermal Toxicity

- OECD Project Number: 

### TABLE I Dosing, Animal Weights, Necropsy Data, and Clinical Observations

Test Substance: Lot 

### 1Batch #: Body Weight (Kg) Weight Clinical Necropsy Animal # Sex Dose Day 0 Day 7 Day 14 Change Observations* Observations gm mL 02/18/04 02/25/04 03/03/04 

### (Kg) 40289 Male 

### N/A 2.72 2.36 2.66 2.80 0.44 Normal Normal 40290 Female 

### N/A 2.65 2.29 2.59 2.69 0.40 Normal Normal 40291 Male 

### N/A 2.55 2.21 2.47 2.60 0.39 Normal Normal 40292 Female 

### N/A 2.62 2.27 2.52 2.66 0.39 Normal Normal 40293 Male 

### N/A 2.57 2.22 2.50 2.70 0.48 Normal Normal 40294 Female 

### N/A 2.66 2.30 2.54 2.68 0.38 Normal Normal 40295 Male 

### N/A 2.69 2.33 2.75 2.83 0.50 Normal Normal 40296 Female 

### N/A 2.84 2.46 2.77 2.88 0.42 Normal Normal 40297 Male 

### N/A 2.35 2.03 2.15 2.30 0.27 Normal Normal 40298 Female 

### N/A 2.40 2.08 2.29 2.49 0.41 Normal Normal 

## C Summary ofClinical Observations, excluding skin scores: Day 0 through Day 14 

### C 

### 0 0 

### 00 00

0) 

### Page 11 of 13

... 00TomonCorporation Acute Dermal Toxicity (Single Exposure) -OPPTS**Projec~**Number: 

### TABLE II Draize Scale Skin Reactions

TEST/CONTROL SITES Test Substance: 

### Lot/Batch#:
(Erythema/Edema)

, 

### Animal # 

### Sex 02118/04

­ 03/03/04 40289 Male 0/0 40290 Female 0/0 40291 Male 0/0 40292 Female 0/0 40293 Male 0/0 40294 Female 0/0 40295 Male 0/0 40296 Female 0/0 40297 Male 0/0 40298 Female 0/0



# o=No reaction



## 000089 

# 87 Pa{!e 12 of 13Tomon Corporation Acute Dermal Toxicity (Single Exposure) -OPPTS Number: APPENDIX I: 

### DRAIZE SCALE FORSCORING SKIN REACTIONS Erythema and Eschar Formation Value No erythema
....................................................................... 0 Very slight erythema (barely perceptible)
.................................... 1 Well>defmed erythema
........................................................'" 2 Moderate to severe erythema
................................................... 3 Severe erythema (beet redness) to slight eschar formation (injuries in depth)
................................................................. 4 Total possible erythema score 

## = 4 Edema Formation No edema
.................................................................................... 0 Very slight edema (barely perceptible)
............................................. 1 Slight edema (edges ofwell defmedby defmite raising) Severe edema (raised more than 1mmand extending beyond ......................2 Moderate edema (raised approximately 1mm)
.................................. 3
area ofexposure)
...........................................................................4 Total possible edema score = 4



## 000090 Page 13 of 13 

# 88Continuation Sheet Contains TSCA**Confi~siness** Information

### ~%J",~ee'

Appendix 8 Buehler SensitizationTest

Page 89 

## 000091TOXIKONFINALREPORT:

BUEHLERSENSITIZATION TEST • OEeD



### Author Laurence Lister, B.S. 

### LAT
Fjnal Report Date

March 05. 2004

MANAGEMENI

### OF THE

### SIUDY ferforming Laboratory



### Toxikon Corporation

15 Wiggins Avenue Bedford. MA 01730 Sponsor



## 000092 

# 90Toxikon Corporation Buehler SensitizationTest

- OECD ProjectNumber: TABLE 

### OF CONTENTS
Title Page Table ofContents StudySununary Quality Assurance Statement Study Director Signature and Verification Dates 

### 1.0 Purpose 

### 2.0 References 

### 3.0 Compliance 

### 4.0 Identification ofTest and Control Substances 

### 5.0 Identification ofTest System 

### 6.0 Justification ofTest System and Route ofAdministration 

### 7.0 Experimental Design 

### 8.0 Dosage 

### 9.0 Evaluation Criteria 

### 10.0 Results 

### 11.0 Conclusion 

### 12.0 Records 

### 13.0 Confidentiality Agreement 

### 14.0 Policy on Pain and Suffering in Animals 

### 15.0 Animal Usage Table I: Preliminary Irritation: Animal Weights, Clinical Observations and SkinReaction Scores Table II: Induction and Challenge: Animal Weights and Clinical Observations Table 

### ill: Induction Phase: SkinExamination Data Table IV: Challenge Phase: Skin Examination Data Appendix I: Magnusson and Kligman Grading Scale for the Evaluation of Challenge Patch Test Reactions



## 000093 

# 91
P:;Uyp ? rtf 17TomonCorporation Buehler SensitizationTest

- OECD

. Project Number: 

### STUDY SUMMARY

The test substance, , was evaluated for its potential to produce allergenic skin reactions following epicutaneous application to albino guinea pigs. The test substance is not considered to be a skin sensitizer since none ofthe test animals exhibited erythema and edema
scores at the challenge exposure following an induction phase. No reactions were observed in the negative control group and 100% reactivity was observed in the positive control group.



## 000094 

# 92
1>':101"**~** of 17Toxikon Corporation Buehler SensitizationTest

- OECD Project Number: QUALITY ASSURANCE STATEMENT
This study was conducted in compliance with OECD Good Laboratory Practices, 1997. The section ofthe regulation not performedby or under the direction ofToxikon Corporation, exempt from this Good laboratory Practice statement, include characterization ofthe test and reference substances, OECD, Section 6.2. The Quality Assurance Unit conducted inspections onthe following dates. The findings were reported to the Study Director and to Toxikon's management.

DATE OF INSPECTIONS INSPECTION CLINICAL OBSERVATIONS 02/14/04 RAW DATA 03/05/04 FINAL REPORT 03/05104



# ..JelJ.Ulfu4Adl**~~~**

Felice Randi LaMadeleine, B.S. Quality Assurance DATE REPORTED STUDY DIRECTOR 02114104 03/05104 03/05104 DATE REPORTED MANAGEMENT 02114/04 03/05104 03/05104 

# oofrdOlf Date



## 000095 

# 93Toxikon Corporation Buehler SensitizationTest

- OECD Project Number: 

### STUDY DIRECTOR SIGNATURE AND VERIFICATION DATES
This stUdy meets the technical requirements ofthe protocol. The study also meets with the requirements ofthe OECD Good Laboratory Practices, with the exemptions as stated in the Quality Assurance Statement.
Protocol Number: CHTIVNO/OO5-03/000 

### Study Director: Laurence Lister, B.S. LAT Company:
Signature: Study Completion Date: Study Supervisor: William Entwistle, A.S., LATG
VERIFICATION DATES: The Toxikon Protocol Effective Date is the GLP Study Initiation Date. The study dates were as follows: Test Substance Receipt: 12124/03 

### Project Log Date: 01108/04 

### Study Initiation Date: 12/29/03 

### Technical Initiation: 01119/04 Primary 

### Irritation: 01119/04
- 01120/04 

### Induction: Week 1

- 01122/04 

### Week 2- 01129/04 

### Week 3- 02/05/04 

### Challenge: 02/19/04 

### Technical Completion: 02122/04



## 000096 

# 94Toxikon Corporation Buehler SensitizationTest

- OBCD Project Number: 

### 1.0 PURPOSE The purpose ofthe test was to evaluate the potential ofa test substance to produce allergic skin reactions following epicutaneous application to albino guinea pigs. 2.0 REFERENCES This study was based on the following references: 2.1 The standards ofthe Organization for Economic Co-operation and Development (OECD), Guidelines for Testing ofChemicals, section 406, July 1992. 2.2 Dennatotoxicology, F.N. Marzulli and H.I. Maibach, editors, 5th edition, 1996, pages 446-447, Taylor & 

### Francis, Washington, DC. 2.3 Magnusson B. and Kligman A.M. (1969). The identification ofcontact allergens by 

### animal assay. The guinea pig maximization test. J. Invest. Dennatol., 

### 52, 268. 3.0 COMPLIANCE The study will conform to all applicable laws and regulations. Specific regulatory requirements include the current OECD GLPs; AAALAC, International; "Guide for the Care and Use of Laboratory Animals," National Research Council, 1996. (NIH) (OLAW), 

### "Public Health Service Policy on Humane Care and Use ofLaboratory Animals," Health Research Extension Act of 1985 (Public Law 99-158 November 20, 1985), Reprinted 1996; USDA, Department of Agriculture, Animal and Plant Health Inspection Service, 9 CFR Ch. 1 (111/95 edition), Subchapter A-Animal Welfare. 4.0 IDENTIFICATION OFTEST AND CONTROL SUBSTANCES The following information was supplied by the Sponsor on a test requisition form or other correspondence wherever applicable; it does not apply to confidential information. The Sponsor was responsible for all test substance characterization data as specified in the GLP regulations. Test and control substances that are mixed with carriers require verification of concentration, homogeneity and stability. Samples oftest and control substance mixtures will be returned to the Sponsor for characterization and verification, wherever applicable. 4.1 Test Substance Test Substance Name: CAS/Code: Not Supplied by Sponsor (N/S) Lot/Batch: Physical State: Color: Expiration Date: 

### 12/23/2005 

# 95 000097TomonCorporatioll Buehler SensitizationTest

- OECD Project Number: 

### Density (liquids): Stability: stable in dark and cool closed containers Solubility: pH: N/S Storage Conditions: Room Temperature Safety Precautions: StandardToxikon Laboratory Safety Precautions 4.2 Control Substance Control Substance Name: Dinitrochlorobenzene (DNCB) Lot/Batch: CSC-02-12-006-

### VN Physical State: Solid Storage Conditions: Room Temperature Safety Precautions: Standard Laboratory Safety Precautions 5.0 IDENTIFICATION OFTHETEST SYSTEM 5. 1 Animals Used in the Test: Number and species: 38 Hartley guinea pigs 

### (Caviaporcellus) Sex: 19 males and 19 females; females were nulliparous and non-pregnant Weight/Age range: 356.2

- 487.1 grams \ 21-67 days old (adUlt). Weighed to nearest 0.1 gm Health status: healthy, not previously used in other experimental procedures. Housing: group housed Animal identification: ear punch 5.2 Animal Care and 

### Maintenance: Acclimatization: minimum 5 days under the same conditions as for the actual test. Animal Purchase: Elm Hill Laboratories, Chelmsford, MA. Bedding: hardwood chips, P.W.l. Industries, St-Hyacinthe, Quebec, Canada (non-contact) Animal room temperature: 68 

### ± 5**~** 

### Animal room relative humidity: 30

- 70% Air exchanges per hour: 10 to 15 

### 000098 

# 96 '0"',...
.. '7 nf 1'7TomonCorporation Buehler SensitizationTest

- ORCn Project Number: 

### Lights: 12-hour light/dark cycle, full spectrum fluorescent lights The laboratory and animal rooms are maintained as limited-access facilities. Animal rations: TEK HI-FIBER Guinea Pig Diet 2041, HarlanTeklad, Madison, WI Water: tap water, adlibitum Animal Selection: chosen from larger pool and examined for lack ofadverse clinical signs There were no known contaminants present in the feed, water, or bedding expected to interfere with the test data. 6.0 

### JUSTIFICATION OF TEST SYSTEM AND ROUTE OF ADMINISTRATION 6.1 Albino guinea pigs were used in this study because they have historically been used in skin sensitization tests and the guidelines have no alternative (non-animal) methods. The number ofanimals was recommended in the reference described in Section 2. 6.2 The test substance was applied topically because it is a likely route ofhuman exposure. 7.0 

### EXPERIMENTAL DESIGN 7.1 Pretreatment Screening Procedure and 

### Selection ofAnimals Animals selected for the study were examined to ensure their skin was free from irritation, trauma, and disease. Animals were weighed prior to initiation ofthe procedure. 7.2 Animal Group Assignment Test animals were distributed into the following groups: (1) Experimental

- 10 malesll0 females (2) Negative Controls - 5 males/5 females (3) Positive Controls - 3 males/2 females (4) Preliminary Irritation - 1male/2 females 7.3 Test Animal Preparation Before each application ofthe test or control substance the animals were clipped and shaved or depilitated on the scapula region, in an area ofabout 3 x 4 cm.


## 000099 

## 97Toxikon Corporation Buehler SensitizationTest

- OECD Project Number: 

### 7.4 Preliminary Irritation Study 7.4.1 A Preliminary Irritation Study was performed with three (3) previously unexposed animals. This phase was run before the start ofthe induction phase. 7.4.2 Four (4) different concentrations (see Section 8.1) oftest substance was applied to the skin for 6 hours. For water soluble test materials, it is was appropriate to use water as the vehicle. 7.4.3 The responses were scored 24 hours after the test substance application and the highest non-irritant and minimal irritant concentrations are determined. 7.5 Induction Phase (Days 0, 7 and 14) 7.5.1 Closed patches for the experimental group were prepared with the test substance per Section 8.2 and was applied directly to the skin and covered with a gauze pad ofapproximately 4 - 6 cm 2• 

### The concentration oftest substance was the highest to cause mild irritation. 7.5.2 The patch was kept inplace with occlusive bandaging. 7.5.3 The patch was removed after 6 hours ofexposure and any residual test substance was washed offwith water. 7.5.4 The test substance was applied once per week for 3 consecutive weeks (Days 0, 7 and 14) on one side ofthe animal. The positive control substance (dinitrochlorobenzene, DNCB) was applied in the same manner. Naive animals, i.e., untreatedduring the induction phase, serve as a negative control group. 7.6 Challenge (Day 28) 7.6.1 Onthe day ofthe challenge, a 4 x 3 cmvirgin skin site was shaved onthe flanks 

### ofthe experimental and control animals. 7.6.2 The challenge test was performed on freshly clipped skin sites oftest and naive control animals in the same way as the 6 hour closed patch test ofthe induction phase. The skin was exposed to the test substance (see Section 8.2) for 6 hours. One virgin site was preparedper animal. The concentration used at challenge was the highest non-irritating dose determined in the Preliminary Irritation Study. 7.6.3 At approximately 21 hours after removal ofthe challenge dose, the area ofthe challenge was marked and the whole back shaved. Approximately three hours after shaving, the test site was examined for erythema and edema. The reaction is was graded according to the Magnusson and Kligman Grading Scale for the Evaluation ofChallenge Patch TestReactions (Appendix I). 

## 0001.00 

# 98TomonCorporation Buehler SensitizationTest

- OECD ProjectNumber: 

### 7.6.4 Reading ofthe skin area is was repeated at approximately 

### 48 hours after removal ofthe challenge dose and the skin reactions are were graded.**7.6~5** The results are surrunarized and expressed inthe following terms: Incidence

- The number ofanimals showing a response of 1 ormore at 

### 24 or

### 48 hours divided by the number oftest animals. Severity

- The sum ofthe test grades divided by the number ofanimals tested. 

### 7.7 Rechallenge 

### 7.7.I 

### Ifconsidered necessary, the sensitized guinea pigs can be rechallenged 

### 1week after the primary challenge (Day 35). 

### 7.7.2 

### Ifthe results ofprimary challenge and rechallenge were unclear and allergenicity ofthe test substance was nevertheless suspected, the test may have been done again athigher sensitizing and challenge concentrations ofthe test substance. 

### 7.8 Moribund or Dead Animals Any animal found dead would have been necropsied 

### as soon as possible, but in no case later 

### than 

### 12 hours after it is was found. 7.9 No therapeutic agents were used in any phase ofthe study. 

### 7. 10 Atthe end ofthe study, all animals were weighed, then sacrificed by C02 inhalation. 

### 8.0 DOSAGE 8.1 Preliminary In-itation Depending on the test substance, up to four (4) different concentrations canbe applied by the closed patch technique described above. The concentrations ran were 

### 100%,50%, 25%, and 10%. 

### 8.2 Induction and Challenge Phase The test substance may be a solid (0.4 g applied), liquid or extract (0.4 mL). Ifit is a liquid, it was applied at the 

### minimal irritating dose for induction and the maximal non-irritating dose for the challenge. Ifthe test substance was non-irritating, it was applied full strength at both induction and challenge. Ifa solid, it was moistened with a vehicle; 

### ifthe test substance applied in this manner produces a marked irritation, it must be diluted withphysiologic saline, water, alcohol, or other suitable material.



## 0001.01. 'Pl'IO'P 10 nf 17 

# 99TomanCorporation Buehler SensitizationTest

- OECD Project Number: 

### During induction. the positive control substance (0.4 

### mL of0.1% 

### DNCB in acetone) was applied inthe same manner. For the challenge. the experimental and negative control animals were exposed to one dose oftest substance on week 05; 0.1

### %DNCB in acetone will be used to challenge the positive control animals. 9.0 EVALUATION CRITERIA 9.1 Evaluation ofData The test substance was graded according to the incidence and severity ofobserved responses (see Section 7.6.5). 10.0 RESULTS 10.1 

### Preliminary Irritation Trial (Table I) 

### Irritation (erythema and 

### edema) was absent from all sites treated with the different test substance 

### concentrations. 10.2Body Weights (Tables I and II) All 

### animals gained weight during the course ofthe study. 10.3 

### Clinical Observations (Tables I 

### and II) 

### No overt signs oftoxicity were evident in any ofthe animals during the course ofthe study. 10.4 Challenge Phase (Table III) No signs oferythema or edema were observed in any ofthe test animals. No signs oferythema or edema were evident inthe negative control group. Both animals in the positive control group exhibited signs oferythema and/or edema. The incidence and severity ofthe responses post-challenge were calculated to be: NEGATIVE TEST POSITIVE 24 hr 48 he 24 hr 48 he 24 hr 48 hr INCIDENCE 0/20 = 0 0/20

## =0 0/20 = 0 0/20 

## =0 2/2:::: 100% 212 :: 100% SEVERITY 0/20 

## =0 0/20:: 0 0/20 = 0 0/20::: 0 7/2 

### = 3.5 5/2 :::: 2.5 

### 11.0 CONCLUSION The test substance. , was evaluated for its potential to produce allergenic skin reactions following epicutaneous application to albino guineapigs. The test substance 

### is not considered to be a skin sensitizer since none ofthe test animals exhibited erythema and edema scores at the challenge exposure following an induction phase. No reactions were observed in the negative control group and 100% reactivity was observed 

### in the positive 

## 000102 

# 100 D
......**~** 1 t nF 1'7Toxikon Corporation Buehler SensitizationTest

- OECD Project Number: control group. 12.0 RECORDS 12.1 Original raw data is archived at Toxikon Corporation. 12.2 A copy ofthe final report and any report amendments is archived at Toxikon Corporation. 12.3 The original final report and a copy ofany protocol amendments ordeviations is forwarded to the Sponsor. 12.4 All unused test substance shall be returned to the Sponsor. 13.0 CONFIDENTIALITY AGREEMENT Statements ofconfidentiality was agreed upon prior to study initiation. 14.0 POllCY ON PAIN AND SUFFERINGIN ANIMALS No evidence ofpain or suffering was observed during the course ofthe study. 15.0 ANIMAL USAGE The Sponsor assures that to the best oftheir knowledge this study did not unnecessarily duplicate previous testing.



## 0001.03 

# 101 1...... f""""Toxikon Corporation Buehler SensitizationTest

- OECD Project Number: TABLE I Preliminary Irritation Animal Weights, Clinical Observations and Skin Reaction Scores
Test Substance: LotlBatch

### #:
Animal Weights and Clinical Observations
Animal 

### # Sex Day 

### 0 01119/04 Day 1 

### 01120/04 Weight Change Signs of Toxicity-­ 36 Male 419.7 426.3 6.6 None 37 Female 464.2 469.1 4.9 None 38 Female 369.1 372.8 3.7 None 

## *. Swnmary ofclinical observation Day 0 through Day 2, excluding skin reactions.
Skin Reaction Scores·
Animal

### # Sex 

## ohour 

### 01119/04 24hour 

### 01120/04 36 Male 

### 0 0 37 Female 

### 0 0 38 Female 

### 0 0

• Sum ofErythema I Edema observations for all concentrations at 0, 24 and 48 hours after test substance application.



## 0001.04 

# 102 Page 13 of 17Tox.ikon Corporation Buehler SensitizationTest

- OECD Protocol Number: TABLE II Animal Weigbts and Clinical Observations TestSubstance: Lot#: Group Animal # Sex BodyWeights (gm) Day 0 Day30 01122/04 02/21104 Change (gm) Clinical Observation·
Test I 2 3 4 5 6 7 8 9 10 Male Male Male Male Male Female Female Female Female Female 367.3 473.2 400.8 414.5 394.0 456.5 393.7 487.1 446.1 442.4 502.3 581.2 539.7 554.5 520.4 574.5 506.8 603.9 563.1 575.8 135.0 108.0 138.9 140.0 126.4 118.0 113.1 116.8 117.0 133.4 None None None None None None None None None None Test 11 12 13 14 15 16 17 18 19 20 Male Male Male Male Male Female Female Female Female Female 356.2 374.1 375.5 477.9 467.6 373.1 454.8 435.0 447.4 473.1 501.4 493.0 505.9 606.5 616.1 521.6 586.2 555.0 593.7 604.4 145.2 118.9 130.4 128.6 148.5 148.5 131.4 120.0 146.3 131.3 None None None None None None None None None None Negative Control 21 22 23 24 25 26 27 28 29 30 Male Male Male Male Male Female Female Female Female Female 379.0 405.7 418.2 416.1 387.3 449.9 4t3.3 382.6 383.0 399.8 499.3 529.2 548.6 515.5 504.3 556.5 523.3 516.9 509.6 507.6 120.3 123.5 130.4 99.4 117.0 106.6 110.0 134.3 126.6 107.8 None None None None None None None None None None Positive Control 31 32 33 34 35 Male Male Male Female Female 374.6 459.8 433.6 462.1 405.7 523.8 567.3 559.9 579.5 535.6 149.2 107.5 126.3 117.4 129.9 None None None None None "'Summary ofclinical observations excluding skin reaction scores - Day0 through Day 30


### 000:105 Pa2:e 14 of 17 

# 103TomonCorporation BuehlerSensitizationTest • OBCD Project Number: 

### TABLEID Challenge Phase

- SkinExaminationData Test Substance: Lot/Batch

### #: Animal # Day 29 02120/04 Day 30 02121104 Test Substance (Erythema/Edema) 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 10 0 0 11 0 0 12 0 0 13 0 0 14 0 0 15 0 0 16 0 0 17 0 0 I 18 0 0 19 0 0 20 0 0



## 000106 D"
....o 

## 1" "f 1'7 

# 104Toxikon Corporation Buehler Sensitization Test

- OECD Protocol Number: 

### TABLEll Challenge Phase

- Skin Examination 

### Data (Cont.) Test Substance: Lot/Batch

### #: Animal# Day 29 11111199 Day 30 11112199 Negative ContrOl 21 0 0 22 0 0 23 0 0 24 0 0 25 0 0 26 0 0 27 0 0 28 0 0 29 0 0 30 0 0 Positive Control 31 2 2 33 3 3 32 3 2 34 3 2 35 2 2 Positive Control Combined Incidence 

## = 515 == 100% Combined Average Severity (24 hr.) 

### = 1315 = 2.6 Combined Average Severity (48 hr.) 

## = 1115 

## =2.2 Negative Control Combined Incidence =0% Combined Severity (24 hr) = 0110 = 0 Combined Severity (48 hr) = 0110 == 0 Test Substance Combined Incidence 

## =0% Combined Severity (24 hr) == 0/20 

### =0 Combined Severity (48 hr) = 0/20 == 0 

## 000107 Page 16 of 17 

# 105TomonCorporation 'Buehler SensitizationTest

- OECD Protocol Number:
___ .__ _ 

### APPENDIX I: Magnusson and Kligman Grading Scale for the Evaluation ofChallenge PatchTestReactions
Reaction No visible change Discrete or patchy erythema Moderate and confluent erythema Intense erythema and swelling 

# o 1 

### 2 3



## 000108 

# 106Continuation Sheet Contains TSCA

### Co~siness Information**~"fI1...-~~**

Appendix 9 Salmonella Typhimurium and Escherichia Coli Reverse Mutation Assay



## 0001.09 Page [07

### TOXIKON FINAL REPORT:



### SALMONELLA TYPHIMURIUMAND 

### ESCHERICHIA COLIREVERSE MUTATION ASSAY -OECD

Author Paul Lezberg. B.A.

Final RmortDate April 6, 2004

MANAGEMENT OF THE STUDY PerfonningLaboratory

ToxikonCorporation

15 Wiggins Avenue Bedford.MA 01730 Sponsor



## 0001.1.0 

# 108Tonkon Corporation Salmonella typhimllrillm and Escherichia coliReverse Mutation Assay

- OECD ProjectNumber: 

### TABLE OFCONTENTS
TitlePage Table ofContents StudySummary QualityAssurance Statement StudyDirectorSignature and VerificationDates
1.0 Purpose 2.0 References 3.0 Compliance 4.0 Identification ofTest and ControlArticles 5.0 Identification ofthe Test System 6.0 Justification ofTest System andRoute ofAdministration 7.0 Experimental Design 8.0 Dosage 9.0 Evaluation Criteria 10.0 Results Table I: Range FindingAssaywithoutMicrosomal Activation TableII: Reverse MutationAssay withoutMicrosomal Activation TableIII: ReverseMutation Assay withMicrosomal Activation TableIV: ReverseMutation Assay withoutMicrosomal Activation

- ConfirmatoryAssay TableV: ReverseMutation AssaywithMicrosomalActivation

- ConfirmatoryAssay 11.0 Conclusion 12.0 Records 13.0 ConfidentialityAgreement

Page 2 of18 

## 000:1:11. 

# 109Toxikon Corporation Salmonella typhimurium and Escherichia coliReverse Mutation Assay

- OECD ProjectNumber: 

### STUDY SUMMARY


### The Salmonella typhimurium 

### and Escherichia coli 

### Reverse Mutation Assay (Ames Assay) evaluated the potential ofthe test substance, ,to induce reverse mutations in histidine (his· to his) and tryptophan (tryp- to tryp) genes in Salmonella typhimurium (S. typhimurium) 

### and Escherichia coli (E. coli) 

### respectively. This direct plate incorporation assay was conducted with four strains of Salmonella typhimurium (S. typhimurium) 

### and one strain ofEscherichia coli (E. coli) 

### in the presence and absence of an exogenous mammalian activation system. All positive controls exhibited a statistically significant increase in the number ofrevertant colonies as compared to the corresponding negative control, validating the functioning ofthe assay. A statistically significant increase in the number ofrevertant colonies
was not observed with the test substance. Under the test conditions, the test substance is not mutagenic inthe test species

Page 3of18 

## 0001.1.2 

# 110Toxikon Corporation Salmonella typhlmurium andEscherichia coliReverse Mutation Assay

- OECD ProjectNumber: QUALITYASSURANCE STATEMENT
This studywasconducted incompliance withU.S. Food and DrugAdministrationregulations set forth in 21 CFR, Part 58. The sectionsofthe regulationsnot performedbyorunderthe directionofToxikon Corporation, exempt from this Good LaboratoryPractice Statement, included characterizationand stabilityof the test article and itsmixturewithcarriers, 21 CFR, Parts 58.105 and 58.113. The QualityAssurance Unit conducted inspections onthe following dates. The findings were reported to the StudyDirectorandto Toxikon's Management.

INSPECTIONS DATE OF DATEREPORTED DATEREPORTED INSPECTION STUDYDIRECTOR MANAGEMENT
SCORlNG 03/01104 03/01104 03/01104 RAWDATA 04/06/04 04106104 04/06/04 FINALREPORT 04/06/04 04/06/04 04/06/04**,~~uui Jayaram~**  B.S
..

.tyAssurance



## 0001.1.3 

# 111 Pae:e 4 of 18Toxikon Cttrporation Salmonella typhimurium and Escherichia coliReverse MutationAssay

- OECD ProjectNumber: 

### STUDY DIRECTORSIGNATURE AND VERIFICATIONDATES
This studymeets withthetechnicalrequirements oftheprotocol. Thestudy also meets the requirements oftheGoodLaboratoryPractice Regulations, 21 CFR,Part 58, withthe exemptions as statedinthe QualityAssurance Statement.
ProtocolNumber: CHTNITROIOO 1-03/000 StudyDirector: PaulLezberg, B.A. Company: Toxikon Corporation
Signature: Date: StudySupervisor: Leigh Bergeron, B.A.
VERIFICATIONDATES: TheToxikonProtocolEffectiveDate is the GLP StudyInitiation Date. The studydates were as follows: ProtocolEffective Date: 12/24/03 TestSubstanceReceipt: 12/24/03 ProjectLogDate: 01108104 Range FindingAssay: 01/27/04
- 01130104 Reverse Mutation AssayTechnical Initiation: 02/27/04 Reverse Mutation Assay Technical Completion: 03/01104 ConfmnatoryAssayTechnicalInitiation: 02/27/04 ConfinnatoryAssay Technical Completion: 03/01104



## 0001.1.4 PaQ'e 5 of 1 R 

# 112Toxikon Corporation Salmonella typhimurium andEscherichia coliReverse Mutation Assay

- OECD ProjectNumber: 

### 1.0 PURPOSE TheSalmonella typhimurium andEscherichia coliReverse Mutation Assay (Ames Assay) evaluated thepotential ofa test substanceto inducereverse mutations in histidine (his

-to his ) andtryptophan (tryp

-to tryp +) genes inthese organisms, respectively. This directplate incorporation assayis conductedwith four strains ofSalmonella typhimurium (s. typhimurium)
.... and one strain ofEscherichia coli (E. coli) inthepresence and absence ofan exogenous mammalian activation system. A confirmatory assaywasperformedinordertoverify the results ofthe ReverseMutationAssay. 

### 2.0 REFERENCES The studywas conductedbased upon the following references: 2.1 Organization forEconomic Co-operationand Development (OECD), Guidelines for TestingofChemicals, "BacterialReverseMutation Test", Section471, adopted: 21 July 1997. 2.2 Maron, D.M., Ames B.N., RevisedMethods for the Salmonella MutagenicityTest, Mutation Res., 113: 173-215, 1983. 

### 3.0 COMPLIANCE Thepresent study conformedto all applicable laws andregulations. Specificregulatory requirements includedthe current GoodLaboratoryPractice for Nonclinical LaboratoryStudies, FDA, 21 CPR., Part 58. 

### 4.0 IDENTIFICATIONOFTHETESTAND CONTROL ARTICLES The following informationwas suppliedbythe Sponsorwherever applicable; it did not applyto confidential information. The Sponsorwas responsible for all test substancecharacterization data as specifiedintheGLP regulations. Test and control articles (exclusive ofextracts) thatare mixedwithcarriers require verification ofconcentration,homogeneity and stability. Samplesof test and control article extracts werenotreturned to the Sponsorfor characterization and verification. 4.1.1 Test SubstanceName: CASICode#: Not SuppliedbySponsor(N/S) LotlBatch#: Physical State: Color: ExpirationDate: 12/23/2005 Density: 1.30 Stability: stable in dark and cool closedcontainers Solubility: :- - .. pH: 

### N/S Storage Conditions: RoomTemperature SafetyPrecautions: StandardToxikonLaboratory SafetyPrecautions 

## 000115 

# 113 Page 

### 6 of18Toxikon Corporation Salmonella typhimurium andEscherichia coliReverse Mutation Assay

- OECD ProjectNumber: 4.2 Control Articles (Toxikon Supplied): 4.2.1 Negative Control Article: Control Article Name: Dimethyl Sulfoxide (DMSO) Toxikon QC #: CSC-03-08-044-MB Physical State: Liquid Color: Colorless Storage: Room Temperature SafetyPrecautions: Standard Laboratory SafetyPrecautions 4.2.2 Positive Control Articles: 4.2.2.1 Control ArticleName: 2-Aminoanthracene Toxikon QC #: CSC-01-01-002-MB Physical State: Powder Color: Fluorescent GreenlYellow Storage: Desiccated,4±2°C SafetyPrecautions: Known Mutagen. Appropriate Laboratory SafetyPrecautions 4.2.2.2 Control Article Name: SodiumAzide (100**~glmL)** Toxikon QC #: AMES-04-01-004-MB Physical State: Liquid Color: Colorless Storage: -20 ± 2°C SafetyPrecautions: Known Mutagen. Appropriate Laboratory SafetyPrecautions 4.2.2.3 Control Article Name: SodiumAzide (5**~glmL)** Toxikon QC #: AMES-04-01-005-MB Physical State: Liquid Color: Colorless Storage: -20 ± 2°C SafetyPrecautions: Known Mutagen. Appropriate Laboratory SafetyPrecautions 4.2.2.4 Control Article: 2-Nitrofluorene Toxikon QC #: AMES-03-05-016-MB Physical State: Liquid Color: Colorless/Clear Storage: -20 ±.2°C SafetyPrecautions: Known Mutagen. Appropriate Laboratory SafetyPrecautions



## 0001.1.6 

# 114 Page 7of18ToxikOlI Corporation Salmonella typhimurium andEscherichia coliReverse MutationAssay

- OECD ProjectNumber: 

### 4.2.2.5 Control ArticleName: 9-Aminoacridine Toxikon QC 

### #: AMES-03-11-04-MB Physical State: Liquid Color: GreenlYeHow Storage: -20

### ±2°C SafetyPrecautions: KnownMutagen. Appropriate Laboratory SafetyPrecautions 4.2.2.6 ControlArticleName: l-Ethyl-3-Nitro-I-Nitrosoguanidine (ENNG) Toxikon QC

### #: CSC-OI-09-0l5-MB Physical State: Powder Color: Beige Storage: -20

## ±2°C SafetyPrecautions: Known Mutagen. Appropriate LaboratorySafetyPrecautions 5.0 IDENTIFICATION OFTHETEST SYSTEM 5.1 TheSalmonella typhimurium 

### strains used inthis assay were TA98, TAlOO, TA1535, and TA1s37. These strains and theEscherichia coli 

### strainWP2 used in this assaywerepurchased from Moltox, Inc. (Boone,NC). 6.0 JUSTIFICATION OFTESTSYSTEMAND ROUTE OFADMINISTRATION 6.1 Historically, the Reverse Mutation Assay inS. typhimurium 

### has been used to detect reverse mutations in a histidine gene evaluated as histidine-independent growth. This test system is recommended inthe references listed in Section2.0. TheReverseMutation Assay in E. coli 

### hasbeenused to detect reverse mutations in atryptophan gene evaluated as tryptophan-independent growth. This test system is recommended in the OECD Guidelines. 6.2 The test substance was extracted in DMSO and administered in vitro, 

### to the test system at various dilutions oftheextract. 7.0 EXPERIMENTALDESIGN 7.1 Method: The bacteriawere exposed to the test substance with and without a metabolic activation system, and directly plated on a minimal medium. After a period ofincubation, revertant colonies were countedand compared to the number ofspontaneous revertants in the negative control group.



## 0001.1.7 

# 115 Page 8of18ToxikonCorporation Salmonella typhimurium and Escherichia coli Reverse Mutation**As~ay** - OECD ProjectNumber: 7.2 TesterStrains: The requirement ofhistidine and tryptophan for growth was demonstrated for the Salmonella typhimurium strains and E. coli, respectively. Other phenotypic characteristics were verified by using crystal violet sensitivity and resistance to ampicillin. Spontaneous reversion frequency was in the range expected either as reported in literature or as established byToxikon'shistorical mean values. 7.3 Stock Cultures: Working stock cultures were grown fresh for the assay. Frozen stock for each strain wasthawed and inoculated into sterile nutrient broth. Cultures were incubated overnight at 37 

### ±2°C. Cultures with an Absorbance >1 at 650 nm, read against a nutrientbroth blank, were used 

### in the assay. 7.4 NegativeControl Article: Tester strains were plated with DMSO that was exposed to the same conditions as the test substance, with and without metabolic activation. This served asthe negative control article and providedreference forbackground lawns andrevertant colonyfonnation. 7.5 Positive Control Articles: The positive control article for the metabolically activated portion of the assay was 2-aminoanthracene inall strains.


### S.typhimurium TA98 2-Aminoanthracene (5 J.1g1mL) S.typhimurium TAIOO 2-Aminoanthracene (10 J.1g1mL) 

### S.typhimurium TAI535 2-Aminoantlrracene (20 J.1g1mL) 

### S.typhimurium TA1537 2-Aminoanthracene (30 J.1g1mL) E.coli WP2 2-Aminoanthracene (200 J.1g1mL)
Positive control articles not requiringmetabolic activation are strain specific. S.typhimurium TA98 2-Nitrotluorene (10 J.1g1mL) S.typhimurium TA100 SodiwnAzide (100 J.1g1mL) 

### S.typhimurium TA1535 SodiwnAzide (5 J.1g1mL) S.typhimurium TA1537 9-Aminoacridine (800 J.1g1mL) 

### E. coli WP2 l-Ethyl-3-NitroI-Nitrosoguanidine (20 J.1g1mL)
The appropriate concentrations for all positivecontrol articles were applied at 100 J.1Uplate.


## 0001.1.8 

# 116 Paile 9 of18Toxikon Corroration Salmonella typhimurium andEscherichia coliReverse Mutation Assay

- OECD ProjectNumber: 7.6 Replication: All controls andtest groups were platedintriplicate. 7.7 Non-activated Assay: Top agar, supplemented with 0.5 mM histidine

- 0.5 mMbiotinper 1.0 mL ofagar, was used as an overlay. Tryptophanwas used inplaceofhistidine for WP2. The agarwasmaintained at 42-48°C until used. The overlayconsistedofsterile tubes containing: 2 mLofmoltentop agar 0.1 mLofthe appropriate tester strain 0.1 mLofthe appropriate concentrationofthe test substance orcontrol article 0.5 mLof0.2 M phosphatebufferpH=7.4 The tubes were vortexed andpouredonto minimal glucose agarplates. All plates intherange finding assaywere incubated at 37± 2°C for 64.5 hours. All plates inthe ReverseMutation Assay and ConfinnatoryAssaywere incubated at 37 ± 2°C for 65.5 hours. Plates were checked for unifonnbackgroundlawns and the numberofrevertant colonies counted. 7.8 MetabolicActivation Assay: Tubes requiringmetabolic activation contained an S9 fraction ofrat liverhomogenateobtained from Aroclor® 1254 treated Sprague-Dawleyrats. The S9 activation system, prepared fresh on the dayofthe assay andkeptrefrigeratedoronice, containedthe following per 10 mL:
0.4M MgCh/1.65M KCI 1 M glucose-6-phosphate 0.1 MNADP 0.2 M phosphatebufferpH=7.4 USP Water for Injection S9Fraction 0.20 mL 0.05 mL 0.40 mL 5.00 mL 3.35 mL 1.00mL
Themethodofpreparation for the top agarwas the same as inthe non-activated assay, withthe exception ofsubstitutingthe 0.5 mLcomplete S9 mix for thephosphatebuffer. 8.0 DOSAGE 8.1 Range Finding Assay: A Range Finding Assay was perfonned without metabolic activation to detennine levels at which the test substance exhibited toxicity. The assaywas conducted onlywithTA100. Thetest substance was assayed at six dilutions in Dimethyl Sulfoxide (DMSO). The concentrations testedwere 5000, 500, 50, 5, 0.5 and 0.05 ).1g/plate.



## 0001.1.9 

# 117 Pae:e 10 of 18Toxikon Corporation Salmonella typhimurium andEscherichia coliRevers-: Mutation Assay**~** OECD Project Number: 

### 8.2 Reverse Mutation Assay: As no toxicity was observed, the same concentrations were tested in the reverse mutation assay. 1

00**~L** 

### ofDMSO containing the appropriate concentration oftest substance was applied per plate. 8.3 ConfirmatoryAssay: The results ofthe Reverse Mutation Assaywere confirmed through a Confirmatory Assay. The ConfirmatoryAssaywas performed as described in Sections 7.0, 8.0 and 9.0. 

### 9.0 EVALUATION CRITERIA 9.1 Evaluation CriteriaoftheRangeFindingAssay: The Range Finding assay is performed with strain TAl00 without microsomal activation. If 

### toxicityis detected, dose levels wouldbechosento bracket toxic and**non~toxic** 

### levels. 9.2 EvaluationCriteriaofthe ReverseMutationAssay: The test results are analyzed using a statistical program such as GrapbPad Prism Software by Analytical Software, Inc. (Analyzing Data with Graph Pad Prism, Harvey Motulsky), ANOVA (analysis ofvariance) and**Newman~Keuls** 

### Test for confirmation ofpairwise comparisons. This statistical method is used to determine ifthere is a significant (p**~0.05)** 

### increase in the mutation frequency causedbythe test substance comparedto the negative control article. 9.3 PositiveResponse: The positive control article assay consists of direct-acting mutagens and mutagens requiring metabolic biotransformation. All positivecontrols must exhibit a statistically significantincrease in the number of mutants as compared to the corresponding negative control, in order to demonstrate that the test system is functional with known mutagens. Thenegative control article plates, for each strain, give a referencepointto comparethe test data. The test substance is considered to have caused a positive response ifthe test substance shows a statistically significant increase in the number of mutants as compared to the corresponding negative control. Biological significance will be taken into consideration in the evaluation of results. Generally aresponse that is twice that ofthe negative control article is consideredto be a mutagenic response. 9.4 ConfirmatoryAssay: The results ofthe ConfirmatoryAssay should verifythe outcome ofthe Reverse Mutation Assay. 

### 10.0 RESULTS 10.1 RangeFindingAssay (Table I) 

### The Range Finding Assay did not show anycytotoxicity orsignificantly increased mutants with anyconcentrationofthe test substance as comparedto the negative control. 

## 000120 

# 118 PaQe 11 of18Toxikon Corporation Salmonella typhimurium andEscherichiscoliReverseMutation Assay

- OECD ProjectNumber: 

### 10.2 Reverse Mutation Assay (Tables II and ill) 

### The mean number ofrevertants per plate was calculated for the test substance and the negative andpositive control articles. All positive controls exhibited a statistically significant increase iil the number ofmutants as compared to the corresponding negative control, demonstrating that the test system was functional withknownmutagens. A statistically significant increase in the number of colonies was not observed with the test substance incomparisonto the negative control group at any tested concentrations. 10.3 ConfirmatoryAssay (Tables IV and V) The results of the Reverse Mutation Assay were confinned through a Confinnatory Assay. Results ofthe ConfinnatoryAssay arepresented inTables IV and V. 10.4 Theresults are summarized inTables I-V.



## 000121**P::lP~** l' of 1 R 

# 119Toxikon Corporation Salmonella typhimurium and Escherichia coli ReverseMutation Assay

- OECD ProjectNumber: TABLE I Range FindingAssay WithoutMicrosomalActivation TestArticle: Lot/Batch#: ..,j
RevertantsIPlate'"
STRAIN
TAIOO 402 376 MEAN 369.0 SO 37.0 5000 58 45 48 50.3 6.8 500 55 63 41 53.0 ILl TEST SUBSTANCE DOSE LEVEL (J.1g1late) 50 47 53 58 52.7 55 5 61 60 42 54.3 10.7 0.5 50 49 44 47.7 3.2 0.05 63 46 48 52.3 9.3 '" Allplates were dosed at 100 J.1Uplate "'''' Thepositive controlusedwas sodiumazide for strainTAl00 "''''''' Thenegative controlused inthe assay was DMSO

Page 13 of 18


## H00122 

# 120To:dkon Corporation Salmonellatyphilnurium andEscherichia coli Reverse Mutation Assay

- OECD ProjectNumber: TABLE II Reverse Mutation AllaY Without**~lIerOiomal** Activation TestArtlde: LotiBatth#: RcvCl1antsIPlatc•

STRAIN CONTROLS TEST SUBSTANCE POSmVE CONTROL·· NEGATIVE CONTROL··· DOSELEVEL (flglplatc) 5000 500 50 S 0.5 0.05 TA98 14S 162 124 10 10 13 13 9 15 14 8 8 IS 11 6 9 9 II 12 12 7 S 7 10 MEAN SO 144.7 19.2 11.0 1.7 12.3 3.1 10.0 3.5 11.7 6.0 9.7 1.2 10.3 2.9 8.3 1,5 TAIOO 6S8 644 686 115 128 III 93 108 114 85 98 102 84 81 132 109 135 102 102 109 124 132 150 96 MEA.'i SO 662.7 21.4 118.0 8.9 105.0 10.8 95.0 8.9 99.0 28.6 115.3 17.4 111.7 11.2 126.0 27.S TAIS3S 119 97 131 8 10 18 II II 14 9 10 10 5 12 10 II 7 7 10 14 6 6 6 II MEAN SO 115.7 17.2 12.0 5.3 12.0 t.7 9.7 0.6 9.0 3.6 8.3 2.3 10.0 4.0 7.7 2.9 TAIS37 IS2 ISS 164 17 22 25 15 18 14 22 16 13 19 IS IS 14 10 28 22 17 20 12 18 21 MEAN SO 167.0 13.7 21.3 4.0 15.7 2.1 17.0 4.6 16.3 2.3 17.3 9.5 19.7 2,5 17.0 4.6 WP2 88 65 71 14 19 17 16 19 21 12 14 14 17 21 14 14 22 10 20 12 IS 16 24 10 MEAN SO 74.7 11.9 16.7 2.5 IS.7 2.5 13.3 1.2 17.3 3.5 15.3 6.1 IS.7 4.0 16.7 7.0 All plates were dosed at 100j1Uplatc •• Thepositive control used was sodium azide for strainsTAIOO and TA1535. 2-nitrofluorene forstrainTA98. 9-aminoacridine for strainTAl537. and ENNG forsninWP2 ••• The negativecontrol used in theassaywas OMSO**Pa~14oflS**



## U00:123 

# 121Toxikon Co,!,uration Salmonella typhlmurlum and Escherichia ((,II Reverse Mutation Assay

- OJ<:CD ProjectNumber: TABLE III Reverse MutationAssay WitII Microsomal Activation TestArticle: LotiBalch#: RevertantsIPlate· 

# ..

STRAlN CONTROLS TEST SUBSTANCE POSlTlVE CONTROL·· NEGATIVE CONTROL··· DOSE LEVEL (l1g{plate) 5000 500 SO S O.S O.OS TA98 180 167 153 18 12 IS 14 10 10 IS 10 10 21 14 14 23 10 10 14 II 16 23 7 II MEAN SO 166.7 I3.S 15.0 3.0 ILl 2.3 11.7 2.9 16.3 4.0 14.3 7.S 13.7 2.S 13.7 8.3 TAIOO 447 502 611 130 118 105 tl2 122 109 99 114 110 8S 142 109 146 100 \03 109 100 125 146 101 112 MEAN SO 520,0 83.5 117.7 12.S 1\4.3 6.8 107.7 7,8 Jl2.0 28,6 Jl6,3 25.7 111.3 12.7 119.7 23.5 TAIS3S 118 145 161 14 8 II 10 12 11 9 9 12 10 6 IS 6 12 12 8 8 17 9 16 8 MEAN SO 141.3 ' 21.7 11.0 3,0 I\.0 \.0 10,0 1.7 10.3 4.5 10.0 ),5 11.0 5,2 11.0 4,4 TAI537 194 171 164 12 20 17 20 18 9 17 20 6 14 10 21 II 16 19 \0 18 16 21 14 15 MEAN SO 176.3 1S.7 16.3 4.0 15,7 5,9 14.3 7,4 15.0 5,6 15.3 4.0 14,7 4.2 16,7 3.8 WP2 85 89 62 \0 19 16 15 15 18 14 22 13 8 26 15 5 II 23 9 17 IS )3,7 4,2 9 )3 

# -r-¥r MEAN SO 78.7 14.6 15.0 4,6 16.0 \,7 16J 4.9 16,3 9.1 13.0 9,2 All platesweredosed at 100J.lUplate •• Thepositivecontrol used was 2·aminoanlhl'acene for all strains ••• The negativecontrol used in the assaywas OMSO

Page IS ofl8



## JvUl.:G4 

# 122Toxikon Corporation Salmonellatyphimurlumand Lscheriehla coil ReverseMutation Assay. OECD ProjectNumber: TABLEIV ReverseMutationAssay WltboutMicrosomalActivation ConnrmatoryAssay TestArticle: LotlBatch#: Revel1alltsIPlale'

STRAIN CONTROLS TESTSUBSTANCE POSmVE CONTROL" NEGATIVE CONTROL-·· DOSE LEVEL (l1wplate) 5000 500 50 S O.S O.OS TA98 lSI 159 In 12 10 14 9 9 \3 II 13 10 9 12 12 11 10 12 14 6 8 9 13 10 MEAN SD 160.7 10.6 12.0 2.0 10.3 2.3 11.3 1.5 11.0 1.7 11.0 1.0 9.3 4.2 10.7 2.1 TAl00 S89 626 648 118 121 116 103 116 119 123 104 III 124 109 113 112 101 118 107 113 101 126 III lIS MEAl"! SO 621.0 29.8 118.3 2.S 112.7 8.5 112.1 9.6 115.3 7.8 112.3 S.S 101.0 6.0 111.3 7.8 TAIS35 128 141 135 10 13 6 5 12 11 12 10 8 S 9 16 12 6 9 5 9 9 8 8 12 MEAN SO 134.1 60S 9.7 3.5 9.3 3.8 10.0 2.0 10.0 5.6 9.0 3.0 7.7 2.3 9.3 2.3 TAIS37 162 178 169 IS 19 27 10 10 16 10 14 18 10 17 19 17 10 IS 12 10 14 14 14 10 MEAN SO 169.7 8.0 20.3 6.1 12.0 3.S 14.0 4.0 15.3 4.7 14.0 3.6 12.0 2.0 12.7 2.3 WP2 93 64 69 12 20 18 13 18 17 13 10 16 IS 15 8 14 12 16 10 10 16 10 13 9 MEAN SO 75.3 15.5 16.7 4.2 16.0 2.6 13.0 3.0 12.7 4.0 14.0 2.0 12.0 3.S 10.7 2.1
• All plales wen: dosed at 100 !,Up1ate

,. Thepositive control used was sodiumazidefor strains TA100andTA1S3S. 2-nitrofluorene for strain TA98. 9·aminoacridine for strain TAlS37, andENNG for slninWP2 ••• Thenegativecontrol used in theassay was OMSO

Page 160fl8



## 0001.25 

# 123Toxikun Corporation SalmonellatyphlmuriumandEscherichiacoli Reverse MutationAssay

- OECD Project Number: TABLEV ReveneMulatlonAssay WithMicrosomalActivation ConfirmatoryAssay TestArticle: LotlBatch II:

STRAIN CONTROLS TEST SUBSTANCE POSITIVE CONTROL·· NEGATIVE CONTROL··· DOSE LEVEL (J,l.gfplatc) SOOO SOO SO S O.S 0.05 TA98 ISS 138 129 21 IS 13 14 10 IS 24 11 10 22 19


### . 9 16 II 14 10 8 18 18 18 5 MEAN SD 140.7 13.2 16.3 4.2 13.0 2.6 15.0 7.8 16.7 6.8 13.7 2.5 12.0 5.3 13.7 7.S TA100 513 702 511 136 122 109 128 141 93 125 118 120 121 109 113 122 129 111 145 108 103 132 118 112 MEAN SO 575.3 109.7 122.3 13.5 120.7 24.8 121.0 3.6 114.3 6.1 120.7 9.1 118.7 22.9 120.7 \0.3 TAIS3S 103 165 157 IS 18 13 19 10 12 8 13 10 10 8 10 II 13 18 10 23 12 18 9 15 MEAN SO 14\.7 33.7 1S.3 2.5 13.7 4.7 10.3 2.5 9.3 1.2 14.0 3.6 15.0 7.0 14.0 4.6 TAIS37 203 165 177 16 19 22 17 17 14 14 10 26 \3 25 12 17 21 12 IS IS 11 16 10 23 MEAN. SO 181.7 \9.4 \9.0 3.0 16.0 1.7 16.7 8.3 16.7 7.2 16.7 4.5 13.7 2.3 16.3 6.5 WP2 103 69 85 17 II 19 10 19 14 12 12 20 13 13 9 14 10 21 21 16 9 21 20 7 MEAN SO 85.7 17.0 15.7 4.2 14.3 4.5 14.7 4.6 11.7 2.3 15.0 5.6 15.3 6.0 16.0 7.8 All plat.were dosed at 100 )lIJplate The positivecontrol used was 2-aminoanthnu::enc forall strains ••• The negative control used in the assay was DMSO

Page 17 of 18



## 000126 

# 124To:dkon Corporation Salmonella typhimurium andEscherichia coliReverse Mutation Assa)

- OECD ProjectNumber: 

### 11.0CONCLUSION TheSalmonella typhimurium 

### and Escherichia coli 

### Reverse MutationAssay (AmesAssay) evaluated the potential ofthe test substance, ,to inducereverse mutations in histidine (his'to his) andtryptophan (tryp" 

### to tryp+) genes inSalmonella typhimurium (S. typhimurium) 

### andEscherichia coli (E. coli) 

### respectively. This direct plateincorporation assaywas conductedwithfour strains ofSalmonella typhimurium (s. typhimurium) 

### and one strain ofEscherichia coli (E. coli) 

### inthe presence and absenceofan exogenous mammalian activation system. Allpositive controls exhibited a statistically significant increase inthe number ofrevertant colonies as comparedto the correspondingnegative control, validatingthe functioning ofthe assay. A statistically significant increase inthe number ofrevertantcolonies was not observed with the test substance. Underthe test conditions, the test substance is not mutagenic inthe test species. 

### 12.0 RECORDS 12.1 Original raw datais archived at Toxikon Corporation. 12.2 A copyofthe final report and anyreport amendments are archived at Toxikon Corporation. 12.3 Theoriginal final report and acopyofanyprotocol amendments ordeviations are forwarded to the Sponsor. 12.4 Allunusedtest substance shall bedisposed ofbyToxikon, perSponsor's specifications. 12.5 Finalreports shallnotbereproduced, except in full, withoutthe written authorization/approval from Toxikon. 

### 13.0 CONFIDENTIALITYAGREEMENT Statements ofconfidentialitywere as agreedupon priorto studyinitiation.



## a00127 Page 18 

### of18