Form Approved. OMJ3. No. 2070-00 J2.· Approval Expires IO-J 1-96 

U.S. ENVIRONMENTAL PROTECTION AGENCY AGENCY USE ONLY

_~_I::DA Unte of ReceIpt PREMANUFACTURE 

~C--I-\ NOTICE 

FOR NEW CHEMICAL SUBSTANCES 

Company Sanitized 

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 the Toxic 

Substances Control Act (TSC A) Information Service by calling 202-554-1404. or faxing 20:'-554-5603) 

• 	 If a 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 have generated. 

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 if YOU 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 [(?I kw will nm begin until EPA r,-,(elves tillS 

inf(lrmJlinn A kacr 111 support of your 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 f there' arc several man ufacturc, processmg. or usc operations to he dcscflb~d In Pan 

11. sectIOn !\ and B ol'this notice, rcproduce the seclions as needed 

Panlll- LIST OF ATTAClIMEJ'.:TS 

Attach additional sheets if there IS mIL enough space to :lnswer H qL.cslion till'y 

Lahd each COllllnU31IO" sheet \\lIh the corresponding scelion headlllg In Part 

Ill. ht these atlJchnWIllS. :m" lest <.lat:l or other data and any optIOnal 

ll1fnrmallon lI1c1uUt'lt In the nOlie;; 

OPTIONAL INFORMAl f()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 IIself prohibit the 'lIhlmtt~r rrom 

later deVl:ltll1g I[om the 1Il1l1l'malion (except chcmical,"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 of the 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 ,If conlrul <md to pro\,ld-: a 

descnpuoll of all other data known to or reasonanle as,cnamahle to you, ir these dala 

arc rdatcd to Ihe health and environmental e:kcls of the manuta<:turc, [lrocessmg. 

dlSmhllll()1l III C(lml1lcrcc'. usc. or dlsposal of the ne\~ chemJcal substallce Standurd 

Ineralurc cllUllOllS may he suhmllled for dma mille oren sClcnl1t1c literature. Complete 

tes! datu IwrutC11111 Em,ilsh), not summafle5 {If data, 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 of tes! data and other dut;]. Data 

should b" suhmllled accllrdlOg to the rCljuiremel1ts of § 72050 of the Premanufactllfe 

Notlficallon Ruk 140 CrR 1':111720) 

Tesi Data IClwck Bdml un\' mcluded in this nOllee) 

Em lronmenlillinte daw Other datu DYes 

• I kalth 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 

or control of Ihe 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 	TMIA (Test Marketing EXclllpuon /\ppl!<:<1110n) 47~f -....... •.. ' - J 


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

D LOREX I Low Rcleasdl.ow Exposurc LxelTIrtion) 7is:jo fc)(2) 

o l.VI- Modillcalloll D LOREX Mod i11catlOn 

lS 1II1S·\ CO!\SOLIDATED PMN') 0 Ycs 

# of chemIcals 

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

EPA FOR M 7710-25 (Rev 5-(5) 	 Replaces previous editions of EPA Form 7710-25 I 
Public reporting burden for this collection of information is estimated to avcrage 110 hours per response, including time for reviewing 

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

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

reducing this burden. to Chief, Information Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M St.. S.W., Washington, D.C. 

20460; and to the Office of Management and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

CERTIFICATION 

I certify that to the best of my knowledge and belief: 

1. 	 The company named in Part I, Section A, subsection Ia ofthis notice form intends to manufacture or import for a commercial purpose, 

other than in small quantities solely for research and development, the substance identified in Part L Section B. 

2. 	 All information provided in this notice is complete and truthful as of the date of submission. 

3. 	 1am submitting with this notice all test data in my possession or control and a description of all other data known to or reasonably 

ascertainable by me as required by §720.50 of the Premanufacture Notification Rule. 

Additional Certification Statements: 

If you are submitting a PMN, Intermediate PMN, Consolidated PMN. or SNUN. check the following user fee certification statement that 

applies: 

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 40 CFR 700.45 (b), or 

o 	 The Company named in Part I, Section A is a small business concern under 40 CFR 700.43 and has remitted a fee of $100 

in accordance with 40 CFR 700.45 (b). 

If you are submitting a low volume exemption (LV E) application in accordance with 40 CFR 723.50 (c (I) or a Low release and 

low exposure exemption (LoREX) application in accordance with 40 CFR 723.50 (c (2), check the following certification 

statements: 

o 	 The manufacturer submitting this notice intends to manufacture or import the new chemical substance for commercial 

purposes, other than in small quantities solely for research and development, under the terms of 40 CFR 723.50. 

o 	 The manufacturer is familiar with the terms of this 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 	 If this application is for an LVE in accordance with 40 CFR 723.50 (cl( I), the manufacturer intends to commence 

manufacture of the exempted substance for commercial purposes within I year of the date of expiration of the 30 day 

review period. 

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


substance described herein. Any knowing lind willful misinterpretation is subject to criminal penalty pllnllantto 18 esc 100I. 
 Confidential 

Signature and Title of Authorized Official (Original Signature Required) Date 

Signature of agent - (if applicable) Date 

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

x 
Part I -- GENERAL INFORMATION 

Section A - SUBMITTER IDENTIFICATION 

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

la. Person Name of authorized ofticial Position 

Submitting 

Notice 

(in U.S.) Company 

Confidential 

X 

Mailing address (number and street) 

City, State, ZIP Code 

b. Agent (if 

applicable) 

Name of authorized official 

Company 

Position 

Mailing address (number and street) 

City, State, ZIP Code Telephone 

C. lfyou are submitting this notice as part of a joint submission, mark (X) this box. 

I 

I 

I 

I 

Area Code I 

I 

I 

I 

Number 

D 

Joint Name of authorized official 


Submitter (if 


applicable) 


Company 

Mailing address (number and street) 

City, State, ZIP Code 

2. 	 Technical Name 

Contact 

(in U.S.) 

Company 

Mailing address (number and street) 

City, State, ZIP Code 

3. 	 If you have had a prenotice communication (PC) concerning this notice and 

EPA assigned a PC Number to this notice, enter the number. 

4. 	 If you have submitted an exemption appl ication for the chemical substance 

covered by this notice, enter the exemption number assigned by EPA. If you 

previously submitted a PMN for this substance, enter the PMN number 

assigned by EPA (i.e. withdrawn or incomplete). 

5. 	 If you have submitted a notice of Bona fide intent to manufacture or import 

for the chemical substance covered by this notice, enter the 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) if none D 

Mark (X) if none D 

X 

Mark (X) ifnone D 

X 

2. 12I Irrp:rt OJIy 

D Binding Option 

Mark (X) 

3. D Both 

X 

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

You must provide a currently correct Chemical Abstracts (CAl name of the 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. 

If another 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 of that person in a continuation sheet. 

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

a. Class of substance - 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 or obtain the specified chemical identity information reported in this notice: 

(check one) 

Confidential 

x 

X 

d. 

Method I (CAS Inventory Expert Service - a copy of the Identification 

report obtained from the CAS Inventory Expert Service must be 

submitted as an attachment to this notice) 

Molecular formula and CAS Registry Number (if a 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 of the reaction or process. (3) Indicate the range of composition 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 if you attach a continuation sheet 

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

Contains TSCA Confidential Business Information 

Section B - CHEMICAL IDENTITY INFORMATION 

l.d 

I.e 

Page4(A) 

000005 
Part I - GENERAL INFORMATION - Continued 

Section B - CHEMICAL IDENTITY INFORMATION - Continued Not Applicable 

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

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

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

1000 absolute molecular weight of that composition. 

Describe the methods of measurement or the bases for your estimates: GPCD Other D (Specify)

i) lowest number average molecular weight: 

ii) maximum weight % below 500 molecular weight: 

D 

iii) maximum weight % below 1000 molecular weight: 

Mark (X) this box if you attach a continuation sheet See attachment, page 14. 

b. You must make separate confidentiality claims for monomer or other reactant 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 (if a number exists) of each monomer or other reactant used in the manufacture of the 

polymer. 

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

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

(4) - Mark (X) the identity column if you want a monomer or other reactant used at two weight percent or less to be listed as part of the polymer description on 

the TSCA Chemical Substance Inventory. 

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

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

purposes. 

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

Monomer or other reactant and CAS Registry Number Contid- Typical Identity Confid- Maximum Confidential Composition Mark (X) ential residual ential 

(I) (2) (3) (4) (5) (6) (7) 

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

c. Please idenOWhiCh method you used to develop or obtain the specitied chemical identity information reported in this notice: (check one) 

Method I (CAS Inventory Expert Service a copy of the 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 similar polymers. 

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

D 

D Mark (X) this box if you attach a continuation sheet. r>.r..",,...,,. 

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

Section B - CHEMICAL IDENTITY INFORMATION - Continued 

3. Impurities 

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

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

(b) - Estimate the maximum weight % of each impurity. If there 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 if you attach a continuation sheet. 

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

X 

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

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

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

6. 	 Generic chemical name - If you 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 guidance on developing generic names. 

Substituted aryl sulfonium polynuorophosphate salts 

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

7. 	 Byproducts - Describe any byproducts resulting from the manufacture, processing, use, or disposal of the new chemical substance at sites you controL Provide i 

the CAS Registry Number if available. 

Byproduct CAS Registry Number Confid­

( I) (2) ential 

X 

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

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

000007 

X 
Part I - GENERAL INFORMATION - Continued 

Section C - PRODUCTION, IMPORT AND USE INFORMATION 

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

I. Production volume - Estimate the maximum production volume during the first 12 months of production. Also estimate the maximum production volume for any 

consecutive 12-month period during the first three years of production. Estimates should be on 100% new chemical substance basis. For a Low Volume Exemption 

applicalion, ((you choose 10 have your nolice reviewed al a IOlrer produclion volume Ihan /a.nnn kg..yr. specify Ihe volume and mark (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 of the category of use, the percent of production volume devoted to each 

category. the formulation orthe new substance, and other use information. Mark (X) the "Confidential" box next to any item you claim as confidential. 

a. (I) Describe each intended category of use of the new chemical substance by function and application. 

(2) Mark (X) this column if entry 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 of total production for the first three years devoted to each category of use. 

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

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

sites under your control associated with each category of use. 

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

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

the information provided in (8) binding. 

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

Category of Use (I) CBI Binding Produc- CSI %in CBI % of substance expected per use 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; 

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

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

which this substance loses its identity in the consumer product. 

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

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

use Manual for examples of generic use descriptions. 

description Catalyst 

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

3. Hazard Information - Include in the notice a copy of reasonable facsimile of any hazard warning statement, label, material safety data sheet, or other Binding 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 of the new substance. List in part III hazard information you include. 

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

- FORM EPA 7710-25 (Rev. 5-9::» Page 7 000008 
Part II - HUMAN EXPOSURE AND 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 of manufacture, processing or use operation involving the new chemical substance at industrial sites you control. Importers do 

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

operations after import. You must describe these operations. 

I. Operation Description 

a. Identity - Enter the identity of the 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 

If the same operation will occur at more than one site. enter the number of sites. Identify the additional sites on a continuation I # ofsites I 

sheet, and ifany of the sites have significantly different production rates or operations. 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 D 	 Processing D 
 Use

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 or kglbatch on a 100% new chemical substance basis), and entry point of all starting materials 

and feedstocks (including reactants, solvents, and catalysts, ete.), and of all products. recycle streams, and wa,tes. Include cleaning ehemicals (note 

frequency if not used daily or per batch). 

(3) 	 Identify by number the points ofrelease, including small or intennittent releases, to the environment ofthe new chemical substance. 

D Mark (Xl this box if you attaeh a continuation sheet. 

FOR~ EPA 7710-25 (Rev. 5-95) 	 Page 8 

000009 
Part 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 of worker activity, physical form oflhe new chemical substance, number 

of workers exposed, and duration of activity. 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 if entry 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) of the new chemical substance (e.g. solid: crystal, granule. powder. or dust) and % new chemical substance (if part ofa mixture) 

at the time of exposure. 

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

(8)-- Estimate the maximum number of workers involved in each activity. 

(9)-- Mark (X) this column if entry 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) th is column if entries 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., bag dumping, 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 of the new chemical substance released 

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

(l )-- Enter the number of each release point identified in the process description, part II, section A subsection I d(3) 

(2)-- Estimate the 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 if entries 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 or incineration, POTW, or other (please 

specify) to which the new substance will be released from that release pom!. 

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

disposed ofon land. characterize the disposal method and state whether it is approved for disposal of RCRA hazardous waste. On a continuation sheet for 

each site describe any additional disposal methods that will be used and whether the waste is subJect to secondary or tertiary on-site treatment. b. Estimate the 

amount released to the environment after control technology (in kg/day). 

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

(7)-- Identify the destination(s) of releases to water. Please supply NPDES (!'Jational Pollutant Discharge Elimination System) #s for direct dischargers or NPDES 

numbers of the POTW (Publiciy Owned Treatment Works). Mark (X) if the POTW name or NPDES # is confidential business information (CBr). 

Release Amount of new 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 

desti nation( 5) of f-­

releases to water. Waterway 

D Mark (X) this box if you attach a continuation sheet. 00001.0 

FORM EPA 7710-25 (Rev. 5-95) Page 9 
Part II - 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 processing or use activities after import. See the Instructions Manual. Complete a 

separate section B/or each type o/processing or use operation involVing the new chemical substance. If the same operation is performed at more than one site, 

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

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 of all feedstocks (including reactants, solvents, and catalysts, etc.) 

and of all products, recycle streams, and wastes. Include cleaning chemicals (note frequency ifnot used daily or per batch). (3) Identify by number the points 

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

identify the locations of these sites on a continuation sheet): 

User Sites [ I 

# of sites 

See continuation page IO(A) 

[3 Mark (X) this box if you 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 number of workers exposed for all sites combined. 

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

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

workers. 

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

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

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

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

(specify) and control technology, if any, 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 if any orthe preceding entries are confidential business information (CBI), 

Letter of 

Activity 

(I) 

# of 

Workers 

Exposed 

(2) 

CBI 

(3) 

Duration of 

Exposure 

CBI 

(5) 

Protective EquipJ 

Engineering Controls! 

Physical Form and % 

new substance 

(6) 

%in 

Formulation 

( 7) 

CHI 

(8) 

Releasc 

Number 

(9) 

Amount of New 

Substance Released 

CBI 

(11 ) 

Control Technology 

(12) 

CBI 

(4a) (4b) (13)

(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 if you attach a continuation sheet. See continuation pages IO(A) 

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

0000:11 
Continuation Sheet 

Contains TSCA Confidential Business Information 

Part II - Human Exposure and Environmental Release 

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 or bracket the specific information that you claim as confidential. 

In this section you may provide infonnation not reported elsewhere in this fonn regarding your 

efforts to reduce or minimize potential risks associated with activities surrounding 

manufacturing, processing, use and disposal of the PMN substance. Please include new 

infonnation pertinent to pollution prevention, including source reduction, recycling activities 

and safer processes or products available due to the new chemical substance. Source reduction 

includes the reduction in the amount of toxicity of chemical wastes by technological 

modification, process and procedure modification, product refonnulation, raw materials 

substitution, and/or inventory control. Recycling refers to the reclamation of useful chemical 

components from wastes that would otherwise be treated or released as air emissions or water 

discharges, or land disposal. Descriptions of pollution prevention, source reduction and 

recycling should emphasize potential risk reduction subsequent to compliance with existing 

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

this infonnation to assess overall net reductions in toxicity of environmental releases and 

exposures, not the shifting of risks to other environmental media or non-environmental areas 

(e.g., occupational or consumer exposure). In addition, infonnation on the relative cost 

or perfonnance characteristics of the PMN substance to potential alternatives may be provided. 

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

substance. See 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 or the environment; (2) a reduction in the volume 

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

toxicity of human exposure and/or environmental release: (5) an increase in product performance. a decease in the cost of production and/or 

improved operation efficiency of the 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 tor an existing substance that poses a greater overall risk to human health or the 

environment. 

[ 

J 

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

FORM EPA 7710-25 (Rev. 5.95) Page 11 VUUU..l.u 
Continuation Sheet 


Contains TSCA Confidential Business Information 


Part III -- LIST OF ATTACHMENTS 

Attach continuation sheets for sections of the 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 of the form to which it relates. if 

appropriate. Number consecutively the pages ofthe attachments. In the column below. enter the inclusive page numbers of each 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 of the notice fonn a sanitized version of any 

attachment in which you claim infonnation as contidential 

Attachment name Attachment page I Conlinumbcrs(s) dential 

Physical and Chemical Properties Worksheet 13 X

Appendix 1 - HPLC 14 - 16 X

Appendix 2 - IES Report with attachments 17 - 23 X

Appendix 3 - Material Safety Data Sheet (MSDS) 24 - 27 X 

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

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

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 

ApPl!ndix 8 - BUl!hler Sl!usitization Tl!st OECD 89-106 X 

ApPl!ndix 9 . Salmonella Typhimurium and Escherichia Coli Reverse Mutation Assay· OECD 107-125 X I 

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

FORM EPA 7710-25 (Rev. 5·95) Page 12 

00001.4 
Continuation Sheet 


Contains TSCA Confidential Business Information 


PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET 


To assist EPA's review of physical and chemical properties data, please complete the following worksheet for data you provide and include it in the notice. Identify 

the property measured, the page of the notice on which the property appears, the value of the property, the units in which the property is measured (as necessary), and 

whether or not the property is claimed as confidential. The physical state of the neat substance should be provided. These measured properties should be for the neat 

(100% pure) chemical substance. Properties that are measured for mixtures or formulations should be so noted (% PMN substance in ~). You are not required to 

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

You should submit this worksheet as a supplement to your submission of test data. This worksheet is not a substitute for submission of test data. 

Property 

(a) 

Mark (X) 

if provided 

Page 

number 

(b) 

Value 

(c) 

Measured 

or 

Extimate 

(M orE) 

Physical state of neat substance 

Vapor pressure 

@ Temperature "C 

__(s) __(I) __(g) 

Torr 

Density I relative 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 

Octanoll water 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 

X 
Continuation 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 x 25 cm 

MeOH I Water 

UV255nm 

10 j.tI 

1.0 mllmin 

10 mg in 5 ml MeOH 

ca. 40 min 

00001.7 15 
Injection 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 16 

Continuation Sheet 

Contains TSCA ~siness Information 


SelM crl-z..eJ 


Appendix 2 

lES Report 

000019 

Page 11 
InventorY.. Exeert Service 


A division of the American 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 

0 

~1 1.. :j 

o _ 

U'I 

0 

0 

PPM 

i 000022 20 
[ Mass Spectrum ] 

Data Date 08-0ct-73 16:40 : FAb-81B9 .~ Sample: 

Note C\t : * 

Inlet: DIrect Ion Mode FAB+ 


Spec trum Type : Regu I ar (MF-Li near] 


RT : .2. 10 min Sc an. : 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 

300 

I 

400 500 

1 I 

600 700 

r 

800 

I 

900 

II 


IOOVl 1100 1200
 100 200 


m/z 
000024 22 

To. CAS Inventory Expert Service Pax: ++1-614-447-3747 

Jennifer Moore 

Da..: 2003111114 

.. CAS No. registration 1 

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

Best Regards, 

000025 23 
Continuation Sheet 

Contains TSCA C~iness Information 

Scvvt ltr2..<.J 

Appendix 3 


Material Safety Data Sheet 


Page 24 000026 
MATERIAL 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 causes eye irritation 

May cause allergic skin reaction 

4. First aid measures 

If InlHlled: 

Remove to fmsh air and summon medical help if respiratory irritation develops or if breathing becomes difficult. 

On skIn contact: 

Wipe away excess material with dry towel and then wash affected areas with plenty of water for several minutes. Get medical 

attention immediately if irritation occurs. 

On contact wlfh eyes: 

Immediately wash affected eyes for several minutes under running water with eyelids held open. Get medical attention 

immediately if irritation occurs. 

If swallowed: 

Wash out mouth with water and then drink plenty of water, and summon physician. Do not give anything by mouth In an 

unconscious or convulsing person. 

5. Fire fighting and measures 

Flash point : >140 °C (284 OF) (Closed Cup) 

Flammable limits : Not applicable 

Decomposition temp. : >100 °C. 

Extinguishing media : Carbon dioxide. foam, dry chemical. water spray. 

Fire fighting procedures-speciat : Use self-contained breathing apparatus. 

REVISED DATE: JANUARY 1.2004 1/3 ~.Q:;121 000027 
6. 

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 

contact with skin, eyes or clothing, 

Handling and storage 

Handling precautions: In accordance with good industrial practice, handle with care and avoid unnecessary personal contact. 


Avoid contact with eyes and prolonged or repeated skin contact. is for industrial use only. 


Shipping and storing precautions: Keep container tightly closed in cool condition INhen not in use and during transport 


Personal hygiene: Wash thoroughly after handling. 


Exposure controls and personal protection 

Protective equipment: 

Eye protection : Safety goggle is recommended. 

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 

Stability and reactivity 

Stability: Stable. 


Incompatibilities: Mineral acid. Avoid contact with excessive heat, open flame, sparks, or ignition source. Avoid exposure to 


sunlight, ultraviolet light or fluorescent light. 


Hazardous decomposition: Toxic fumes of carbon dioxide, carbon monoxide, sulfur oxides, and hydrogen fluoride gas. 


Toxicological information 

Acute effects 

May be harmful by inhalation, ingestion, or skin absorption. 

May cause eye and skin irritation. 

May be irritating to mucous membranes and upper respiratory tract. 

Ecological information 

Data not yet available. 

Disposal considerations 

Waste disposal method: 


Incinerate in a chemical incinerator equipped with an afterbumer and scrubber. Follow all federal, state and local regulations, 


REVISED DATE: JANUARY 1 , 2004 2/3 000028 WI-MS1~6
MATERIAL SAFETY DATA SHEET 


14. 	 Transport information 

DOT proper shipping name: 

Not regulated as a hazardous material by the U.S. dept of transportation (DOT) 49 CFR 172.101 HAZADOUS MATERIAL 

TABLE. 

DOT class: None. 


DOT number: None. 


HMIS Rating Health: 2 Flammability: 1 Rea ctivity : 1 Protective equipment: B 


NFPA Rating Health: 2 Flammability: 1 Reactivity : 1 Special: ­

Emergency TEL: 


15. 	 Regulatory information 

15.1 FEDERALI NATIONAL 


RCRA status: Not a hazardous waste under RCRA (40 CFR 261). 


CERCLA status: Not listed. 


SARAITITLE III - TOXIC CHEMICALS LIST: This product does not contain 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 believed to be correct However. no guaranty 

or warranty of any klnd expressed or implied is made with respect to the information contained here. 

REVISED DATE JANUARY 1.2004 	 3/3 000029 W'-MS1227 
Continuation Sheet 

Contains TSCA Co~usiness Information 


SaM\.1.'"l.-e.J 


Appendix 4 


Acute Dennal Irritation/Corrosion - OECD 


Page 28 000030 
TOXIKON FINAL REPORT: 

ACUTE DERMAL IRRITATION/CORROSION - OEeD 

Autbor 


Shirley Lister. B.S .• LATG 


Final Report Date 


March 02, 2004 


MANAGEMENT OF THE STUDY 

Performing Laboratory 

T oxikon Corporation 

15 Wiggins Avenue 

Bedford. MA 01730 

Sponsor 

000031 29 

Toxikon Corporation 

Acute Dermal Irritation/Corrosion - OBeD 

Project Number: 

TABLE OF CONTENTS 

Title Page 

Table of Contents 

Study Summary 

Quality Assurance Statement 

Study Director Signature and Verification Dates 

1.0 Purpose 

2.0 References 

3.0 Compliance 

4.0 Identification of Test and Control Substance 

5.0 Identification of Test System 

6.0 Justification of Test System and Route of Administration 

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 13 
Tomon Corporation 

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 of New 

Zealand White rabbits. The test substance is considered a negligible irritant. 

000033 31 

Toxikon 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 in 21 CFR. Part 58 and OECD GLPs. 1982. 

The sections of the regulations not performed by or under the direction of Toxikon 

Corporation, exempt from this Good laboratory Practice statement, include characterization 

and stability of the 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 32 

Tomon Cor, 'oration 

Acute Dermal Irritation/Corrosion - OECD 

Project Number: 

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, 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., LA TG 

Company: T ox ikon 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 13 
Toxikon Corporation 

Acute Dermal Irritation/Corrosion - OBCD 

Project Number: 

1.0 PURPOSE 

The purpose of the test is to evaluate the potential of the test substance to produce primary 

dermal irritation or corrosion after a single topical 4 hour application to the skin of albino 

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 of Chemicals, "Acute Dermal Irritation/Corrosion". Section 404, 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) (OLA W). "Public Health 

Service Policy on Humane Care and Use of Laboratory 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 (1-1-95 edition), 

Subchapter A-Animal Welfare. 

4.0 IDENTIFICATION OF THE TEST AND CONTROL ARTICLES 

The following information was supplied by 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 GLP regulations. Test and control substances (exclusive of extracts) 

that are mixed with carriers require verification of concentration. homogeneity, and stability. 

Samples of test and control substance mixtures shall be returned to the Sponsor for 

characterization. 

4.1 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/S 


Storage Conditions: Room Temperature 


Safety Precautions: Standard Toxikon Laboratory Safety Precautions 


000036 34 

Page 6 of 13 
Toman Corporation 

Acute Dermal Irritation/Corrosion - OECD 

Project Number: 

4.2 A control article was not utilized. Untreated skin of each animal served as it's own control. 

5.0 IDENTIFICATION OF THE TEST SYSTEM 

5.1 	 Animals Used in the Test: 

Number and species: 3 New Zealand White rabbits (Oryctolagus cuniculus) 

Sex: 1 male 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 of 5 days under the same conditions as for the actual test. 

Animal selection: selected from a larger pool of animals 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, ad libitum 

000037 

Page 7 of n 

35 
Tomon Corporation 

Acute Dermal Irritation/Corrosion - OECD 

Project Number: 

Water: tap water, ad libitum 

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 In vivo irritation testing was not undertaken until all available data relevant to the potential 

dennal corrosivity/irritation of the 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 of corrosivity/irritation of one or more structurally related substances or 

mixtures of such substances, strong acidity or alkalinity of the substance and results from 

validated and accepted in vitro or ex vivo tests. It was the Sponsor's responsibility that these 

data, if available be provided to Toxikon prior to testing. 

6.2 Rabbits was used in this study because they have historically been used in dennal safety 

evaluation studies. Dennal application corresponds to the likely route of human exposure. 

The species and number of animals used was recommended by the OECD guidelines. 

7.0 EXPERIMENTAL DESIGN 

7. 1 Pretreatment Screening Procedure and Selection of Animals 

Animals selected for the study were examined to ensure that their skin is free from irritation, 

trauma, and disease. 

7.2 Preparation of Test Animals 

The application sites were prepared by clipping the skin of the trunk free of hair approximately 

24 hours before application of the test substance. The site of application was not abraded 

intentionally or accidentally during preparation. Not less than 10% of the body surface was 

clear for application of the test substance. 

7.3 Test Substance Application 

7.3.1 The test substance was applied to a small area (approximately 6 cm2

) of skin. Each 

application area was covered with a gauze patch, which was held in place with non-irritating 

tape. The patch was loosely held in contact with the skin for the duration of the exposure 

period. Care was taken to prevent access by the animal to the patch. which may result in 

accidental ingestion/inhalation of the 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 

36 
Tomon Corporation 

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 of the 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 of 4 hours. 

7.4 After the 4 hour exposure period, animals were observed for signs of erythema and edema 

at 60 minutes, and then at 24, 48, and 72 hours after patch removal. Observations were scored 

according to the "Grading of Skin Reaction" (Appendix I). Daily clinical observations include 

all toxicologic and pharmacologic signs. 

7.5 No therapeutic agents were used in any phase of the study. 

7.6 Animals were weighed at the end of the observation period 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 of the 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 of Animal Data 

Erythema and edema scores (see Appendix I) were determined for each of 3 individual animals 

at 60 minutes, 24, 48, and 72 hours after patch removal (at a minimum). 

9.2 Evaluation of Test Results 

Dermal irritants are those test substances that produce reversible changes in the derma. Those 

test substances that destroy the structure of the intact skin or change it irreversibly are 

considered corrosive. The dermal irritation scores were evaluated in conjunction with the 

nature and reversibility or otherwise of the responses observed. 

10.0 RESULTS 

10.1 Body Weights (Table I) 


All of the test animals exhibited a gain in body weight during the study. 


37 000039 
Tomon Corporation 

Acute DermallrritationlCorrosion - OECD 

Project Number: 

10.2 Clinical Observations (Table I) 


No overt signs of toxicity were evident in any of the animals during the course of the study. 


10.3 Dermal Irritation (Table II) 


In both the initial test and the confirmatory test there were slight signs of erythema at one hour 


after the four hour exposure period. No signs of erythema or edema were present at the 24,48 


or the 72 hour observation points in any of the animals. None of the control sites of any 


animal at any of the observation periods showed signs of erythema or edema. 


11.0 CONCLUSION 


The test substance, , was evaluated for its potential to produce primary 


dermal irritation or corrosion after a single topical 4 hour application to the skin of New 


Zealand White rabbits. The test substance is considered a negligible irritant. 


12.0 RECORDS 


12.1 	 Original raw data is archived at Toxikon Corporation. 


12.2 	 A copy of the final report and any report amendments is archived at Toxikon 

Corporation. 

12.3 	 The original final report and a copy of any protocol amendments or deviations is 

forwarded to the Sponsor. 

12.4 	 All unused test substance shall be disposed of by 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 of confidentiality may be agreed upon prior to study initiation. 


14.0 POLICY ON PAIN AND SUFFERING IN ANIMALS 


No evidence of pain and suffering was observed in the animals. 


15.0 ANIMAL USAGE 


The Sponsor assures that to the best of their knowledge this study does not unnecessarily 

duplicate previous testing. 

000040 38 

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Toxikon Corporation 

Acute Dermal Irritation/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._UJ jJ\.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 

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• 

Toxik.on Corporation 

Acute Dermal Irritation/Corrosion- OECD 

Project Number: 

APPENDIX 1: Grading of Skin 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 

Slight edema (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 of exposure) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 FINAL REPORT: 

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 A venue 

Bedford, MA 01730 

Sponsor 

000045 43 
Toxikon Corporation 

Acute Eye Irritation/Corrosion - OECD 

Project Number: 

Title Page 

Table of Contents 

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 of the Test and Control Substances 

5.0 Identification of the Test System 

6.0 Justification of Test System and Route of Administration 

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 of 20 000046 44 
Toxikon 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 of irritation (conjunctival redness) were 

noted in the treated eyes of two of the animals at the 1 hour time point. All signs resolved in 

all animals by the 48 hour observation point. No signs of irritation were present in any of the 

control eyes. Based on the methods employed, the test substance is considered to be a slight 

irritant to the ocular tissue of New Zealand White rabbits. 

000047 45 

Page 3 of 20 
Toxikon Corporation 

Acute Eye IrritatioDlCorrosion - OECD 

Project Number: 

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 of the regulations not perfonned by or under the direction of Toxikon 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 of 20 46 
Toxikon Corporation 

Acute Eye Irritation/Corrosion - OECD 

Project Number: 

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: CHTNIVO/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 

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Project Number: 

1.0 PURPOSE 

The purpose of this test was to evaluate the potential of the test substance to produce an 

irritating and/or corrosive effect on the ocular tissue of albino 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 

of Chemicals, "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) (OLA W), "Public Health 

Service Policy on Humane Care and Use of Laboratory 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 (1/1195 edition), 

Subchapter A-Animal Welfare. 

4.0 IDENTIFICAnON OF THE TEST 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 will be responsible for all test substance characterization data as specified in the GLP 

regulations. Test and control substances (exclusive of extracts) that are mixed with carriers 

require verification. of concentration, homogeneity and stability. Samples of test 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 

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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 OF THE TEST SYSTEM 

5.1 	 Animals Used in the Test: 

Number and species: 3 New Zealand White rabbits (Oryctolagus cuniculus) 

Sex: 1 male 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 70f20 
Toxikon Corporation 

Acute Eye Irritation/Corrosion - OECD 

Project Number: 

Animal rations: TEK 8630 Rabbit Food, Harlan Teldad, Madison, WI 

Water: tap water, ad libitum 

Animal Selection: chosen from larger pool and examined for lack of adverse 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 system justification 

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 of human exposure. 

6.2 Definition of irritation and corrosion 

Eye irritation is the production of reversible changes in the eye following the application of a 

test substance to the anterior surface of the eye. Eye corrosion is the production of irreversible 

tissue damage in the eye following application of a test substance to the anterior surface of the 

eye. 

6.3 Testing and Evaluation Strategy for Eye IrritationlCorrosion 

The OECD guideline recommends weight-of-the-evidence analysis of all available relevant data 

as to the potential eye corrosivity/irritation of the test substance prior to vivo testing. A 

summary of the 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 of Animals 

Animals selected for the test were not subjected to any previous experimental procedures. 

Animals selected for the study were chosen from a larger pool of animals and were examined 

within the 24 hours before administration of the test substance to insure that the eyes were free 

of abnormality, damage, and disease. 

000052 50 

Page 8 of20 
Toxikon Corporation 

Acute Eye Irritation/Corrosion - OECD 

Both eyes of each 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 of Eye Test Parameters 


The left eye of each 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 24 hours, the eyes of all rabbits were 

further examined with the aid of fluorescein to further characterize corneal opacity (Appendix 

Ill). Reading of reactions was facilitated by the use of a slit lamp. The eyes were rinsed with 

20 rnL of 0.9% 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 of the study. 


7.9 Therapeutic Agents 


No therapeutic agents were used in any phase of the study. 


7.10 Study Termination 


At the end of the study, all animals were weighed and then: sacrificed by injectable barbituate. 


000053 51 

Page 9 of 20 
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Acute Eye Irritation/Corrosion - OECD 

Project Number: 

8.0 DOSAGE 

The test substance was dosed neat. Each animal received a dose of 0.1 mL of the test 

substance. The test substance was placed in the conjunctival sac of one eye of each 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 of the scores observed. 

A test substance was rated satisfactory if the ocular changes of the test are equivalent to or less 

than those for the untreated (control) eyes. 

The determination of the irritation and/or corrosive potential of the test substance was based on 

the interpretation of the results by the Study Director. 

10.0 RESULTS 


10.1 Animal Weights (Table I) 


All of the animals exhibited an increase in body weight during the course of the study. 


10.2 Clinical Observations (Table I) 


No overt signs of toxicity other than the ocular effects were evident during the course of the study 


in any of the animals. 


10.3 Eye Scores (Table II) 


Corneal Opacity: No corneal opacity was seen in any of the rabbits at any observation point. 


Iris: No iris involvement was seen in any of the rabbits at any observation point. 

Conjuctiva: Slight conjunctival redness was present at the 1 hour observation in two of the treated 

eyes. These findings resolved by the 48 hour observation point. 

10.4 Fluorescein Staining (Table II) 


No Fluorescein Staining was seen in any of the rabbits at any observation point. 


000054 

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11.0 CONCLUSION 

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 of irritation (conjunctival redness) were 

noted in the treated eyes of two of the animals at the 1 hour time point. All signs resolved in 

all animals by the 48 hour observation point. No signs of irritation were present in any of the 

control eyes. Based on the methods employed, the test substance is considered to be a slight 

irritant to the ocular tissue of New Zealand White rabbits. 

12.0 RECORDS 

12.1 Original raw data is archived at Toxikon Corporation. 

12.2 A copy of the final report and any amendments is archived at Toxikon Corporation. 

12.3 The original final report and a copy of any protocol amendments or deviations is 

forwarded to the Sponsor. 

12.4 All unused test article shall be returned. 

13.0 CONFIDENTIALITY AGREEMENT 


Statements of confidentiality were as agreed upon prior to study initiation. 


14.0 POUCY ON PAIN AND SUFFERING IN ANIMALS 


No evidence of pain or suffering was observed in the animals during the study. 


15.0 ANIMAL USAGE 


The Sponsor assures that to the best of their knowledge this study does not unnecessarily 

duplicate previous testing. 

000055 53 

Page 11 of 20 
Toxikon Corporation 

Acute Eye Irritation/Corrosion - OECD 

Project Number: 

TABLE I 

ANIMAL WEIGHTS 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 of cluucal observations on Day 0 through Day 3 excluding Eye Scores. 

000056 54 

Page 12 of20 
Toxikon 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 

OCULAR LESION 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 

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Project Number: 

Table II 

OCULAR LESION 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 

OCULAR LESION 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 

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Project Number: __ _ . 

Table IT 

OCULAR LESION SCORES AND FLUORESCEIN EXAMINATION (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 12 hr 

CORNEA 

OCULAR LESION 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 

FLUORESCEIN EXAM 

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 


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APPENDIX I 


TESTING AND EVALUA nON STRATEGY FOR EYE IRRITATION/CORROSION 


OECD/OCDE 405 Supplement Summary 


Step 1: Evaluation of existing 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 of animals, 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 of non-corrosivity and nonirritancy from previously performed ocular studies should also not be tested in in vivo eye 

studies. 

Step 2: Analysis of structure activity relationships (SAR) 

When available, the results of test of structurally related substances should be considered. 

Human and/or animal data on the potential eye irritationlcorrosivity of structurally related 

substances or mixtures of such substances should be evaluated; if these 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 of structurally 

related substances or mixtures of such substances do not constitute sufficient evidence to noncorrosivity /non-irritancy of a 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 of a validated and accepted in vitro or in 

vivo test (see Steps 5 and 6). 

Step 4: Consideration of other existing information 

All data relating to systemic toxicity via the dermal route should be evaluated. Acute dermal 

toxicity of the test substance should also be considered. Ifthe test substance has been 

demonstrated to be highly toxic by the dermal route of administration, it may not need to be 

tested in the eye. It can be presumed that if a test substance is highly toxic via the dermal 

route, it will also exhibit high toxicity when instilled into the eye. 

000060 58 

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Project Number: 

APPENDIX I (Cont.) 


TESTING AND EVALUAnON STRATEGY FOR EYE IRRITATION/CORROSION 


OECD/OCDE 40S Supplement Summary 


Steps 5 and 6: Results from in vitro and ex vivo tests 

Test substances that have demonstrated corrosive or severe irritant properties in an in vitro or 

ex vivo test that has been validated and is accepted for the assessment specifically of eye 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 or corrosivity of the substance 

When insufficient evidence exists with which to perform a conclusive weight-of-the-evidence 

analysis of the potential eye irritation/corrosivity of a substance based 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. If the 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. If the 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 of this 

test indicate the substance to be a severe irritant or corrosive to the eyes, further testing should 

not be performed. If that 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 of 20 
Toxikon Corporation 

Acute Eye Irritation/Corrosion - OECD 

Project Number: 

APPENDIX II 

GRADES FOR OCULAR LESIONS 

CORNEA* 

OPACITY -degree of density (areas most dense taken for reading) 

No ulceration or opacity o 

Scattered or diffuse areas of opacity 

(other than slight dulling of normal luster), 

details of iris clearly visible 1 

Easily discernible translucent area, 

details of iris slightly obscured 2 

Nacrous area, no details of iris visible, 

size of pupil 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 of these or combination of any thereof, 

iris reactive to light (a sluggish reaction is positive score) 1 

No reaction to light. hemorrhage, 

gross destruction (any or all of these) 2 

* The area of corneal opacity should be noted. 

000062 60 


Page 18 of20 
Tomon Corporation 

Acute Eye Irritation/Corrosion - OECD 

Project Number: 

APPENDIX II (Cont.) 

GRADES FOR OCULAR LESIONS 

CONJUNCTIV AE 

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 of lids 2 

Swelling with lids about half closed 3 

Swelling with lids more than half closed 4 

O(J0063 61 

Paee 19 of 20 
Tomon Corporation 

Acute Eye Irritation/Corrosion - OECD 

Project Number: 

APPENDIXll 

FLUORESCEIN STAINING GRADING SCALE 

A. INTENSITY OF STAINING 

Absence of fluorescein staining o 

Slight fluorescein staining confined to a small focus. With diffuse 

illumination the underlying structures are easily visible. The outline 

of the pupillary margin is as if there 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 of detail. 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 INVOL VEO 

No area of fluorescein o 

One-quarter of less. 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 of20 
Continuation 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 64 

Toxikon Corporation 

Acute Oral Toxicity Test: Up and Down Procedure - OECD 

Number: 

TABLE OF CONTENTS 


Title Page 

Table of Contents 

Study Summary 

Quality Assurance Statement 

Study Director Signature and Verification Dates 

1.0 Purpose 

2.0 References 

3.0 Compliance 

4.0 Identification of the Test Substance 

5.0 Identification of the Test System 

6.0 Justification of Test System and Route of Administration 

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 65 
Tomon Corporation 

AcUte Oral Toxicity 1 cst: 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 of greater than 2000 mg/kg body weight. 

000068 66 

Paae 3 of 11 
Toxikon Corporation 

Acute Oral Toxicity 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 of the regulations not performed by or under the direction of Toxikon 

Corporation, exempt from this Good laboratory Practice statement, include characterization 

and stability of the test substance and its mixture with carriers, 40 CFR. Part 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 Toxikon's management. 

INSPECTIONS DATE OF DATE REPORTED DATE REPORTED 

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 11 
Toxikon Corporation 

Acute Oral Toxicity Test: Up and Down Procedure - OECD 

Project Number: 

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, 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 11 
Toxikon Corporation 

Acute Oral Toxicit} Test: Up and Down hocedure - OECD 

Project Number: 

1.0 	 PURPOSE 

The test was designed to assess the acute oral toxicity of a test substance via oral administration. 

The up and down test procedure as described in OECD 425 was used. This procedure minimized 

the number of animals used, allowed the observation of signs of toxicity and provided 

information both for hazard assessment and for hazard classification purposes. 

2.0 	 REFERENCES 

The test was based on the following reference: 

2.1 	 Based upon the standards of the Organization for Economic Co-operation and 

Development (OECD), Guideline for Testing of Chemicals, Acute Oral Toxicity, Upand-Down Procedure, Section 425,2001. 

3.0 COMPLIANCE 

The study conformed 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; (Nlli) (OLAW), "Public Health Service 

Policy on Humane Care and Use of Laboratory 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 (1/1/95 edition), 

Subchapter A-Animal Welfare. 

4.0 	 IDENTIFICATION OF THE TEST SUBSTANCE 

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. 

4.1 Test Substance 


Test Substance Name: 


CAS/Code #: Not Supplied by Sponsor (N/S) 


LotlBatch #: 


Physical State: 


Color: 


Expiration Date: 12/23/2005 


Density: 


Stability: stable in dark and cool closed containers 


Solubility: 


pH: Not Applicable 


Storage Conditions: Room Temperature 


Safety Precautions: Standard Toxikon Laboratory Safety Precautions 


000071. 69 
Tomon Corporation 

Acute Oral Toxicity Test: Up and Down Procedure - OECD 

Project Number: 

5.0 IDENTIFICATION OF THE TEST SYSTEM 

5.1 	 Animals Used in the Test: 

Number and Species: 5 Outbred Albino Rats (Rattus norvegicus) 

Sex: Female; females were nulliparous and 

non-pregnant 

Weightl Age 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 % of the 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 

ad libitum 


Water: Tap water, ad libitum 


000072 70 

Page 7 of 11 
Tomon Corporation 

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 of Animals: Randomly selected from a larger pool and examined for a 

lack of adverse clinical signs 

6.0 	 JUSTIFICATION OF TEST 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 of animals used was recommended by the 0 ECD guidelines. Oral 

administration corresponded to a likely route of human exposure. The test was based on 

a up and down procedure with the use of a minimum number of animals for the entire 

study, yet enabling sufficient information to be obtained on the acute toxicity of the 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 of the test substance by intragastric intubation by means of a 

ball tip gavaging needle and a syringe. After dosing, the animals were returned to their 

cages and supplied with feed and water ad libitum. 

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 of a liquid that can be administered at one time 

depends on the size of the 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 of 5 


animals were tested. Reference is made to OECD 425 and the statistical computer 


program (AOT425StatPgm) will be used to determine the termination point of the 


study. 


7.4 	 Post Treatment Procedures 

Careful clinical observations were made at least twice on the day of dosing. Special 

attention was given during the first 4 hours. The frequency was determined by the 

71 Page 8 of 11 	 000073 
Toxikon Corporation 

Acute Oral Toxicity Test: Up and Down Procedure - OECD 

Project Number: 

response of the test animals to the treatment. Animals were observed daily for 14 days 

for clinical manifestations. 

Cageside observations included changes in the 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 of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma. 

The time of death 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 of the study and all gross pathological changes was recorded. 

7.6 	 No therapeutic agents were used in any phase of the 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 of the 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 1for actual dose and volume. 

9.0 	 EVALUATION CRITERIA 

An evaluation of acute toxicity data included the relationship, if any, between the 

animals exposed to the test substance and the incidence and severity of all 

abnormalities, including behavioral and clinical abnormalities, gross lesions, 

bodyweight changes, effects on mortality, and any other toxic effects. Calculation of 

the LD50 and the confidence intervals of the test substance were detennined by using 

the maximum likelihood method. A statistical computer program (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 of the study gained 

weight. 

72 Page 9 of 11 
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Acute Oral Toxicity Test: Up and Down Procedure - 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 of toxicity were observed 

in any of the animals for the duration of the study. 

lOA 	 Input of the 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 of greater than 2000 mg/kg body weight. 

12.0 RECORDS 

12.1 	 Original raw data will be archived at Toxikon Corporation. 

12.2 	 A copy of the Final Report and any amendments will be archived at Toxikon 

Corporation. 

12.3 	 All unused test substance shall be disposed of by Toxikon, per Sponsor's specifications. 

13.0 CONFIDENTIAUTY AGREEMENT 


Statements of confidentiality were as agreed upon prior to study initiation. 


14.0 POLICY ON PAIN AND SUFFERING IN ANIMALS 


There was no evidence of pain and suffering during the course of the study. 


15.0 	 ANIMAL USAGE 

The Sponsor assures that to the best of their knowledge this study did not unnecessarily 

dup licate previous testing. 

000075 Page 10 of 11 	 73 
3 

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 74 
Continuation 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 76 
Toxikon Corporation 

Acute Dermal Toxicity - OECD 

Project Number: 

TABLE OF CONTENTS 


Title Page 

Table of Contents 

Study Summary 

Quality Assurance Statement 

Study Director Signature and Verification Dates 

1.0 Purpose 

2.0 References 

3.0 Compliance 

4.0 Identification of Test and Control Articles 

5.0 Identification of Test System 

6.0 Justification of Test System and Route of Administration 

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 13 
Tomon Corporation 

Acute Dermal Toxicity - 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 3 of 13 
Toxikon 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 of the regulations not perfonned by or under the direction of Toxikon 

Corporation, exempt from this Good laboratory Practice statement, include characterization 

and stability of the 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 fmdings were 

reported to the Study Director and to Toxikon1

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 13 
Tomon Corporation 

Acute Dermal To~jty - OECD 

Project Number: 

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, 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: William Entwistle, 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 

Project Log Date: 01108/04 

Technical Initiation: 02/18/04 

Technical Completion: 03/03/04 

000082 

Pae:e 5 of 13 

80 
Toxikon Corporation 

Acute Dermal Toxicity· OEeD 

Project Number: 

1.0 PURPOSE 

The purpose of the test is to evaluate the potential of the test substance to produce systemic . 

toxicity or death after a single topical 24 hour application to the skin of albino 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 of Chemicals, Section 402, 2002. 

2.2 	 Draize, J.H., "Dermal Toxicity", Appraisal of the Safety of Chemicals in Food, 

Drugs and Cosmetics-Dermal Toxicity, pp. 49-52. Association of Food and 

Drug Officials of the 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) (OLA W), "Public Health 

Service Policy on Humane Care and Use of Laboratory 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 OF THE TEST AND CONTROL SUBSTANCES 

The following information was supplied by 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 GLP regulations. Test and control substances (exclusive of extracts) 

that are mixed with carriers require verification of concentration, homogeneity, and stability. 

Samples of test and control substance mixtures shall be returned to the Sponsor for 

characterization. 

4.1 Test Substances Name: 


CAS/Code #: Not Supplied by 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 81 
Toxikon Corporalicn 

Acute Dermal Toxicity - OECD 

Project Number: 

4.2 A control substance was not utilized. Untreated skin of each animal served as it's own 

control. 

5.0 IDENTIFICATION OF THE TEST 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: ear marker 

Health status: healthy, previously unused in other experimental procedures 

Animal purchase: Millbrook Breeding Labs, Amherst, MA 

Acclimatization: minimum of 5 days under the same conditions as for the actual test. 

Animal selection: selected from a larger pool of animals 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 13 
Toxikon Corporation 

Acute Dermal Toxicity - OECD 

Project Number: 

Animal rations: TEK 8630 Rabbit Diet. Harlan Teklad. Madison. WI. ad libitum 

Water: tap water. ad libitum 

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 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 of animals used is recommended by the OECD 

guidelines. Dermal application corresponds to the likely route of human exposure. 

6.2 	 Acute dermal toxicity is the adverse effects occurring within a short time of dermal 

application of a single dose of a substance or multiple doses given within 24 hours. 

The albino rabbit is preferred because of its size. skin permeability. and extensive 

database. 

7.0 EXPERIMENTAL DESIGN 

7.1 	 Pretreatment Screening Procedure and Selection of Animals 

Animals selected for the study were examined to ensure that their skin was free from 

irritation. trauma and disease. 

7.2 	 Preparation of Test Animals 

The application sites were prepared by clipping the skin of the trunk free of hair 

approximately 24 hours before application of the test substance. The site of application 

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 skin of the body surface (approximately 10%) of each of ten 

animals. Animals were immobilized and the patches were secured in place by 

wrapping the entire trunk of the animal with an impervious bandaging. The test sites 

were secured to prevent the animals from ingesting the test substance. 

7.4 	 Post -Treatment Procedures 

At the completion of the exposure period, 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 of erythema 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 13 
Toxikon Corporation 

Acute Dermal Toxicity - aBeD 

Project Number: 

7.5 	 Moribund and Dead Animals 

No animals died during the course of the study. 

7.6 	 No therapeutic agents were used in any phase of the study. 

7.7 	 Animals were weighed on Day 0, Day 7, and Day 14; at the end of the study animals 

were sacrificed by an injectable barbiturate. 

8.0 DOSAGE 

8.1 	 Test Substance Preparation 

A weighed amount of the test substance was first mixed with USP Water for Injection 

into a paste prior to dosing. The test substance was dosed directly onto the skin of the 

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 

If there is no toxicity at the 2000 mg/Kg dose 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 of systemic toxicity were evident during the course of the study 

(Table n. 

10.3 	 Skin Reactions 

No signs of erythema/edema or necrosis were noted at the application sites of any of 

the animals (Table II). 

000086 84 

Page 9 of 13 
Toxikon 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 of the 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 of the fmal report and any report amendments is archived at Toxikon 

Corporation. 

12.3 	 The original final report and a copy of any protocol amendments or deviations is 

forwarded to the Sponsor. 

12.4 	 All unused test article shall be disposed of by 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 of confidentiality may be agreed upon prior to study initiation. 


14.0 POLICY ON PAIN AND SUFFERING IN ANIMALS 


No evidence of pain and suffering was observed in the animals. 


15.0 ANIMAL USAGE 

The Sponsor assures that to the best of their knowledge this study does not unnecessarily 

duplicate previous testing. 

000087 85 

Page 10 of 13 
Toxikon Corporation 

Acute Dermal Toxicity - OECD 

Project Number: 

TABLE I 


Dosing, Animal Weights, Necropsy Data, and Clinical Observations 


Test Substance: 


Lot 1 Batch #: 


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 of Clinical Observations, excluding skin scores: Day 0 through Day 14 

C 

0 

0 

00 

00 

0) Page 11 of 13 ... 

00 
Tomon Corporation 

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 13 
Tomon Corporation 

Acute Dermal Toxicity (Single Exposure) -OPPTS 

Number: 

APPENDIX I: 


DRAIZE SCALE FOR SCORING 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 of well defmed by defmite raising) 

Severe edema (raised more than 1 mm and extending beyond 


...................... 2 


Moderate edema (raised approximately 1 mm) .................................. 3 


area of exposure)...........................................................................4 


Total possible edema score = 4 


000090 Page 13 of 13 

88 
Continuation Sheet 

Contains TSCA Confi~siness Information 

~%J",~ee' 

Appendix 8 


Buehler Sensitization Test 


Page 89 000091 
TOXIKON FINAL REPORT: 

BUEHLER SENSITIZATION TEST • OEeD 

Author 


Laurence Lister, B.S. LAT 


Fjnal Report Date 


March 05. 2004 


MANAGEMENI OF THE SIUDY 

ferforming Laboratory 

Toxikon Corporation 

15 Wiggins A venue 

Bedford. MA 01730 

Sponsor 

000092 90 

Toxikon Corporation 

Buehler Sensitization Test - OECD 

Project Number: 

TABLE OF CONTENTS 

Title Page 

Table of Contents 

StudySununary 

Quality Assurance Statement 

Study Director Signature and Verification Dates 

1.0 Purpose 

2.0 References 

3.0 Compliance 

4.0 Identification of Test and Control Substances 

5.0 Identification of Test System 

6.0 Justification of Test System and Route of Administration 

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 Skin Reaction 

Scores 

Table II: Induction and Challenge: Animal Weights and Clinical Observations 

Table ill: Induction Phase: Skin Examination 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 17 
Tomon Corporation 

Buehler Sensitization Test - 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 of the 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 17 
Toxikon Corporation 

Buehler Sensitization Test - OECD 

Project Number: 

QUALITY ASSURANCE STATEMENT 

This study was conducted in compliance with OECD Good Laboratory Practices, 1997. 

The section of the regulation not performed by or under the direction of Toxikon Corporation, 

exempt from this Good laboratory Practice statement, include characterization of the test and 

reference substances, OECD, Section 6.2. 

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 INSPECTIONS INSPECTION 

CLINICAL 

OBSERV ATIONS 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 93 

Toxikon Corporation 

Buehler Sensitization Test - OECD 

Project Number: 

STUDY DIRECTOR SIGNATURE AND VERIFICATION DATES 

This stUdy meets the technical requirements of the protocol. The study also meets with the 

requirements of the 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 94 
Toxikon Corporation 

Buehler Sensitization Test - OBCD 

Project Number: 

1.0 PURPOSE 

The purpose of the test was to evaluate the potential of a 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 of the Organization for Economic Co-operation and Development 

(OECD), Guidelines for Testing of Chemicals, 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 of contact 

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) (OLA W), "Public Health 

Service Policy on Humane Care and Use of Laboratory 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 OF TEST 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 of test 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 

000097 
Tomon Corporatioll 

Buehler Sensitization Test - OECD 

Project Number: 

Density (liquids): 

Stability: stable in dark and cool closed containers 

Solubility: 

pH: N/S 

Storage Conditions: Room Temperature 

Safety Precautions: Standard Toxikon 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 OF THE TEST SYSTEM 

5. 1 Animals Used in the Test: 

Number and species: 38 Hartley guinea pigs (Cavia porcellus) 

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 1'7 
Tomon Corporation 

Buehler Sensitization Test - 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, Harlan Teklad, Madison, WI 

Water: tap water, ad libitum 

Animal Selection: chosen from larger pool and examined for lack of adverse 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 of animals was recommended in the reference described in Section 2. 

6.2 The test substance was applied topically because it is a likely route of human exposure. 

7.0 EXPERIMENTAL DESIGN 

7.1 Pretreatment Screening Procedure and Selection of Animals 


Animals selected for the study were examined to ensure their skin was free from irritation, 


trauma, and disease. Animals were weighed prior to initiation of the procedure. 


7.2 Animal Group Assignment 


Test animals were distributed into the following groups: 


(1) Experimental - 10 malesll 0 females 


(2) Negative Controls - 5 males/5 females 


(3) Positive Controls - 3 males/2 females 


(4) Preliminary Irritation - 1 male/2 females 


7.3 Test Animal Preparation 


Before each application of the test or control substance the animals were clipped and shaved or 


depilitated on the scapula region, in an area of about 3 x 4 cm. 


000099 97 
Toxikon Corporation 

Buehler Sensitization Test - 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 of the 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 of approximately 

4 - 6 cm2

• The concentration of test substance was the highest to cause mild irritation. 

7.5.2 The patch was kept in place with occlusive bandaging. 

7.5.3 The patch was removed after 6 hours of exposure and any residual test substance was 

washed off with water. 

7.5.4 The test substance was applied once per week for 3 consecutive weeks (Days 0, 7 and 

14) on one side of the animal. The positive control substance (dinitrochlorobenzene, DNCB) 

was applied in the same manner. Naive animals, i.e., untreated during the induction phase, 

serve as a negative control group. 

7.6 Challenge (Day 28) 

7.6.1 On the day of the challenge, a 4 x 3 cm virgin skin site was shaved on the flanks of the 

experimental and control animals. 

7.6.2 The challenge test was performed on freshly clipped skin sites of test and naive control 

animals in the same way as the 6 hour closed patch test of the induction phase. The skin was 

exposed to the test substance (see Section 8.2) for 6 hours. One virgin site was prepared per 

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 of the challenge dose, the area of the 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 of Challenge Patch Test Reactions 

(Appendix I). 

0001.00 98 
Tomon Corporation 

Buehler Sensitization Test - OECD 

Project Number: 

7.6.4 Reading of the skin area is was repeated at approximately 48 hours after removal of the 

challenge dose and the skin reactions are were graded. 

7.6~5 The results are surrunarized and expressed in the following terms: 


Incidence - The number of animals showing a response of 1 or more at 24 or 48 hours divided 


by the number of test animals. 


Severity - The sum of the test grades divided by the number of animals tested. 

7.7 Rechallenge 

7.7. I If considered necessary, the sensitized guinea pigs can be rechallenged 1 week after the 

primary challenge (Day 35). 

7.7.2 If the results of primary challenge and rechallenge were unclear and allergenicity of the 

test substance was nevertheless suspected, the test may have been done again at higher 

sensitizing and challenge concentrations of the 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 of the study. 

7. 10 At the end of the 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 can be 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. If the test substance was non-irritating, it was applied full strength at both 

induction and challenge. If a solid, it was moistened with a vehicle; if the test substance 

applied in this manner produces a marked irritation, it must be diluted with physiologic saline, 

water, alcohol, or other suitable material. 

0001.01. 'Pl'IO'P 10 nf 17 

99 
Toman Corporation 

Buehler Sensitization Test - OECD 

Project Number: 

During induction. the positive control substance (0.4 mL of 0.1 % DNCB in acetone) was 

applied in the same manner. For the challenge. the experimental and negative control animals 

were exposed to one dose of test 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 of Data 


The test substance was graded according to the incidence and severity of observed 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. 


10.2 Body Weights (Tables I and II) 


All animals gained weight during the course of the study. 


10.3 Clinical Observations (Tables I and II) 


No overt signs of toxicity were evident in any of the animals during the course of the study. 


10.4 Challenge Phase (Table III) 


No signs of erythema or edema were observed in any of the test animals. No signs of erythema 


or edema were evident in the negative control group. Both animals in the positive control 


group exhibited signs of erythema and/or edema. 


The incidence and severity of the 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 guinea pigs. The test substance 

is not considered to be a skin sensitizer since none of the 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 1'7 
Toxikon Corporation 

Buehler Sensitization Test - OECD 

Project Number: 

control group. 

12.0 RECORDS 

12.1 Original raw data is archived at Toxikon Corporation. 

12.2 A copy of the final report and any report amendments is archived at Toxikon 

Corporation. 

12.3 The original final report and a copy of any protocol amendments or deviations is 

forwarded to the Sponsor. 

12.4 All unused test substance shall be returned to the Sponsor. 

13.0 CONFIDENTIALITY AGREEMENT 


Statements of confidentiality was agreed upon prior to study initiation. 


14.0 POllCY ON PAIN AND SUFFERING IN ANIMALS 


No evidence of pain or suffering was observed during the course of the study. 


15.0 ANIMAL USAGE 


The Sponsor assures that to the best of their knowledge this study did not unnecessarily 

duplicate previous testing. 

0001.03 101 


1...... f"""" 
Toxikon Corporation 

Buehler Sensitization Test - 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 of clinical observation Day 0 through Day 2, excluding skin reactions. 

Skin Reaction Scores· 

Animal # Sex ohour 

01119/04 

24 hour 

01120/04 

36 Male 0 0 

37 Female 0 0 

38 Female 0 0 

• Sum of Erythema I Edema observations for all concentrations at 0, 24 and 48 hours after test substance 

application. 

0001.04 102 

Page 13 of 17 
Tox.ikon Corporation 

Buehler Sensitization Test - OECD 

Protocol Number: 

TABLE II 


Animal Weigbts and Clinical Observations 


Test Substance: 

Lot #: 

Group Animal # Sex 

Body Weights (gm) 

Day 0 Day 30 

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 - Day 0 through Day 30 

000:105 

Pa2:e 14 of 17 

103 
Tomon Corporation 

Buehler Sensitization Test • OBCD 

Project Number: 

TABLEID 

Challenge Phase - Skin Examination Data 

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 

D" ....o 1" "f 1 '7 000106

104 
Toxikon 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 

105 
Tomon Corporation 

'Buehler Sensitization Test - OECD 

Protocol Number: ___ .__ _ 

APPENDIX I: 


Magnusson and Kligman Grading Scale for the Evaluation 


of Challenge Patch Test Reactions 

Reaction 

No visible change 

Discrete or patchy erythema 

Moderate and confluent erythema 

Intense erythema and swelling 

o 

1 

2 

3 

000108 106 
Continuation Sheet 

Contains TSCA Co~siness Information 

~"fI1...-~~ 

Appendix 9 

Salmonella Typhimurium 

and 

Escherichia Coli 

Reverse Mutation Assay 

Page [07 0001.09
TOXIKON FINAL REPORT: 

SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI REVERSE 

MUTATION ASSAY -OECD 

Author 


Paul Lezberg. B.A. 


Final Rmort Date 


April 6, 2004 


MANAGEMENT OF THE STUDY 


Perfonning Laboratory 


Toxikon Corporation 


15 Wiggins Avenue 


Bedford. MA 01730 


Sponsor 


0001.1.0 108 

Tonkon Corporation 

Salmonella typhimllrillm and Escherichia coli Reverse Mutation Assay - OECD 

Project Number: 

TABLE OF CONTENTS 

Title Page 

Table of Contents 

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 of the Test System 

6.0 Justification of Test System and Route of Administration 

7.0 Experimental Design 

8.0 Dosage 

9.0 Evaluation Criteria 

10.0 Results 

Table I: Range Finding Assay without Microsomal Activation 

Table II: Reverse Mutation Assay without Microsomal Activation 

Table III: Reverse Mutation Assay with Microsomal Activation 

Table IV: Reverse Mutation Assay without Microsomal Activation - Confirmatory Assay 

Table V: Reverse Mutation Assay with Microsomal Activation - Confirmatory Assay 

11.0 Conclusion 

12.0 Records 

13.0 Confidentiality Agreement 

Page 2 of 18 000:1:11. 109 
Toxikon Corporation 

Salmonella typhimurium and Escherichia coli Reverse Mutation Assay - OECD 

Project Number: 

STUDY SUMMARY 

The Salmonella typhimurium and Escherichia coli Reverse Mutation Assay (Ames Assay) 

evaluated the potential of the 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 of Escherichia 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 of revertant colonies as compared to the corresponding negative control, validating the 

functioning of the assay. A statistically significant increase in the number of revertant colonies 

was not observed with the test substance. Under the test conditions, the test substance is not 

mutagenic in the test species 

Page 3 of 18 0001.1.2 110 
Toxikon Corporation 

Salmonella typhlmurium and Escherichia coli Reverse Mutation Assay - 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. 

The sections ofthe regulations not performed by or under the direction ofToxikon Corporation, 

exempt from this Good Laboratory Practice Statement, included characterization and stability of 

the test article and its mixture with carriers, 21 CFR, Parts 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. 

INSPECTIONS DATE OF DATE REPORTED DATE REPORTED 

INSPECTION STUDY DIRECTOR MANAGEMENT 

SCORlNG 03/01104 03/01104 03/01104 

RAW DATA 04/06/04 04106104 04/06/04 

FINAL REPORT 04/06/04 04/06/04 04/06/04 

,~~uui Jayaram~ B.S.. 

.ty Assurance 

0001.1.3 111 

Pae:e 4 of 18 
Toxikon Cttrporation 

Salmonella typhimurium and Escherichia coli Reverse Mutation Assay - OECD 

Project Number: 

STUDY DIRECTOR SIGNATURE AND VERIFICATION DATES 

This study meets with the technical requirements ofthe protocol. The study also meets the 

requirements ofthe Good Laboratory Practice Regulations, 21 CFR, Part 58, with the 

exemptions as stated in the Quality Assurance Statement. 

Protocol Number: CHTNITROIOO 1-03/000 

Study Director: Paul Lezberg, B.A. 

Company: Toxikon Corporation 

Signature: 

Date: 

Study Supervisor: Leigh Bergeron, B.A. 

VERIFICATION DATES: 

The Toxikon Protocol Effective Date is the GLP Study Initiation Date. The study dates were as 

follows: 

Protocol Effective Date: 12/24/03 

Test Substance Receipt: 12/24/03 

Project Log Date: 01108104 

Range Finding Assay: 01/27/04 - 01130104 

Reverse Mutation Assay Technical Initiation: 02/27/04 

Reverse Mutation Assay Technical Completion: 03/01104 

Confmnatory Assay Technical Initiation: 02/27/04 

Confinnatory Assay Technical Completion: 03/01104 

0001.1.4 PaQ'e 5 of 1 R 

112 
Toxikon Corporation 

Salmonella typhimurium and Escherichia coli Reverse Mutation Assay - OECD 

Project Number: 

1.0 PURPOSE 

The Salmonella typhimurium and Escherichia coli Reverse Mutation Assay (Ames Assay) 


evaluated the potential of a test substance to induce reverse mutations in histidine (his - to his ) 


and tryptophan (tryp -to tryp +) genes in these organisms, respectively. This direct plate 


incorporation assay is conducted with four strains ofSalmonella typhimurium (s. typhimurium) .... 


and one strain ofEscherichia coli (E. coli) in the presence and absence of an exogenous 


mammalian activation system. A confirmatory assay was performed in order to verify the results 


ofthe Reverse Mutation Assay. 


2.0 REFERENCES 

The study was conducted based upon the following references: 

2.1 	 Organization for Economic Co-operation and Development (OECD), Guidelines for 

Testing ofChemicals, "Bacterial Reverse Mutation Test", Section 471, adopted: 21 July 

1997. 

2.2 	 Maron, D.M., Ames B.N., Revised Methods for the Salmonella Mutagenicity Test, 

Mutation Res., 113: 173-215, 1983. 

3.0 COMPLIANCE 

The present study conformed to all applicable laws and regulations. Specific regulatory 

requirements included the current Good Laboratory Practice for Nonclinical Laboratory Studies, 

FDA, 21 CPR., Part 58. 

4.0 IDENTIFICATION OF THE TEST AND CONTROL ARTICLES 

The following information was supplied by the Sponsor wherever applicable; it did not apply to 

confidential information. The Sponsor was responsible for all test substance characterization 

data as specified in the GLP regulations. Test and control articles (exclusive of extracts) that are 

mixed with carriers require verification of concentration, homogeneity and stability. Samples of 

test and control article extracts were not returned to the Sponsor for characterization and 

verification. 

4.1.1 Test Substance Name: 


CASICode #: Not Supplied by Sponsor (N/S) 


LotlBatch #: 


Physical State: 


Color: 


Expiration Date: 12/23/2005 


Density: 1.30 


Stability: stable in dark and cool closed containers 


Solubility: :- - .. 


pH: N/S 

Storage Conditions: Room Temperature 

Safety Precautions: Standard Toxikon Laboratory Safety Precautions 

000115 113 

Page 6 of 18 
Toxikon Corporation 

Salmonella typhimurium and Escherichia coli Reverse Mutation Assay - OECD 

Project Number: 

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 

Safety Precautions: Standard Laboratory Safety Precautions 

4.2.2 Positive Control Articles: 

4.2.2.1 Control Article Name: 2-Aminoanthracene 

Toxikon QC #: CSC-01-01-002-MB 

Physical State: Powder 

Color: Fluorescent GreenlY ellow 

Storage: Desiccated,4±2°C 

Safety Precautions: Known Mutagen. Appropriate Laboratory Safety Precautions 

4.2.2.2 Control Article Name: Sodium Azide (100 ~glmL) 

Toxikon QC #: AMES-04-01-004-MB 

Physical State: Liquid 

Color: Colorless 

Storage: -20 ± 2°C 

Safety Precautions: Known Mutagen. Appropriate Laboratory Safety Precautions 

4.2.2.3 Control Article Name: Sodium Azide (5 ~glmL) 

Toxikon QC #: AMES-04-01-005-MB 

Physical State: Liquid 

Color: Colorless 

Storage: -20 ± 2°C 

Safety Precautions: Known Mutagen. Appropriate Laboratory Safety Precautions 

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 

Safety Precautions: Known Mutagen. Appropriate Laboratory Safety Precautions 

0001.1.6 114 

Page 7 of 18 
ToxikOlI Corporation 

Salmonella typhimurium and Escherichia coli Reverse Mutation Assay - OECD 

Project Number: 

4.2.2.5 Control Article Name: 9-Aminoacridine 

Toxikon QC #: AMES-03-11-04-MB 

Physical State: Liquid 

Color: GreenlY eHow 

Storage: -20 ±2°C 

Safety Precautions: Known Mutagen. Appropriate Laboratory Safety Precautions 

4.2.2.6 Control Article Name: l-Ethyl-3-Nitro-I-Nitrosoguanidine (ENNG) 

Toxikon QC #: CSC-OI-09-0l5-MB 

Physical State: Powder 

Color: Beige 

Storage: -20 ±2°C 

Safety Precautions: Known Mutagen. Appropriate Laboratory Safety Precautions 

5.0 IDENTIFICATION OF THE TEST SYSTEM 

5.1 The Salmonella typhimurium strains used in this assay were TA98, TAlOO, TA1535, and 

TA1s37. These strains and the Escherichia coli strain WP2 used in this assay were purchased 

from Moltox, Inc. (Boone, NC). 

6.0 JUSTIFICATION OF TEST SYSTEM AND ROUTE OF ADMINISTRATION 

6.1 Historically, the Reverse Mutation Assay in S. typhimurium has been used to detect reverse 

mutations in a histidine gene evaluated as histidine-independent growth. This test system is 

recommended in the references listed in Section 2.0. 

The Reverse Mutation Assay in E. coli has been used to detect reverse mutations in a tryptophan 

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 of the extract. 


7.0 EXPERIMENTAL DESIGN 


7.1 Method: 


The bacteria were exposed to the test substance with and without a metabolic activation system, 


and directly plated on a minimal medium. After a period of incubation, revertant colonies were 


counted and compared to the number of spontaneous revertants in the negative control group. 


0001.1.7 115 

Page 8 of18 
Toxikon Corporation 

Salmonella typhimurium and Escherichia coli Reverse Mutation As~ay - OECD 

Project Number: 

7.2 Tester Strains: 


The requirement of histidine 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 by Toxikon's historical 


mean values. 


7.3 Stock Cultures: 


Working stock cultures were grown fresh for the assay. Frozen stock for each strain was thawed 


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 nutrient broth blank, were used in the 


assay. 


7.4 Negative Control 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 as the negative control article and 


provided reference for background lawns and revertant colony fonnation. 


7.5 Positive Control Articles: 


The positive control article for the metabolically activated portion of the assay was 


2-aminoanthracene in all 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 requiring metabolic activation are strain specific. 

S.typhimurium TA98 2-Nitrotluorene (10 J.1g1mL) 

S.typhimurium TA100 Sodiwn Azide (100 J.1g1mL) 

S. typhimurium TA1535 Sodiwn Azide (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 positive control articles were applied at 100 J.1Uplate. 

0001.1.8 116 

Paile 9 of18 
Toxikon Corroration 

Salmonella typhimurium and Escherichia coli Reverse Mutation Assay - OECD 

Project Number: 

7.6 Replication: 


All controls and test groups were plated in triplicate. 


7.7 Non-activated Assay: 


Top agar, supplemented with 0.5 mM histidine - 0.5 mM biotin per 1.0 mL of agar, was used as 


an overlay. Tryptophan was used in place ofhistidine for WP2. The agar was maintained at 


42-48°C until used. The overlay consisted ofsterile tubes containing: 


2 mL ofmolten top agar 

0.1 mL ofthe appropriate tester strain 

0.1 mL ofthe appropriate concentration ofthe test substance or control article 

0.5 mL of 0.2 M phosphate buffer pH=7.4 

The tubes were vortexed and poured onto minimal glucose agar plates. All plates in the range 

finding assay were incubated at 37 ± 2°C for 64.5 hours. All plates in the Reverse Mutation 

Assay and Confinnatory Assay were incubated at 37 ± 2°C for 65.5 hours. Plates were checked 

for unifonn background lawns and the number ofrevertant colonies counted. 

7.8 Metabolic Activation Assay: 


Tubes requiring metabolic activation contained an S9 fraction ofrat liver homogenate obtained 


from Aroclor® 1254 treated Sprague-Dawley rats. The S9 activation system, prepared fresh on 


the day ofthe assay and kept refrigerated or on ice, contained the following per 10 mL: 


0.4 M MgCh/1.65M KCI 

1 M glucose-6-phosphate 

0.1 MNADP 

0.2 M phosphate buffer pH=7.4 

USP Water for Injection 

S9 Fraction 

0.20 mL 

0.05 mL 

0.40 mL 

5.00 mL 

3.35 mL 

1.00mL 

The method ofpreparation for the top agar was the same as in the non-activated assay, with the 

exception ofsubstituting the 0.5 mL complete S9 mix for the phosphate buffer. 

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 assay was conducted only with TA100. The test 

substance was assayed at six dilutions in Dimethyl Sulfoxide (DMSO). The concentrations 

tested were 5000, 500, 50, 5, 0.5 and 0.05 ).1g/plate. 

0001.1.9 117 

Pae:e 10 of 18 
Toxikon Corporation 

Salmonella typhimurium and Escherichia coli Revers-: 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 of DMSO containing the appropriate concentration of test substance was applied per 


plate. 


8.3 Confirmatory Assay: 


The results of the Reverse Mutation Assay were confirmed through a Confirmatory Assay. The 


Confirmatory Assay was performed as described in Sections 7.0, 8.0 and 9.0. 


9.0 EVALUATION CRITERIA 

9.1 Evaluation Criteria ofthe Range Finding Assay: 


The Range Finding assay is performed with strain TAl00 without microsomal activation. If 


toxicity is detected, dose levels would be chosen to bracket toxic and non~toxic levels. 


9.2 Evaluation Criteria ofthe Reverse Mutation Assay: 


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 of variance) and Newman~Keuls Test for confirmation of pairwise comparisons. This 


statistical method is used to determine if there is a significant (p ~0.05) increase in the mutation 


frequency caused by the test substance compared to the negative control article. 


9.3 Positive Response: 


The positive control article assay consists of direct-acting mutagens and mutagens requiring 


metabolic biotransformation. All positive controls must exhibit a statistically significant increase 


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. The negative control article 


plates, for each strain, give a reference point to compare the test data. 


The test substance is considered to have caused a positive response if the 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 a response that is twice that ofthe negative control article is considered to be a 

mutagenic response. 

9.4 Confirmatory Assay: 


The results ofthe Confirmatory Assay should verify the outcome ofthe Reverse Mutation Assay. 


10.0 RESULTS 

10.1 Range Finding Assay (Table I) 


The Range Finding Assay did not show any cytotoxicity or significantly increased mutants with 


any concentration ofthe test substance as compared to the negative control. 


000120 118 

PaQe 11 of18 
Toxikon Corporation 

Salmonella typhimurium and Escherichis coli Reverse Mutation Assay - OECD 

Project Number: 

10.2 Reverse Mutation Assay (Tables II and ill) 


The mean number of revertants per plate was calculated for the test substance and the negative 


and positive control articles. 


All positive controls exhibited a statistically significant increase iil the number of mutants as 

compared to the corresponding negative control, demonstrating that the test system was 

functional with known mutagens. 

A statistically significant increase in the number of colonies was not observed with the test 

substance in comparison to the negative control group at any tested concentrations. 

10.3 Confirmatory Assay (Tables IV and V) 


The results of the Reverse Mutation Assay were confinned through a Confinnatory Assay. 


Results of the Confinnatory Assay are presented in Tables IV and V. 


10.4 The results are summarized in Tables I-V. 


000121 

P::lP~ l' of 1 R 

119 
Toxikon Corporation 

Salmonella typhimurium and Escherichia coli Reverse Mutation Assay - OECD 

Project Number: 

TABLE I 

Range Finding Assay 

Without Microsomal Activation 

Test Article: 

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 

'" All plates were dosed at 100 J.1Uplate 

"'''' The positive control used was sodium azide for strain TAl00 

"''''''' The negative control used in the assay was DMSO 

Page 13 of 18 

H00122 120 

To:dkon Corporation 

Salmonella typhilnurium and Escherichia coli Reverse Mutation Assay - OECD 

Project Number: 

TABLE II 


Reverse Mutation AllaY 


Without ~lIerOiomal Activation 


Test Artlde: 

LotiBatth #: 

RcvCl1antsIPlatc • 

STRAIN 

CONTROLS TEST SUBSTANCE 

POSmVE 

CONTROL·· 

NEGATIVE 

CONTROL··· 

DOSE LEVEL (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 100 j1Uplatc 

•• The positive control used was sodium azide for strains TAIOO and TA1535. 

2-nitrofluorene for strain TA98. 9-aminoacridine for strain TAl 537. and ENNG for snin WP2 

••• The negative control used in the assay was OMSO 

Pa~14oflS 

U00:123 121 
Toxikon Co,!,uration 

Salmonella typhlmurlum and Escherichia ((,II Reverse Mutation Assay - OJ<:CD 

Project Number: 

TABLE III 


Reverse Mutation Assay 


WitII Microsomal Activation 


Test Article: 

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 plates were dosed at 100 J.lUplate 

•• The positive control used was 2·aminoanlhl'acene for all strains 

••• The negative control used in the assay was OMSO 

Page IS ofl8 

JvUl.:G4 122 
Toxikon Corporation 

Salmonella typhimurlum and Lscheriehla coil Reverse Mutation Assay. OECD 

Project Number: 

TABLE IV 


Reverse Mutation Assay 


Wltbout Microsomal Activation 


Connrmatory Assay 


Test Article: 

LotlBatch #: 

Revel1alltsIPlale' 

STRAIN 

CONTROLS TEST SUBSTANCE 

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 

TAl 00 

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 


,. The positive control used was sodium azide for strains TA100 and TA1S3S. 


2-nitrofluorene for strain TA98. 9·aminoacridine for strain TAlS37, and ENNG for slnin WP2 

••• The negative control used in the assay was OMSO 

Page 160fl8 

0001.25 123 
Toxikun Corporation 

Salmonella typhlmurium and Escherichia coli Reverse Mutation Assay - OECD 

Project Number: 

TABLE V 


Revene Mulatlon Assay 


With Microsomal Activation 


Confirmatory Assay 


Test Article: 

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 positive control used was 2-aminoanthnu::enc for all strains 

••• The negative control used in the assay was DMSO 

Page 17 of 18 

000126 124 
To:dkon Corporation 

Salmonella typhimurium and Escherichia coli Reverse Mutation Assa) - OECD 

Project Number: 

11.0 CONCLUSION 

The Salmonella typhimurium and Escherichia coli Reverse Mutation Assay (Ames Assay) 

evaluated the potential of the 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 ofSalmonella 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 in the test species. 

12.0 RECORDS 

12.1 	 Original raw data is archived at Toxikon Corporation. 

12.2 	 A copy of the final report and any report amendments are archived at Toxikon 

Corporation. 

12.3 	 The original final report and a copy of any protocol amendments or deviations are 

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 of confidentiality were as agreed upon prior to study initiation. 


a00127 

Page 18 of 18