# ft

# ~EPA \\**Jl~t1** UHl1pktcd**'~lld** thl' 101111 to Enter the total number ofpages in the Premanufacture Notice Ires 10-3 I-96.!

• You must provide all information requested in this form to the extentthat it is known to orreasonably ascertainable by you. Make reasonable estimates ifyou do not have actual data. • Befure you complete this form, you should read the "Instructions Manual for Premanulacture :-.1otification" (the Instructions Manual is available from the Toxic Substances Control Act (TSCA) Information Service bycalling 202·554.1404, or faxing 202·554·5603). Ifa user fee has been remitted for this notice (40 CFR 700.45), indicate in the boxes above the TS,user fee identification number you have generated. Remember, your user fee ID number must also appear on your corresponding fee remittance, which is sent to EPA, HQ Accounting Operations Branch (PM·226), P,O. U.S. ENVIRONMENTAL PROTECTION AGENCY AGENCY USE ONLY**RECE!\f["I~** 

## C

DDT " I I 

### Company Sanitized 

## PREMANUFACTURE NOTICE FORNEW CHEMICAL SUBSTANCES DOCUMENT CONTROL OFFICER OFFICE OF POLLUTION PREVENTION AND TOXIC SUBSTANCES, 7407 U.S. E.P.A. 401 M STREET, SW WASHINGTON, D,C. 204 

# so

360399M, Pittsburgh, PA 15251·6399, Attn. TSCA eser fee.
Pan 1- GENERAL lNFORMAnON You must provide the currently correctChemical Abstracts (CAl Name ofthe new chemical substance, even ifyou claimthe identity as confidentiaL You may authorize another person to submitchemical identity information for you, but your submission will notbe complete and the review will notbegin until EPA receives this information. A letter in supportofyour submission should reference your TS user fee identification number, You must submit an original and two copies ofthis notice including all test data. Ifyou claimed any information as confidential, a single sanitized copy must also be submitted. Part II HUMAN EXPOSURE A."ID ENVIRO:-.IMENTAL RELEASE Ifthere are several manufacture, processing, oruse operations to be described in Part II, sections A and B ofthis notice, reproduce the sections as needed.
Part III - LIST OF A TTACHMENTS Attach additional sheets ifthere is not enough space to answer a question fully. Label each continuation sheet with the corresponding section heading. In Pan III, list these attachments, any test data or other data and any optional information included in the notice. opnONAL INFORMATION You may include any information that you want EPA to consider in evaluatingthe new substance. On page II ofthis form, space has been provided for you to described pollution prevention and recycling information you may have regarding the new substance. So-called "binding" boxes are included throughout this form for you to indicate your "ilIingness to be bound to certain statements you make in this section, such as use, production volume, protective equipment, .. This option is intended to reduce delays Ihat routinely accompany the development ofconsent orders or SignificantNew Use Rules. Except in the case ofexemption applications (such as TMEA, L VE, LOREX) where certain information provided in such notification is binding on the submitter when the Agency approves the exemption application, checking a binding box in this notice does not by itselfprohibitthe submitter from laterdeviating from the information (except chemical identity) reported in the form. CONFIDENTIALITY CLAIMS You may claimany information in this notice as confidential. To assert a claimon the form, mark (X) tne confidential box next to the information that you claim as confidential. To assert a claim in an attachment, circle or bracket the information you claim as confidential. Ifyou claim information in the notices asconfidential, you must also provide a sanitized version oOhe notice, (including attachments). For additional instructions on claiming information as confidential, read the Instructions Manual. Mark (x) ifany information in this notice is claimed as confidential. TEST DATA AND OTHER DATA You are required to submit all test data in your possession orcontrol and to provide a description ofall otherdata known to or reasonably ascertainable by you, ifthese data are related to the health and environmental effects on the manufacture, processing, distribution in commerce, use, or disposal ofthe new chemical substance. Standard literature citations maybe submitted for data in the open scientific literature. Complete test data (written in English), not summaries ofdata. must be submitted ifthey do not appear in the open literature. You shouldclearly identitY whether test data is on the substance oron an analog. Also, the chemical composition ofthe tested material should becharacterized. Following are examples oftest data and otherdata. Data should be submitted according to the requirements of§720.50ofthe Premanufacture Notification Rule (40 CFR Part 720).

Test Data (Check.Below any included in this notice) Environmental fate data 0 Yes Other data 0 Yes Health effects data t!iI Yes Risk assessments Environmental effects data 0 Yes Structure/activity relationships PhysicaliChemical Properties· t!iI Yes Test data not in the possession orcontrol oftne submitter Aphysical andchemical properties worksheet is located on the last page ofthis form. TYPE OF NOnCE (Check OnlyOne) 

### [8J 

## D D D 

## D D D PMN (Premanufacture Notice) INTERMEDIATE PMN (submitted in sequence with final productPMN) SNUN (SignificantNew Use Notice) TMEA (TestMarketing Exemption Application) -T'1 

### nQ LVE (Low Volume Exemption)@40 CFR**723.5~1)** Q;:O -QPl -00 N -lrn LOREX (Low Release/Low Exposure ExemptiOn)!f/:iO**CF1t'7l~(c)(2)** -0 C>fT\ LVE Modification 

## D LOREX Mtiiucati.;:)0 IS THIS ACONSOLlDATED PMN? Yes # ofchemicals (Prenotice Communication # required, enter # on page 3)
EPA FORM 7710·25 (Rev. 5·95) Replaces previous editions ofEPA Fonn 7710·25. 

## 00000:1Public reporting burden for this collection ofinformation is estimated to average 110 hours per response, including time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and revIewing the collection of information. Send comments regarding the burden estimate or any other aspect ofthis collection ofinformation, including suggestions for reducing this burden, to Chief, Information Policy Branch, PM-223, U.S. Environmental ProtectIOn Agency, 401 M. St., S.W., Washington, D.C. 20460; and to the Office ofManagement and Budget, Paperwork Reduction Act (2070-00!2), Washington, D.C. 20503. 

### CERTIFICATION I certify that to the best ofmy knowledge and belief: 1. The company named in Part I, section A, subsection laofthis notice form intends to manufacture or import for a commercial purpose, other than in small quantities solely for research and development, the substance identified in Part I, Section B. 2. All informationprovided inthis notice is complete and truthful as ofthe date ofsubmission. 3. I am submitting with this notice all test data in mypossession orcontrol and a description ofall other data known to or reasonably ascertainable by me as required by §720.50 ofthe'Premanufacture NotificationRule. Additional Certification Statements: Ifyou are submitting a PMN, Intermediate PMN, Consolidated PMN, or SNUN, check the following user fee certification statement that applies: 

## L8J The Company named in Part I, SectionA has remitted the fee of$2500 specified in 40 CFR 700.45(b), or 

# o The Company named in Part I, SectionA has remitted the fee of$1000 for anIntermediate PMN (defmed @ 40 CFR 700.43) in accordance with40 CFR 700.45(b), or 

# o The Company named in Part I Section A is a smallbusiness concern under40 CFR 700.43 and has remitted a fee of$100 in accordance with 40 CFR 700.45(b). Ifyou are submitting a low volume exemption (LVE) application in accordance with 40 CFR 723.50(c)(1) or a Low release and low exposure exemption (LoRex) application in accordance with 40 CFR723.50(c)(2), checkthe following certification statements: 

# o Themanufacturer submitting this notice intends to manufacture or import the new chemical substance for commercial purposes, otherthan in small quantities solely for research and development, under the terms of40 CFR 723.50. The manufacturer is familiar with the terms ofthis section and will comply with those terms; and 

# o The new chemical substance for which the notice is submitted meets all applicable exemptionconditions. Ifthis application is for an LVE in accordance with 40 CFR 723.50(c)(I), the manufacturer intends to commence manufacture ofthe exempted substance for commercialpurposes within 1 year ofthe date ofthe expiration ofthe 30 day review period.
Confidential 

### x Signature ofagent - (ifapplicable) Date

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

## xConfidential la. Person Submitting Notice (in U.S.)

City, State,
b. Agent (if applicable) Company
M and street)

Name authorized official Company
mber and street) City, State, ZIP Code Telephone

c. Ifyou are submitting this notice as part ofajointsubmission, mark (Xl this box.

# -----------------------itJo Joint Submitter (if applicable) Name

2

3. Contact (in U.S.) Company Mailing address (number and street) City. State. ZIP Code
Name ofauthorized official Company Mailing address (number and street) City. State, ZIP Code [fYOll have had aprenotice connmmication (PC)concerning this notice and EPA assigned a PC Number to the notice, entertbe number.
4. IfYOll previously submitted an exemption application for the cbemical sllbslance covered by this notice, enter tbe exemption number assigned by EPA. [fyou previously submitted a PMN for this subSlance enterthe PMN number assigned by EPA (i.e. withdrawn or incompiel£). II for the chemical subslance covered by this notice. enterthe notice nllmber assigned by EPA. .. Telephone Area Code

Telephone

Mark (Xl**~** ifnone Mark (Xl**~** ifnone Mark (Xl**~** ifnone 

## 0 0 0 X X X \. Manufacture 2. Import 6. Type ofNotice
- Mark (X) 3. Both 

# o Only Only 

## 0 Binding Option BindingOption 

## 0 Mark Mark EPA FORM 7710-25 (Rev. 5-95) Page 3I . b. Chemical name (Currently correct Chemical Abstracts (CAl Name consistent with TSCA Inventory listings for similar substances. For Class I substances a CA Index Name must be provided. For Class 2 substances either a CA Index Name or CA Preferred Name must 

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

notice: (check one). 

## D Method 2 (Other Source) Sci-Finder c.

d.

CAS#

e. For a class 1 substance, provide a complete and correct chemical structure diagram. For a class 2 substance
- (I) List the immediate precursor substances with their respective CAS Registry Numbers. (2) Describe the nature ofthe reaction or process. (3) Indicate the 

## x range ofcomposition and the typical composition (where appropriate). (4) Provide a correct representative orpartial chemical structure diagram, as complete as can be known, ifone can be reasonably ascertained. f. Complete either item I

Class I or 2 a. Class ofsubstance
- Mark (X) 

## D Class I or 2 Class 2 

## x

rg] Mark (X) this box ifyou attach a continuation sheet. (See Page 15)
EPA FORM 7710-25 (Rev. 5-95) 

### Page4aIfanother person will submit chemical identity information for you (foreither Item I or2). mark (X) the box at the right. ofthat in a continuation sheet. 1. Class 1or 2 chemical substances (for definitions ofclass I and class 2 substances, see the Instructions Manual) a. Class ofsubstance Mark (X) I 

## D Class I or 2 

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

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

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

## D Method 2 (Other Source) Sci-Finder report obtained from the CAS Inventory Expert Services must be submitted as an attachment to this d. Molecular formula and CAS Registry Number (ifa number already exists for the substance)
CAS#

e. correct structure (I) List
f precursor substances with their respective CAS Registry Numbers. (2) Describe the nature ofthe reaction orprocess. (3) Indicate the range ofcomposition and the typical composition (where appropriate). (4) Provide a correctrepresentative or partial chemical structure diagram, as complete as can be known, ifone can be reasonably ascertained. Contidential



## x x

x

attach a continuation sheet. (See 16) 

### Page4b EPA FORM 7710-25 (Rev. 5-95) 

## 000005Class I or 2 chemical substances (for definitions a. Class ofsubstance· Mark (X) 1 b. Chem1cal name (Currentlycorrect Chemical Abstracts (CA) Name that is consistent with TSCA Inventory listings for substances.

c. For Class I substances a CA Index Name must be provided. For Class 2 substances eithera CA Index Name or CA Preferred Name must be provided, which ever is appropriate based on CA 9C1 nomenclature rules and conventions).

which method you used to develop or obtain the specified chemical identity infonnation Method I (CAS Inventory Expert Service·a copy ofthe Identification 

## 0 Method 2 (Other Source) Sci·Finder based on



## x x t.

report obtained from the CAS Inventory Expert Services must be submitted as an attachment to this substance) d. Mo
CAS#

e. For a class I substance, provide a complete and correct chemical structure diagram. For a class 2 substance· (1) List the immediate precursor substances With theirrespective CAS Registry Numbers. (2) Describe the nature ofthe reaction or process. (3) Indicate the range ofcomposition and the typical composition (where appropriate). (4) Provide a correct representative or partialchemical structure diagram, as complete as can be known, ifone can be reasonably ascertained. f.

attach a continuation sheet. Page 4c EPA FORM 7710·25 (Rev. 5·95) 

## 000006 

## xsubstance based on

Ifanother person will submit chemical identity information for you (for either Item I or2), mark (X) the box at the right. the ofthat in a continuation sheet. I, Class I or 2 chemical substances (for delinitions ofclass I and class 2 substances, see tlie Instructions Manual) a, Class ofsubstance
- Mark (X) 

## 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 CA Index Name or CA Preferred Name must be provided, which ever is appropriate based on CA 9C1 nomenclature rules and conventions).

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

### [gJ Method 1 (CAS Inventory Expert Service - a copy ofthe Identification 

## D Method 2 (Other Source) Sci-Finder report obtained from the CAS Inventory Expert Services must be as an to this d, Molecular formula and CAS Registry Number (ifa number already exists for the substance)

e, 1substance, a complete and correct precursor substances with their respective CAS Registry Numbers, (2) Describe the nature ofthe reaction or process. (3) Indicate the range ofcomposition and the typical composition (where appropriate). (4) Provide a correct representative or partial chemical structure diagram, as complete as can be known. ifone can be reasonably ascertained. Conndential



## x x

CAS#

structure 2 substance
- (I) List the immediate 

## x

this box ifyou attach a continuation sheet. Page 4d EPA FORM 7710-25 (Rev. 5-95) 

## 00000'(substance based on Section B - CHEMICAL IDENTITY Complete enher item 1(Class 1or 2 substances) or 2 (Polymers) as appropriate, Complete all other items, Ifanother person will submit chemical identity information for you (for either Item I or 2), mark (Xl the box at the right Contildenti the and in a sheet. dential I, Class 1or 2 chemical substances (for definitions of I and class 2 substances. see the Instructions Manual) a, Class ofsubstance
- Mark (X) I Class 1 or 2 Class 2 

## 0 

## x b, Chemical name (Currently correct Chemical Abstracts (CAl Name that is consistent with TSCA Inventory listings for similar substances. For Class I substances a CA Index Name must be provided, For Class 2 substances either a CA Index Name or CA Preferred Name must 

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

c, Please identify which method you used to develop or specified chemical identity information 

### [8J Method I (CAS Inventory Expert Service - a copy ofthe Identification 

## 0 report obtained from the CAS Inventory Expert Services must be d. (check one). Method 2 (Other Source) Sci-Finder

CAS#

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



## [8J Mark (X) this box ifyou attach a continuation sheet. (See Page 19)
EPA FORM 7710-25 (Rev. 5-95) 

## 000008 

## xConfidential a. [ndicate the number-average weight ofthe lowest molecular weight composition ofthe polymer you intend to manufacture.

d. lndicate maximum weight percent oflow molecularweight species (not including residual monomers, reactants, or solvents) below 500 and below 1,000 absolute molecular weight ofthat composition. Describe the methods ofmeasurement or the basis for your estimates: GPC

## 0 Other 

## 0: (Specify) i) lowest number average molecular weight: ii) maximum weight % below 500 molecular weight: iii) maximum weight % below 1000 molecular weight: attach a continuation sheet. 

## x

ou must separate contidentiality claims for monomer or other reactant identity, composition information, and residual information. Mark (X) the "Confidential" box next to any item you claim as confidential (I) Provide the specific chemical name and CAS Registry Number (ifa number exists) ofeach monomer or other reactant used in the manufacture of the polymer. (2) Mark (Xl this column ifentry in column (I) is confidential. (3) Indicate the typical weight percent ofeach monomer or other reactant in the polymer. (4) Mark (X) the identity column ifyou want a monomer orother reactant used at two weight percent or less to be listed as part ofthe polymer description on the TSCA Chemical Substance Inventory. (5) Mark (X) this column ifentries in columns (3) and (4) are confidential. (6) Indicate the maximum weight percent ofeach monomer orotherreactant that may be present as a residual in the polymer as manufactured for commercial purposes.

% % X % X % X X % X % X % % % % % reported in notice (check one). Method 2 (other source)
the polymer that is consistent TSCA Inventory listings for similar polymers.

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

D Page 5a 

## 000009XContidential a. Indicate the number-average weight ofthe lowest molecular weight composition ofthe polymer you intend to manufacture. Indicate maximum weight percent oflow molecular weight species (not including residual monomers, reactants, or solvents) below 500 and below 1,000 absolute molecular weight ofthat composition. Describe the methods ofmeasurement orthe basis for yourestimates: GPC

## 0 Other 

## 0: (Specify) I) lowest number average molecular weight: ii) maximum weight % below 500 molecular weight: iii) maximum weight % below 1000 molecular weight: attach a continuation sheet.



# o Mark

obtained from CAS Inventory Expert Service must be submitted as as attachment to this X Typical

%
% % % % X % X % X % % % %

d. The currentlycorrect Chemical Abstracts (CAl name for the polymer thatis consistent with TSCA Inventory listings for similar polymers.

e. Provide a correctrepresentative orpartial chemical structure diagram, as complete as can ascertained. X You must make separate contidentiality claims for monomer or other reactant identity, composition information, and residual information. Mark (X) the "Confidential" box next to any item you claimas confidential (I) Provide the specitic chemical name and CAS Registry Number (ifa number exists) ofeach monomer or other reactant used in the manufacture of the polymer. (2) Mark (X)this column ifentry in column (I) is contidentiaL (3) Indicate the typical weight percent ofeach monomer orotherreactant in the polymer. (4) Mark (X) the identity column ifyou want a monomer orother reactant used at two weight percent or less to be listed as part ofthe polymer description on the TSCA Chemical Substance Inventory. (5) Mark (X) this column ifentries in columns (3) and (4) are confidential. (6) Indicate the maximum weight percent ofeach monomer orother reactant that may be present as a residual in the polymer as manufactured for commercial purposes.

attach a continuation sheet. EPA FORM 7710-25 (Rev. 5-95) 

### Page 5b 

## 00001.0Contidential a. Indicate the number-average weight ofthe lowest molecular weight composition ofthe polymer you intend to manufacture. Indicate maximum weight percent oflow molecular weight species (not including residual monomers, reactants, or solvents) below 500 and below 1,000 absolute molecular weight ofthat composition. Describe the methods ofmeasurement or the basis for your estimates: GPC 

## D Other 

# D: (Specify) i) lowest number average molecular weight: ii) maximum weight % below 500 molecular weight: iii) maximum weight % below 1000 molecular weight: attach a continuation sheet. 

### x

b. You must separate confidentiality claims for monomer or other reactant identity, composition information, and residual information. Mark the "Confidential" box next to any item you claimas confidential (I) Provide the specific chemical name and CAS Registry Number (ifa number exists) ofeach monomer or otherreactant used in the manufacture of the polymer. (2) Mark (X) this column ifentry in column (I) is confidential. (3) Indicate the typical weight percent ofeach monomer or other reactant in the polymer. (4) Mark (X) the identitycolumn ifyou want a monomer or other reactant used attwo weight percent or less to be listed as part ofthe polymer description on the TSCA Chemical Substance Inventory. (5) Mark (X) this column ifentries in columns (3) and (4) are confidential. (6)
- Indicate the maximum weight percent ofeach monomer or other reactant that may be present as a residual in the polymer as manufactured for commercial purposes. Mark

method you X 

### x

(CAS Inventory Expert Service - a copy ofthe identification report obtained from CAS Inventory Expert Service must be submitted as as attachment to this % % X % X % % % (check one).
currentlycorrect name for the polymer thatis consistent with TSCA Inventory listings for similar polymers. % % X % X % % %

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

Mark (X) this box if attach a continuation sheet. EPA FORM 7710-25 (Rev. 5-95) 

### Page 5c 

## 000011Part I - GENERAL INFORMATION ­ Continued Section 8 - CHEMICAL IDENTITY INFORMATION - Continued 2, Polymers (Fora delinition ofpolymer, see the Instructions Manual.) a, Indicate the number-average weight ofthe lowest molecular weight composition ofthe polymer you intend to manufacture, Indicate maximum weight percent oflowmolecular weight species (not including residual monomers, reactants, or solvents) below 500 and below 1,000 absolute molecular weight ofthat composition, Describe the methods ofmeasurement or the baSIS for your estimates: GPC 

## D Other 

# D: (Specify) i) lowest number average molecular weight: ii) maximum weight % below 500 molecular weight: iii) maximum weight % below 1000 molecular weight: attach a continuation sheet. Contidential



### x

You must make separatecont1dentiality claims for monomer orotherreactant identity, composition information, and residual information, Mark (X)the "Cont1dential" box next to any item you claim as confidential (I) Provide the specific chemical name and CAS Registry Number (ifa number exists) ofeach monomeror otherreactant used in the manufacture of (2) - (3) (4) the polymer, Mark (X) this column ifentry in column (1) is confidential. Indicate the typical weight percent ofeach monomerorother reactant in the polymer. Mark (X) the identity column ifyou want a monomer orother reactant used at two weight percent or less to be listed as partofthe polymer description on the TSCA Chemical Substance Inventory. Mark (X) this column ifentries in columns (3) and (4) are confidential. (5) (6) Indicate the maximum weight percent ofeach monomer or other reactant that may be present as a residual in the polymer as manufactured fur commercial purposes. Mark this column if

% X % X X % X % % % % % X % X % % %Confic.
d,

e.

attach a continuation sheet. (See Page 18) EPA FORM 7710-25 (Rev. 5-95) Please identify which method you used to develop orobtain the specified chemical identity information reported in this notice (check one). 

## D Method I (CAS Inventory Expert Service - a copyofthe identification report 

## D Method 2 (other source) obtained from CAS Inventory Expert Service mustbe submitted as as The currently correct Chemical Abstracts (CA) name for the polymer that is with TSCA Invenf,flrv

Prov' a correct orpartial chemical structurediagram, as complete as can beknown, ifone can be reasonably ascertained. X



## 00001.2Contidential a. Indicate the number-average weight ofthe lowest molecular weight composition ofthe polymer you intend to manufacture. Indicate maximum weight percent oflow molecular weight species (not including residual monomers, reactants, or solvents) below 500 and below 1,000 absolute molecular weight ofthat composition. Describe the methods ofmeasurement or the basis for your estimates: OPC 

## D Other 

# D: (Specify) i) lowest number average molecular weight: ii) maximum weight % below 500 molecular weight: Iii) maximum weight % below 1000 molecular weight: attach a continuation sheet. 

## x

You must make separate confidentiality claims for monomer or otherreactant identity, infonnation, and residual infonnation. Mark (X) the "Confidential"box next to any item you claim as confidential (I) Provide the specific chemical name and CAS Registry Number (ifa number exists) ofeach monomer or other reactant used in the manufacture of the poIymer. (2) Mark (X) this column ifentry in column (I) is confidential. (3) Indicate the typical weight percent ofeach monomer or other reactant in the polymer, (4) Mark (X) the identity column ifyou want a monomer orother reactant used at two weight percent or less to be listed as part ofthepolymer description on the TSCA Chemical Substance Inventory. (5) Mark (X) this column ifentries in columns (3) and (4) are confidential. (6) Indicate the maximum weight percent ofeach monomer or other reactant that may be present as a residual in the polymer as manufactured for commercial purposes, Mark this column if

% % X % X % X X % X % X % % % % % reported in this notice (check one). Method 2 (other source)

known, ifone can be reasonably ascertained. X obtained from CAS Inventory Expert Service must be submitted as as attachment d. The currently correctChemical Abstracts (CA) name for the polymer that is consistent with TSCA Inventory

e. Provide a correct representative orpartial as complete as can

attach a continuation sheet. EPA FORM 7710-25 (Rev. 5-95) 

### Page 5e 

## 0000:13

### Part I -- GENERAL INFORMATION -- Continued Section 8 - CHEMICAl IDt.;rHTlY INFORMATION3. Impurities (a) Identify each impurity that may be reasonably anticipated to be present in the chemical substance as manufactured for commercial purpose. Provide the CAS Registry Number ifavailable. [fthere are unidentified impurities, enter "unidentified." (b) 

### . Estimate the maximum weight % ofeach impurity. [fthere are unidentified' . estimate their total weight %. Impurity and CAS Registry Number Maximum Contipercent dential (a) (b) % X % X
% X
% X % % % 

### [ ] Mark (X) this box ifyou attach a CI',,!inIOMinn sheet. 4. Synonyms
- Enter any chemical synonyms for the new chemical identified in subsection lor 2.

## ;e~~~~ X 

## 0 Mark (X) this box ifOyou attach a continuation sheet. 5. Trade identilication
- List trade names for the new chemical substance identified in subsection I or 2. X 

## 0 Mark (X) this box ifyou attach a continuation sheet. 6. Generic chemical name- Ifyou claim chemical identifyas w"'".....ltial, you must provide a generic name for your substance thatreveals the specific chemical identity ofthe new chemical substance to the maximum extent possible. Refer to the TSCA Chemical Substance Inventory, 1985 Edition, Appendix B for guidance on developing generic names. Polyoxyalkylene, alkylene succinate polyester 

## D Mark (X) this boxifyou attach a continuation sheet. 7. Byproducts • Describe any byproducts resulting from the manufacture. processing, use, or disposal ofthe new chemical substance. Provide the CAS Registry Number ifavailable. ""rMIll'! CAS RegisyY Number**~JI'(I)~~'**

### ;;~j (2) X

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

## 000014: 

## 0

### PartI - GENERAL INFORMATION
-- Continued Section C - PRODUCTION, IMPORT, AND USE INFORMATION: Mark (X) the "Confidential" box next to any item you claimas confidential. l. Production volume
- Estimate the maximum production volume during the first 12 months ofproduction. Also estimate the maximum production volume for any consecutive 12-month period during the first three years ofproduction. Estimates should be on 100% new chemical substance basis. For a Low Volume Exemption application, ifyou choose to have your notice reviewed at a lower production volume than 10,000 kg/yr, specify the volume and mark (x) in the binding box. Ifgranted, you are bound to this volume Maximum first 12-monthproduction (kg/yr) Maximum 12-monthproduction (kglyr) Confi· Binding (100% new chemical substance basis) (100% new chemical substance basis) dential Option Mark (x) X 2. Use Information
- You must make separate confidentialityclaims for the description ofthe category ofuse, the percent ofproduction volume devoted to each category, the formulation of the new substance, and other use information. Mark (X) the "Confidential" Box next to any item you claim as confidential. a. (I) -­ Describe each intended category ofuse ofthe new chemical substance by function and application.. (2)
-­ Mark (X) this column ifentry column (1) is confidential business information (CBI). (3)
-­ Indicate your willingness to have the information provided in column (I) binding. (4)
-­ Estimate the percent oftotal production for the first three years devoted to each category ofuse. (5)
-­ Mark (X) this column ifentry in column (4) is confidential business information (CBI). (6)
-­ Estimate the percent ofthe new substance as formulated in mixtures, suspensions, emulsions, solutions, or gels as manufactured for commercial purposes at sites under your control associated with each category ofuse. (7)
-­ Mark (X) this column ifentry in column (6) is confidential business information (CBI). (8)
-­ Indicate % ofproduct volume expected for the listed "use" sectors. Mark more than one box ifappropriate. Mark (X) to indicate your willingness to have the use type provided in (8) binding. (9) -­ Mark (X) this column ifentry(ies) in column (8) is (are) confidential business information (CBI). Category ofuse (I) CBl Binding Produc- CSI %in CBI %ofsubstance expected peruse CBI Option tion% Form­ (8) (by function and application i.e, adispersive dye Marl< (x) ulation Site- Con-· Indus- Com- Binding for finishing polyester fibers) (2) (3) (4) (5) (6) (7) limited sumer trial mercial Option (9) X % X X X % 

## - % % % % % % % % % % 

# H % % • Ifyou have identified a"consumer" use, please provideon acontinuation sheet adetailed description ofthe use(s) ofthis chemical substance in consumerproducts,

In addition include estimates ofthe concentration ofthe new chemical substance as expected in consumerproducts and describe the chemical reactions by which this substance loses its identity in the consumerproduct. DMark (X)this box ifyou attach acontinuation sheet. b, Generic Ifyou claimany category ofuse description in subsection 2aas confidential, enter ageneric description ofthat category. Read the use Instructions Manual for examples ofgeneric use descriptions, description Colorant for aqueous ink applications

D Mark (X)this box ifyou attach acontinuation sheet. 3. Hazard Information
- Include in the notice acopy ofreasonable facsimile ofany hazard warning statement, label, material safety data sheet, orother

# r=

information which will be provided to any person who is reasonably likely to be exposed to this substance regarding protective equipmentorpractices for the safe handing, transport, use, or disposal ofthe new substance. List in part III hazard information you include, 

### [gJ Mark (X) this box ifyou attach hazard information, (See pages 25-29)
EPA FORM 7710-25 (Rev, 5-95) Page 7 

# 000015

# x Complete section A for each type ofmanufacture, processing, or use operation . new 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 or use after· You must describe these See instructions manual I. Confiwill occur. ofthe site at which the dential

Site address (number and street) City, County, State, ZIP Ifthe same operation will occur at more than one site, enter the number ofsites. Identify the additional sites on a continuation sheet, and ifany ofthe sites have significantlydifferent production rates oroperations, include all the information requested in this section for those sites as attachments. 

### Part 11-- HUMAN EXPOSURE AND

Batches/year L Batch (100010 new chemical Hourstbatch Batches/year substance) 2. Continuous (I) Diagramthe major unit operation steps and chemical conversions. Include interim storage and transport containers (specify- e.g. 5 gallon pails, 55 gallon drum, rail car, tank truck. etc.). (2) Provide the identity, the approximate weight (by kg/day or kgibatch on a 100010 newchemical substance basis), and entrypoint ofall starting materials and feedstocks (including reactants, solvents, catalysts, etc.), and ofall products, recycle streams, and wastes. Include cleaning chemicals (note frequency ifnot used daily or perbatch.). (3) Identify by number the points ofrelease, including small or intermittent releases, to the environmentofthe newchemical substance. 

# See Process Description 

# on Page 30

Mark (X) this box ifyou attach a continuation sheet. (See page30) d. Process description Mark (X) to indicate your willingness to have your process description binding. 

# x x

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

## 0000:16

### Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE
-- Continued Section A
-- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER
- Continued 

## I 2. Occupational Exposure
­ You must make separate confidentiality claims for the description ofworker activity, physical form ofthe new chemical substance, number ofworks exposed, and duration ofactivity. Mark (X) the "Confidential" box next to any item you claim as confidential. (\) Describe the activities (i.e. bag dumping. tote filling, unloading drums, sampling, cleaning, etc.) in which workers may be exposed to the substance. (2)
-- Mark (X) this column ifentry in column (I) is confidential business information (CBI). (3)
-- Describe any protective equipment and engineering controls used to protect workers. (4) and (6)
-­ Indicate your willingness to have the information provided in column (3) or (5) binding. (5) Indicate the physical formes) ofthe new chemical substance (e.g., solid: crystal, granule. powder, or dust) and % new chemical substance (ifpart ofamixture) at the time ofexposure. (7)
-- Mark (X) this column ifentry in column (5) is confidential business information (CBI). (8)
-- Estimate the maximum number ofworkers involved in each activity for all sites combined. (9)
-- Mark (X) this column ifentry in column (8) is confidential business information (CBI). e I0) and (II)
-­ Estimate the maximum duration ofthe activity for any worker in hours per day and days per year. (12)
-­ Mark (X) this column ifentries in columns (10) and (II) are confidential business information ICBI). Worker activity CBI Protective Equipment! Binding Physical forms(s) Binding CBI #of CBI Maximu duration CBI m (i.e., bag dumping, tilling drums) Engineering Controls Option and % new Option Workers Hrsiday Dayslyr Mark (x) substance Mark (x) Exposed (I) (3) (4) (5) (6) (7) (8) (9) (10) (II) (12) X X X X X X X X X X X 

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

3. Environmental Release and Disposal
-­ You must make separate contldentiality claims for the release number and the amount ofthe new chemical substance released and otherrelease and disposal information. Mark (X) the "Confidential"box nextto each item you claim as confidential. (I)
-- Enter the number ofeach release point identitled in the process description, part II, section A. subsection Id(3). (2)
-- Estimate the amount ofthe new substance released (a) directly to the environment or (b) into control technology (in kg/day or kglbatch). (3) Mark (X) this column ifentries in columns (I)and (2) are confidential business information (CBI). (4) --Identify the media (stack air. fugitive air (optional-see Instruction Manual). surface water. on-site oroff-site land orincineration. POTW. orother (specify» to which the new substance will be released from that release point (5) --a. Describe control technology. ifany, and control efficiency that will be used to limit the release ofthe new substan'ce to the environment Forreleases disposed ofon land. characterize the disposal method and state whether it is approved for disposal ofRCRA hazardous waste. On a continuation sheet, for each site describe any additional disposal methods that will be used and whether the waste is subject to secondary or tertiary on-site treatment b. Estimate the amountreleased to the environment after control technology (in kg/day). (6) Mark (X) this column ifentries in columns (4) and (5) are confidential business information (CBI). (7) Identify the destination(s) ofreleases to water. Please supply NPDES (National Pollutant Discharge Elimination System) numbers for direct discharges or NPDES numbers ofthe POTW (PublicI Owned Treatment Works). Mark (X) ifthe POTW name or NPDES # is confidential business information (CBI). Release Amount ofnew substance CBI Media of Control technology and efficiency (you may wish to optionally attach efficiency data) CBl Number released release

(1) (2a) (2b) e·l·stacku Binding (3) (4) (Sa) Mark (X) (5b) (6) X X 

# H
(7) Mark (X) the 

# o POTWprovide name(s) below: CBI 

# o Navigable 

# o Other
- Specify provide NPDES # CBI ,-­ destination(s) of waterway releases to water. Mark (X) this box ifyou attach a continuation sheet.

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

## 0000:1'7

## OPTIONAL POLLUTION PREVENTION INFORMATION To claim information in this section as confidential circle orbracket the specific information that youclaimas confidential. 

### In this section you mayprovide information not reported elsewhere in this form regarding your efforts to reduce orminimizepotential risks associated with activities surroundingmanufacturing, processing, use and disposal ofthe PMN substance. Please include new information pertinent to pollution prevention, including source reduction, recycling activities and saferprocesses orproducts available due to the new chemical substance. Source reduction includes the reduction in the amount ortoxicity ofchemical wastes bytechnological modification, process and procedure modification, productreformulation, raw materials substitution, and/or inventory control. Recycling refers to the reclamation ofuseful chemical components from wastes that would otherwise be treated orreleased as air emissions orwater discharges, or land disposal. Descriptions ofpollution prevention, sourcereduction and recycling should emphasize potential risk reduction subsequent to compliance with existingregulatory requirements and canbe either quantitative orqualitative. The EPA is interested in the information to assess overall net reductions in toxicityor environmental releases and exposures, not the shifting ofrisks to other environmental media ornon-environmental areas (e.g., occupational orconsumer exposure). 

### In addition, information onthe relative cost orperformance characteristics ofthe PMN substance to potential alternatives maybeprovided. 

### All information provided in this section will be taken into consideration duringthe review ofthis substance. See Instructions Manual and Pollution Prevention Guidance manual for guidance and examples. Describe the expected net benefits, such as (1) an overall reduction inrisk to humanhealth or the environment; (2) a reduction in the volume manufactured; (3) a reduction inthe generation ofwaste materials through recycling, source reduction orother means; (4) a reduction inpotential toxicity orhuman exposure and/or environmental release; (5) an increase inproductperformance, a decrease in the cost ofproduction and/or improved operation efficiencyofthe new chemical substance incomparisonto existing chemical substances used in similar application; or (6) the extent to which the new chemical substance maybe a substitute for an existing substance thatposes a greater overall riskto humanhealth or the environment.

# CONF\DEN1\~1**~**

I'2J Mark (X) this box ifyou attach a continuation sheet. (See StudyReports 00 Pages 32-175)

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

## 00001.8

## Part III -- LIST OF ATTACHMENTS Attach continuation sh\':ets for sections ofthe fonn and test data and otherdata (including physical/chemical properties and structure/activity infonnation), and optional infonnation after this page. Clearly identify the attachment and the section ofthe fonn to which it relates, ifappropriate. Number consecutively the pages ofthe attachments. In the column below, enter the inclusive page numbers ofeach attachment. Mark (X) the "Confidential" box next to any attachment name you claim as confidential. Read the Instructions Manual for guidance on how to claim any infonnation in an attachment as confidential. You must include with the sanitized copy ofthe notice fonn a sanitized version ofany attachment in which vou claim information as confidential. Attachmentname Attachment Conlipagenumber(s) dential X X
I X X X X X Continuation Sheet for Part 

### I. Section C.3 Material Safety Data Sheet (MSDS) 25-29 X X X X X X X

I



# tnMark (X) this box ifyou attach acontinuation sheet. Enter the attachmentname and number. EPA FORM 7710-25 (Rev. 5-95) Page 12 

## 000019PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET To assist EPA's review ofphysical and chemical properties data, please complete the following worksheet for data you provide and include it in the notice. Identify the property measured, the page ofthe notice on which the property appears, the value ofthe property, the units in which the property is measured (as necessary), and whether or not the property is claimed as confidential. The physical state ofthe neat substance should be provided. These measured properties should be for the neat (100% pure) chemical substance. Properties that are measured for mixtures or formulations should be so noted (% PMN substance in

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

(a) Mark (X) tf provided Page number

(b) Value (c) lMeasured 0 Estimate (M or E) Confidenti.1 Mark (X)

(d) Physical state ofneat substance X 28 (s) 

## .x (1) (g) M Vapor pressure (a) Temperature 

### °C Torr Density/relative density X 28 0.905 glcm3 M Solubility @ Temperature



### --°C

Solvent giL Solubility in water @ Temperature 25 

### °C X 28 soluble giL M Melting temperature 

### °C Boiling I sublimation temperature(a) torrpressure 

### °C Spectra X Dissociation constant Particle size distribution Octanol/ water partitioncoefficient Herny's Law constant Volatilization from water Volatilizationfrom soil pH@ concentration 60%in water X 28 

## I 7.5 M Flammability X 26 > 212 OF M Explodability Adsorption/ coefficient Other
- Specify

ATTACHMENT 

# ooo'WZrl

### ANALYSIS OF PRODUCT 

# CONFJDENTIAL

PAGE 14 

## 000021Page l5 

# Cffi InventoryExpert Service
A division of the American Chemical Society Phone: 800-631-1884.**61~1-3600** (ext. 2308)**Fax:61~1-3141** E-mail: answers@cas.org '.' 

# CONFIDENTIAL

please print the above CA Index N ... on the appropri.t. p.g. of your PMN.

If this box i. checked, CAS h•• mad. correction(.) marked in red to

## ~ your IES ord.r. Pl•••• make the .... corr.ction(.) to your PMN before submitting it to the EPA. .



## 000022 p.o. Box 3343 Columbus. OhIo43110-0334Page 16 

# ern InventoryExpert Service
A division of theAmerican Chemical Society Phone: 800-631.1884, 61 .........7·3600 (ext 2308) Fax: 61 .........7·37.. 7 E-mail: allSWerS@as.org

,. 

# CONFIDENTIAL

Pleas. print the above CA Index Kame on the .ppropri.t. p.g. of your PHR.

If this box is ch.ck.d, CAS h•• mad. corr.ction(s) mark.d in r.d to your IES ord.r. Pl•••• make the same correction(.) to your PHR before submitting it to the EPA.



### p.o. .. 3J4l CoIumbuI. Ohio43110.0334 

## 000023

### Page 17 

# ern InventoryExpert Service
A division oftheAmerican Chemical Society Phone: 800-631.1884, 61-4-+47·)600 (ext. 2308) 

### Fax: 61-4-+47.3747 E·mail: answen@as.org 

# CONFIDENTIAL

Please print the above CA Index Ham. on the appropriate page of your PMH. 

# Tty If this box is checked, CAS has made correction(s) marked in red to your IES order. please make the sam. correction(s) to your PMH before submitting it to the EPA.



## 000024 P.O." 3343 CoIumbuI. Ohio43210-0334Page 18 

# ern InventoryExpert Service
A division oftheAmerican Chemical Society Phone: 800-631.1884, 614-447·3600 (ext. 2308) Fax: 614-447-3747 E-mail: answen@cas.org

....... 

# CONFIDENTIAL

Please print the above CA Index Name on the .ppropri.t. p.g. of your PMH.



### ,

# -~If thi. boa i. checked. CAS ha. made 

### correction(.) marked in red 

### to

# ~**~our** IES ord.r. Pl•••• make the .ame corr.ction(.) to your PMH b.for. submitting it to the EPA.

P.O."3343 Cok.wnbus. Ohio43210.0334 

## 000025Page 19 

# InventoryExpert Service
A division of the American Chemical Society Phone: 800-631-1884, 61...........7-3600 (ext. 2308) Fax: 61...........7-3747 E-mail: answers@cas.org 

# CONFIDENTIAL

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

## ~If this box is checked, CAS has made correction(s) marked in red to

### ~ your IES order. Pleas. make the same correction(s) to your PMN before submitting it to the EPA.



## 000026 P.O.Bax ]]4] Columbus. Ohio 4]210-0]]4Page 20 Qnent0IIte 1011912000 

# CONFIDENTIAL 10f5



## 000027Page 21



# CONFIDENTIAL

000028Page 22



## 000029Page 23Page ')4



# CONFIDENTIAL

000031

### Page 25 LiquiTone® Cyan 703 MSDS Number: PP6305 (Water) Page 1 January 22, 2001 

# MATERIAL SAFETY DATA SHEET 1. CHEMICAL PRODUCT AND COMPANY IDENTIFICATION Product Identification 

### Product Name: LiquiTone® Cyan 703 (Water) Chemical Family: Polymeric Colorant Blend CAS Number: Blend 

# A'.. Company Identification 

### Milliken Chemical 

# MILLIKEN P.O. Box 817 1440 Campton Road 

# CHEMICAL Inman, SC 29349 USA 1-864-472-9041 (For questions and emergencies) 1-800-424-9300 or 1-703-527-3887 (CHEMTREC) PRODUCT USE: 

### Colorant. 

### 2. COMPOSITION/INFORMATION ON INGREDIENTS Chemical Name Amount CAS Number WATER 45.0 % POLYMERIC COLORANT 55.0 % 

### Confidential LiquiTone® Cyan 703 (Water) samples are manufactured for research and development purposes only. This product may not be listed on the Toxic Substance Control Act (TSCA) chemical substances inventory. Any use in a capacity other than research and development may constitute a violation of TSCA. (See Section 8 for exposure guidelines) (See Section 15 for regulatory information)


# I UNCONTROLLED DOCUMENT 

# I 1111""1111111111111111111111111111111111111 

## 000032Page 26 LiquiTone® Cyan 703 MSDS Number: PP6305 (Water) Page 2 January 22, 2001 

### 3. HAZARDS IDENTIFICATION ***************** EMERGENCY OVERVIEW 

### ***************** 

## * * * The health hazards of this product should be low 

## * * under normal industrial and commercial uses. 

## * 

## * * ********************************************************** HMIS Ratinq - Health: 1 Flammability: 1 Reactivity: 0 POTENTIAL HEALTH EFFECTS EYE: May cause eye irritation. SKIN: Prolonged or repeated skin contact may cause irritation. 

### 4. FIRST AID MEASURES EYE CONTACT FIRST AID: In case of contact, immediately flush eyes with plenty of water for at least 15 minutes. Get immediate medical attention. SKIN CONTACT FIRST AID: Immediatly flush skin with plenty of water for at least 15 minutes. Get medical attention if irritation develops or persists. Remove contaminated clothing and shoes. Wash contaminated clothing before reuse. INGESTION FIRST AID: If swallowed, immediately give 2 glasses of water. Do not induce vomiting. Never give anything by mouth to an unconscious person. Contact a physician. 

### 5 . FIRE FIGHTING MEASURES FLAMMABLE PROPERTIES TCC Flash Point: >100 C (> 212.0 F) Autoignition Temperature: N/A FLAMMABLE LIMITS IN AIR LEL: N/A UEL: N/A 

# I UNCONTROLLED DOCUMENT 

# I 11111111111111111111111111"1111111111111111I 

## 000033

### Page 27 LiquiTone® Cyan 703 MSDS Number: PP6305 (Water) Page 3 January 22, 2001 (section 5 continued) EXTINGUISHING MEDIA: Water, carbon dioxide, foam or dry powder. Water may be effective for cooling, but may not effect extinguishment. Use water spray to cool fire exposed containers. FIRE FIGHTING INSTRUCTIONS: As in any fire, wear self-contained breathing apparatus pressure-demand MSHA/NIOSH (approved or equivalent) and full protect'ive gear. Contain runoff water. Evacuate non-emergency personnel to a safe area. Avoid breathing smoke, fumes, and decomposition products. 

### 6 . 

### ACCIDENTAL RELEASE MEASURES SPILL PROCEDURE: This material is a concentrated colorant. Water will increase the amount of colorant contamination. Do not allow material to enter soil or surface water. Clean up area by absorbent material. Take up and place in secure closed containers. All waste materials should be packaged, labeled, and transported in accordance with all national, state/provincial, and local requirements. 

### 7 . HANDLING AND STORAGE HANDLING (PBRSONNEL): Wear appropriate protective equipment. Do not get in eyes, on skin or clothing. Wash hands thoroughly after handling. Use only in the presence of a safety shower and eye bath. STORAGE PRECAUTIONS: Avoid extreme temperatures. Protect containers from physical damage. Store in a well ventilated place. 

### 8 . EXPOSURE CONTROLS / PERSONAL PROTECTION BYE / FACB PROTECTION REQUllU!:MBN'l'S: Chemical goggles are recommended to avoid contact with eyes. SKIN PROTECTION REQUIREMENTS: For' brief contact, normal work attire should be sufficient. When prolonged or frequently repeated contact could occur, use protective clothing impervious to this material. RESPIRATORY PROTECTION REQUllU!:MBN'l'S: Atmospheric levels should be maintained below the exposure guideline. 

# I UNCONTROLLED DOCUMENT 

# I**1111111I1111111Iltl~IW I~** **~!III"** 

# IIII

### Page 28 LiquiTone® Cyan 703 (Water) Page 4 MSDS Number: PP6305 January 22, 2001 

### (section 8 continued) EXPOSURE GUIDELINES: 

### No Information Available. 

### 9. PHYSICAL AND CHENcrCAL PROPERTIES FORM ..••••..•••.•••••• : 

### Liquid COLOR ................. : Dark Blue BOILING POINT ......... : 100 C @

### 760 

### mm Hg SOLUBILITY IN WATER ... : Soluble SPECIFIC GRAVITy ...... : 0.905 @ 

### 24 C (Water 1) PH .••...••............ : 

### 7.5 % 

### VOLATILES ..........• : 45 % @ 

### 100 C VISCOSITy ............. : 200-600 CPS 

### 10. STABILITY AND REACTIVITY S'l'ABILITY: 

### This product is 

### stable at ambient conditions. POLYMERIZA'l'ION: 

### Hazardous polymerization will not occur. INCOMPA'l'IBILITY WITH O'l'HER MATERIALS: 

### Avoid contact with strong oxidizing agents. DECOMPOSI'l'ION: 

### Decomposition may produce fumes, smoke, oxides of carbon and hydrocarbons. 

### 11. TOXICOLOGICAL INFORMATION No information available. 

### 12 . ECOLOGICAL INFORMATION No information available. 

### 13. DISPOSAL CONSIDERATIONS 

# I uNCON'l'ROLLBD DOctJMZN'l' 

# I 111111111111111111111111111111111111111111111 

## 000035

### Page 29 LiquiTone® Cyan 703 (Water) Page 5 MSDS Number: PP6305 January 22, 2001 (section 13 continued) WASTE DISPOSAL: All waste materials should be packaged, labeled, and transported in accordance with all national, state/provincial, and local requirements. Incineration is recommended. 

### 14. TRANSPORTATION INFORMATION PRODUCT LABEL .......... : LiquiTone® Cyan 703 (Water) D.O.T. SHIPPING NAME ... : Not Regulated 

### 15. REGULATORY INFORMATION No information available. 

### 16. OTHER INFORMATION REASON FOR ISSUE ... : New PREPARED By ........ : Phil Harris APPROVED By ........ : Ginger Ford TITLE .............. : Development Associate APPROVAL DATE ...... : January 22, 2001 SUPERCEDES DATE .... : February 21, 2000 MSDS NUMBER ........ :. PP6305 

### **********************************************************************

The information contained in this Material Safety Data Sheet is furnished without warranty, expressed or implied, except that it is accurate to the best knowledge of Milliken Chemical. The data on this sheet are related only to the specific material designated herein. Milliken Chemical assumes no legal responsibility for use or reliance upon these data. 

### ********************************************************************** END OF MSDS 

### **********************************************************************



# I UNCONTROLLED DOCO'Ml!:NT 

# I 111111III 111111111111111II1111111111111111111 

## 000036

### Page 30 

# CONFIDENTIAL PART 11- HUMAN EXPOSURE AND ENVIROMENTAL RELEASE SECTION 

## A.1.d - INDUSTRAL SITES CONTROLLED BY THE SUBMITTER



## 00003',

## Page 31 

# CONFIDENTIAL Part II
- Human Exposure and Environmental Release Section B - Industrial Sites Controlled by 

## Others .



## 000038

# I Page 

### 32 

# I 

# CONFIDENTIAL

000039**Pag~** 34



### SUBJECT: Acute Oral Toxicity Study in Rats Project No. 2106-138 We, the undersigned, hereby declare that the work was performed under our supervision according to the procedures herein described.

Study Director: Study Coordinator:

# ~./f;fh Life Sciences Division**oooo··~o Pag~**  35



### SAMPLE NUMBER: 70600017 Rainbow Blue X7967-65, a liquid, received June 1, 19B7, and stored at room temperature. ACUTE ORAL TOXICITY STUDY IN RATS Project Number: 2106-13B Test Animal: Young adult male and female albino rats (weighing between 200-250 g) of the Sprague-Dawley strain were procured, maintained in individual cages in temperature-controlled and humidity-monitored quarters, provided continuous access to Purina Certified Rodent Chow8 #5002 and water, and held for an acclimation period of at least 7 days. Acclimated animals were chosen by computer-generated random numbers, housed individually, and identified by individual animal numbers. Food and water were available ad libitum throughout the study, except for an overnight period just before test material administration when food, but not water, was withheld. Method: Five male and five female rats weighing between 202.6 and 231.2 9 

### and 204.5 and 227.7 g, respectively, were dosed at a level of 5000 mg/kg of body weight. 'The dose factor was 10 ml/kg. Preparation and Administration of Test Material: The test material was shaken well before formulation to insure homogeneity. The desired amount of the test material was weighed into a precalibrated beaker ,on an appropriate (mg) balance. Distilled wat.r was added to achieve desired volume. The solution was then stirred on a magnetic stirring plate for approximately 5 minutes. The solution was also stirred while dosing. The test material was assumed to be 100_ pure. Information on method of synthesis, stability, as well aa data on composition or other characteristics which define the test material are on file with the sponsor. Observations: Each animal was observed for signs of toxic and pharmacologic effects at 1, 2 and 4 hours after test material administration and once daily thereafter for 14 days. Mortality/moribundity were recorded twice daily. All animals were weighed just before test material administration, at Day 7, and at termination. At the end of the study, an acute oral LD50 was estimated for each sex, and for both sexes combined. Pathology: At study termination, all the animals were sacrificed and necropsied. Observations were recorded.



# 0000412 Pag.:: 36



### SAMPLE NUMBER: 70600017 PAGE Rainbow Blue X7967-65, a liquid, received June 1, 1987, and stored at room temperature. ACUTE ORAL TOXICITY STUDY IN RATS (CONTINUED) SUMMARY Test Animal: Albino Rats
- Crl: CD

## e BR strain Source: Charles River Laboratories, 'Inc., Kingston, New York Vehicle: Distilled Water Date Animals Received: June 17, 1987 Date**T~st** 

### Started: June 26, 1987 Date Test Completed: July 10.. 1987 Method of Administration: Gavage Estimated Oral LD50: Male
- Greater than 5000 mg/kg of body weight Female
- Greater than 5000 mg/kg of body weight Combined male and female
- Greater than 5000 mg/kg of body weight. Animal INDIVIDUAL BODY WEIGHTS (g) BODY WEIGHT CHANGE Number Initial Day 7 Terminal. Initiation-Termination Male.
- 5000 mg/kg D05744 231.2 303.5 342.3 111.1 D05745 221.4 287.5 313.6 . 92.2 D05746 209.6 301.2 336.8 127.2 D05747 202.6 294.5 328.7 126.1 D05748 203.9 289.1 321.1 117.2 !'emale.
- 5000 mg/kg D05819 204.5 251.4 256.2 51.7 D05820 227.7 266.1 281.5 53.8 D05821 211.8 254.1 268.3 56.5 D05822 205.1 241.0 258.4 53.3 D05823 207.2 254.0 274.5 67.3



## 000042**Pag~** 37



### SAMP['E NUMBER: 70600017 PAGE 3 Rainbow Blue X7967-65, a liquid, and stored at room temperature. received June 1, 1987, ACUTE ORA[' TOXICITY STUDY IN RATS (CONTINUED)
OBSERVATION _HOURS_ 1 2 4 1 2 C['INICA[' OBSERVATIONS DAYS 3 4 5 6 7 8 9 10 11 12 13 14 Males
- 5000 mg/kg No. observed No. found dead Normal Urine stains Soft feces Compound-colored urine Compound-colored feces 5 0 2 3 3 2 3 5 0 1 3 4 2 4 5 0 0 5 4 4 4 5 0 0 0 0 5 0 5 0 0 0 0 1 

### 4 5 0 0 0 0 2 5 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 Females

- 5000 mg/kg No. observed No. found dead Normal Urine stains Soft feces Compound-colored urine Compound-colored feces 5 0 2 2 0 0 1 

### 5 0 2 2 0 1 

### 2 5 0 0 5 1 

### 5 3 5 0 0 0 0 5 0 5 0 0 0 0 2 3 5 0 0 0 0 3 5 5 0 3 

### 0 0 2 0 5 0 5 .0

0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 

### O· 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 0



## 000043**Pag~** 38



### SAMPLE NUHBER: 70600017 PAGE Rainbow Blue X7967-65, a liquid, received June 1, 1987, and stored at room temperature. ACUTE ORAL TOXICITY STUDY IN RATS (CONTINUED) SUHMARY OF NECROPSY FINDINGS Dose Level Hales Females OBSERVATION (mg/leg) : 5000 5000· No. observed 5 5 No. with no gross lesions 5 4 

### INTESTINES Contained yellowish fluid 0 1 All of the animals dosed at 5000 mg/leg of body weight survived to study termination. Soft feces and urine stains were clinical signs noted on the day of dosing_ There were minimal clinical observations noted subsequently, most of which were attributable to compound staining_ All animals were normal by Day 5 of the study. Only one female exhibited any gross pathological findings and this was limited to yellowish fluid in the intestines. All of the animals gained weight throughout the study. Based upon the results of this study, the acute oral LDSO of this compound is estimated to be greater than 5000 mg/leg of body weight in male and female rats_



## 0000445**Pag~** 39



### PAGE SAMPLE NUMBER: 70600017 

### Rainbow Blue X7967-65, a liquid, received June 1, 1987, 

### and stored at room temperature. METHOD REFERENCES ACUTE ORAL TOXICITY STUDY IN RATS Code of Federal Regulation., Volume 16, Part 1500.30



## 000045

# I Page 40 

### I 

# CONFIDENTIAL

00004:6Page 41 

## CONFIDENTIAL 

# .CONFIDENTIAL 

# I REVERSE

# ~ION·**~SAY II~·** **~EsJ** USING SALMONELLA IYPHIMYRIUM 

## AND ESCHERICHIA 

## COLI PROJECT NUMBER 656/2

Date Started: 2 March 1994 Experimental Procedures: Date Completed: 21 March 1994 

# CONFIDENTIAL

STUDY SpoNSOR: 

# Issum BY: Safephar. Laboratories L1a1ted 

## P.O. Box 

## No. 45 DERBY DEl 

## 2BT 

## U.K. Telephone: DERBY (0332) 792896 Facsi.ile: (0332) 799018 PAGE 1 OF 33 PAGES Telex: 377079 SAFMI G 

## 00001:'(

### -2­ Page 42



### . PROJECT HUM8ER: 656/2 • 

## QUALITY ASSURANCE REPORT



## The routine inspection of short term studies at Safepharm laboratories is carried out as a continuous process designed to encompass all major phases of each study type once per month. Dates of the most recently completed series of monthly inspections relevant to the study type(s) in th;s report are given below. Date(s} of Inspection and Reporting: 

# oa, 09, 10, 14, 21, 24 February 1994 This report has been audited by Safepharm Laboratories Quality Assurance Unit. It is 

## considered to be an accurate account of the data generated and of the procedures followed. Date of Report Audit: 31 March 1994



## J.R. Pateaan C. 8iol., M.I. 8iol. 

## FOR SAFEPHARM QUALITY ASSURANCE UNIT


## OAIE: 

# ....................................... 000048

### Page 43 

## PROJECT NUMBER: 656/2

GLP**C~PLlANCE** 

## STATEMENT
I, the undersigned, hereby declare that the objectives laid down in the protocol were achieved and as nothing occurred to adversely affect the quality or integrity of the study, I consider the data generated to be valid. This report fully and accurately reflects the procedures used and data generated. The work described was performed in compliance with the UK Principles of Good Laboratory Practice (The United Kingdom Compliance Programme, Department of Health 1989). These Principles are in accordance with GLP standards published by OECD in 1982; .and are in conformity with, and implement, the requirements of Directives 87/18/EEC and 88./320/EEC. These international standards are acceptable to the United States Environmental Protection Agency and Food and Drug Administration, and fulfil the requirements of 40 CFR Part 160, 40 CFR Part 792 and 21 CFR Part 58 (as 'amended); and to the Japanese Ministry of Agriculture, Forestry and Fisheries

(59 NohSan, Notification No. 3850, AgricultUral Production Bureau)
­ confirmed by an Arrangement between the Ministry and the UK Department of Health; the Japanese Ministry of Health and**W~lfare** 

## (Notification No. 313, Pharmaceutical Affairs Bureau
- as amended t 

## and Kanpogyo No. 39 Environmental Agency, Yakuhatsu No. 229); and the Japanese Ministry of International Trade and Industry (Chemical Substances Control Law t 

## Kanpogyo No. 39 Environmental Agency Kikyoku No. 85) •



# ........**~Sl>(.~=** 

# .............. DATE: .•••**l-~** 

## ..

# t\-r
....**~.~~';t.~:** 

### .•.... 

## P.W. Thompson H.N.C. Study Director for Safepharm Laboratories


## 00004:9

### -4­ Page 44 

## PROJECT NlItBER: 656/2 CONTENTS
SlII4ARY 5 

## INTRODUCTION 6 

## TEST MATERIAL 7 

## METHODS 7 1. 

## Tester Strains 7 

## 2. Preparation of Test and Control Materials 7 

### 3. 

## Microsomal Enzyme Fraction 8 

## 4. 

## S9 Mix and Agar 8 

## S. Test Procedure 9 a) Preliminary Toxicity Study 9 b) Mutation Study 9 

### 6. 

## Interpretation of Results 11 

## RESULTS a) Preliminary Toxicity Study 12 

## b) Mutation Study 12 

## CONCLUSION 13 

## ARCHIVES 13 

## REFERENCES 13 

## KEY TO TABLE OF TEST RESULTS 14 

## TABLES 1 Experilllent 1 - Without Metabolic Activation 15 2 Experiment 1 . With Metabolic Activation 16 3 Experiment 2 - Without Metabolic Activation 17 4 Experiment 2 - With Metabolic Activation 18 APPENDICES 19 I Report of Results of Reverse Mutation Assay in Bacteria 20 

## II Dose Response Curve 29 

## III GLP Certificate of Compliance 33


## 0000:)0

### Page 45 

## PROJECT N1.ttBER: 656/2 

# 1...----·-..... 1 

# CONFIDENTIAL REVERSE MUTATION ASSAY -AMES TESTUSING SALMONELLA TYPHIMURIYM AND ESCHERICHIA 

## COLI SUft1ARY 1. Salmonella tVDhimyr1um strains TA1535, TA1537, TAgS and TA100 and Escherichia coli strain WP2uvrA- were treated with Undiluted Reactint Blue X3LV by the Ames plate incorporation method at five dose levels, 1n triplicate both with and without the addition of a rat liver homogenate metabo11sing system at lOS in standard co-factors. This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, tIN·, 

## MOL and MAFF. It also meets the requirements of the DECO, EEC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity**~** 

## assay and was S to 5000**~g/plate** 

## in the first experiment. The experiment

# ~ was repeated on a separate day using different cultures of the bacterial

# ~ strains and fresh ch.-ical formulations. In this case the dose range of 

# #- '

u'_' mu •• 

### Iwas 

### 312.5 to 

### 5000 l'9/phte. 

## 2. The solvent (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. 3. All positive control chemicals produced marked increases in the numbers of revertant colonies, both with and without t'he IIItabol1sing ,systell. 4. 

# I '**Ica~sed** no reduction in the growth of the bacterial lawn at any dose level either with or without metabolic act1 vation.

## ~ -.. - -----]Was therefore tested up to the maximum recommended dose level of 5000 pg/plate.**5~** 

## No Significant increase in the numbers of revertant colonies was recorded 

## for a.y of the bacterial strains with any dose 

# 0:J' . .. -- 

# J**.~er** with or without .tabol1c activat on. 

# Ii . - ...**~was** 

## found to be non-mytagenic under the conditions of this test. 

## 000051Page 

### 46 

## PROJECT UBER: 656/2 

# I
.......--.....**~-·**

# ...... CONFIDENTIAL**··~-·** 

# ,I REVERSE 

## MUTATION ASSAy -AMES TESTUSING 

## SAlfQtElLA DPHIMURltII 

## AND ESCHERICHIA COLI 

## IKIROOllCTION This study was conducted .according to Safepharm Standard Method Number JMOL OIG and was designed to assess the mutagenic potential of the test material using a bacterial/microsome test systeM. The study was based on the in vitro technique described by Ames and his co·workers (1. 2. 3) and Garner et a] (4) in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella tYDh1mur1um to various concentrations of the test material. This method conforms to the guidelines for bacterial .utagenicity testing published by the major Japanese Regulatory Authorities including MITI. MHW. MOL and MAFF. This method also conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471. Method B14 in Commission Directive 92/69/EEC and the USA, EPA (TSCA) guidelines. These mutant strains of Salmonella are incapable of synthesising histidine and are. therefore, dependent for growth on an external source of this particular amino acid. When exposed to a mutagenic agent these bacteria may undergo a reverse mutation to histidine independent forms which are detected by their ability to grow on a histidine deficient ..diu.. Using various strains of this organisa, revertants aay be produced ifter exposure to a chemical mutagen which have arisen as a result of a base-pair substitution in the genetic material' or as a fr...·shift IUtation in which genetiC ..terial is either added, deleted or aiscoded. In order to aake the bacteria .ore sensitive to mutation by chemical and physical agents several additional traits have been introduced. These include a deletion through the excision repair gene (uvrB) which renders the organism incapable of DNA excision repair and a deep rough IUtation (rfa) which increases the permeability of the cell wall. In addition, a mutant strain of Escherichia coli (VP2uvrA·), which requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence was used to ca.pl...nt the Salmonella sirains. Since aany compounds do not exert a autagenic effect until they have been metabolised by enz,.. systems not available in the bacterial cell, the test material and the bacteria were also incubated in the presence of a liver .icrosomal fraction (S9) obtained from rats previously treattd with a compound known to induce an elevated level of these enzYll8s. 000052

### -7­ Page 47 

## PROJECT NlImER: 656/2 

# CONFIDENTIAL JEST MATERIAL Sponsor's identification 

# : I**>:~.** 

# I

_on .. _-­ 

## Batch number Purity Date Received 28 February 1994 Description dark purple viscous _liquid Container clear glass jar Storage conditions room temperature Data relating to the identity, purity and stability of the test .ateria1 are the responsibility of the sponsor. "ETHODS 

## 1. Jester Strains Salmonella tYDhimyriym TAIS3S, TAIS37, TA9a and TAI00 Escherichia coli WP2uvrAThese strains were obtained from the British Industrial Biological Research Association on 14th August 19a7 and were stored at ·196-C in a Statebourne liquid nitrogen freezer, .adel SXR 34. Prior to being used, characterisation checks were carried out to deter.ine the amino-acid requirement, presence of rfa, R factors, uvr MUtation and. the spontaneous reversion rate. In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Liaited) and incubated at 37-C for 10 hours. 2. Preparation of Test Ind Control "ater111. 

# t . . --_.. 'was accurately weighed and dissolved in

dimethyl sulphoxide and appropriate dilutions aade. The concentration, homogeneity and stability of the test .aterial foc.ulations were not deter.ined by analysis. Negative and positfve controls were used fn parallel with the test .aterfal. Asolvent treatlent group was used as the negatfve control and the positfve control aaterfals were as follows: 000053Page 48 

### -8­ 

## PROJECT NUMBER: 656/2 • 

## METHODS (contd) 2. preparation of Jest and Control Materi.ls (contd) N-ethy1-N'-nitro-N-nitrosoguanidine (ENNG) 2**~g/plate** 

## for WP2uvrA-,

3 pg/p1ate for TAIOO .nd 5**~g/p1.te** 

## for TAI535 . 9·Aminoacridine (9AA) 80 pg/plate for TA1537 4-Nitroqu;noline-l-oxide (4NQO) 0.2 pg/p1ate for TA98 In addition the materials 2-Am1noanthracene (2AA) and benzo(a)pyrene (BP) 

## which are non-mutagenic in the absence of metabolising enzymes were used in the S9 series of plates at the following concentrations: 2AA at 2**~g/plate** 

## for TAI535 and 10 pg/pl.te for WP2uvrABP at 5pg/plate for TAIOO, TA1537 and TA98 3. 

## M1crosOlil EnzyPe Fr.ct1on Lot No. Aro. S9/03/02/94, prep.red on 03/02/94, .nd Aro. S9/03/03/94, prepared on 03/03/94, were obt.ined from the British Industri.1 Biological Rese.rch Association on 22/02/94 and 15/03/94 respectively. They were prepared from the livers of male Sprague-Dawley rats weighing

- 200g. These had received. single i.p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 prepar.tton. The S9 was stored at -196·C 1n a St.tebourne liqu1d nitrogen freezer, IOde1 SXR 34. 4. 

## S9 M1x .nd Agar The S9 Mix was prepared at 4·C as follows: S9 5.0 .1 

## 1.65" KC1/0.4 " MgC12 1.0 .1 

## 0.1 " G1ucose-6-phosphate 2.5 .1 

## 0.1 " HADP 

## 2.0 .1 

## 0.2 " Sodium phosphate buffer (pH 7.4) 25.0 .1 

## Sterile distilled w.ter 14.5 .1 

## 000054\ 

### -9­ Page 49 

## PROJECT fINER: 656/2 MEIHOOS (contd) 4. 59 Mfx 

## and Agar (contd) Top agar was prepared using 0.6S Difco Bacto agar (lot number 23050 12/97) and O.SS sodium chloride. For the Salmonella strains 5 ml of 1.0 

## mM histidine/l.O 

## mM biotin solution was added to each 100 ml of top agar and for the Escherichia col i 

## strain 5 m1 ·of 1.0 mM 

## tryptophan was added to each 100 m1 of top agar. Base agar plates were prepared using**l.~** 

## Oxoid Agar Technical No.3 (lot number B279 73194 9/98) with Voge1­ Bonner Medium Eand 20 mg/m1 D-glucose. 

# s. Test procedure a) preliminary Toxicity Study In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxiCity of the test material to the tester organisms. 0.1 m1 of bacterial suspension (TAIOO or WP2uvrA-), _2 m1 of molten, trace histidine or tryptophan supplemented media (histidine/biotin or tryptophan and top agar). 0.1 m1 of test solution and 0.5 m1 of phosphate buffer were overlayed onto sterile plates of Vogel-Bonner minimal agar (30 ml/p1ate). Five doses Of. the test material and a solvent control (dimethyl su1phoxide) were tested in duplicate. After 48 hours incubation at 37·C the plates were scored for revertant colonies and examined for a thinning of the background lawn. b) "utation Study 

## EXPERIMENT 1 (RAHGE·FINDING STUDY) Five concentrations of the test material were assayed in triplicate against each tester strain using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria. JestMaterial 

# Ind Negatiye Controls 0.1 m1 a1iquots of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 

## .1 of molten trace histidine (or tryptophan in the case of WP2uvrA-) supplemented top agar at 4S·C. These sets comprised two test tubes for each bacterial tester strain. 0.1.1 of the appropriately diluted test material or negative control solution was also added to each of the 

## 000055

### -10­ Page 50 

## PROJECT NUMBER: 656/2 METHODS (contd) 5. 

## Test Procedure (contd) b) Mutation Study (contd) Jest Materl11 and Negatiye Controls (contd) two tubes, followed by either 

## 0.5 .1 of the S9 ltver microsOie lix or 0.5 

## ml of pH 7.4 buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material. Positlye Controls 1) Without Actiyation 0.1 

## .1 of one of the positive control solutions (ENNG, 9AA or 4NQO) WIS Idded to I test tube contlining 2.0 

## .1 of .alten, trice histidine or tryptophan supplemented top Iglr Ind 0.1 .1 

## of the appropriate bacterial suspension. Finilly, 0.5 

## .1 of pH 7.4 buffer was added to the tube, the contents mixed and poured onto aglr plates. This procedure WIS then repeated in triplicate, for each of the posttive controls. 11) With ActlVltion 

## 0.1 .1 of 2AA 

## or BP solution WIS Idded to I test tube contain1ng 2.0 

## .1 of 101ten. trace histid1ne or tryptophan supplemented top aglr Ind 0.1 

## .1 of one of the test blcter111 suspens10ns. F1nllly. 

## 0.5 .1 of S9 

# .,x WIS Idded to the test tube, the contents .1xed Ind poured onto In Iglr pllte. Th1s procedure WIS then repelted. 1n tripliclte, for elch tester strlin. The plltes were lncublted It 37-C for 48 hours Ind the number of revertlnt colon1es counted. EXPERIMENT 2 

## (MAIN STUDY) The second .experlment WIS performed as descr1bed for Exper1 ..nt 1 using fresh blcterlal cultures, test .Iterlal and control solutions In triplicate. 000056

### Page 51 

## ·11· 

## PROJECT NUMBER: 656/2 • 

## METHODS (contd) 6. Interpretation of Results For a substance to be considered positive in this test system, it should have induced a dose·related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the Sg microsomal**en~ymes** 

## in both experiments at sub·toxic dose levels. If the two experiments give conflicting results then a third experiment may be used to confirm the correct response. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity or solubility or up to the maximum recommended dose of 5000**~g/plate.** 

## In this case the limiting factor was the maximum recommended dose.

00005'7

### Page 52 

## -12­ PROJECT HUMBER: 656/2 RESULTS 

# CONFIDENTIAL a) preliminarY Toxicity Study The dose 

### rang.

# 01 iUSed in the 

### prelillinary 

## toxicity study was 0, 312.5, 625, 1250, 

# 200 and 5000 pg/plate. 

# I twas non-toxic in the strains of bacteria

used (TAIOO and WP2uvrA-). The mean numbers of revertant colonies for the tox;c;ty assay were: Dose (pg/plate) Strain 

# o . 312.5 625 1250 2500 5000 TAl00 103.0 111.0 113.5 103.5 96.0 116.5 WP2uvrA- 29.0 36.5 31.0 31.5 28.0 31.0 b) Mutation Study The results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be 

## satisfactory. Individual plate counts together with the mean number of revertant colonies obtained for each tester strain following incubation with the test material, with and without letabolic activation, are shown in Tables 1 and 2 for Experiment 1 and Tables 3 and 4 for Experiment 2. The results are also expressed graphically as Appendix II. The results for positive controls used in this assay are shown in Tables 1 to 4. Inforllation regarding the equ1piltnt and lethods used in these experiments and the details of the personnel involved, as required by the Japanese MITI and MHV, are given as Appendix I. No toxicity was exhibited to any of the strains of bacteria used. No significant increases in the**nu~rs** 

## of revertant colon1ls of bacteria were recorded for any of the strains of bacteria used, at any dose level, either with or without ..taboltc activation. 000058

### Page 53 

## -13­ PROJECT NUMBER: 656/2
RESULTS (contd) b) Mutation Study (contd) The pos;tive control substances all produced .arked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory. 

# CONFfDENTIAL CONCLUSION The test material, 

# 1.

_______________ 

# 1

was found to be MD..:. 

## mutagenic under the conditions of this test. ARCHIVES Unless instructed otherwise by the sponsor, all original data and a copy of the f;na1 report will be retained in the archives of Safepharm laboratories for a period of ten years. After this period, the sponsor's instructions wi 11 be sought. REFERENCES 1. Ames, B.N., Durston, W.E., Yamasaki, E., and Lee, F.D. Proc. Nat. Acad. Sci. USA (1970), 

## 12, 2285 2. Ames, B.N., McCann, 

## J. and Yamasaki, E., Mutation Research (1975), 

## 11, 347. 3. McCann, 

## J., Coi, E., Yamasaki, E., and Ames, B.N. Proc. Nat. Acad. Sci. USA (1975) 

## li, 5135. 4. Garner, R.C., Miller, E.C., and Miller, J.A. Cancer Res. (1972), 

## Jl, 2058.



## 000059Page 54 

## -14­ 

## PROJECT NUMBER: 656/2 •


## KEY TO TABLE OF TEST RESULTS
NOTES; 1. When bacterial growth inhibition is found, the applicable value is marked with an asterisk. 2. The average number of colonies for each concentration is recorded in parenthesis. 3. wNumber of revertants- - The observed values and average value are shown in order, beginning with the lowest concentration of the test substance.



## - 4. The following postfixes are used when required:­ C • contaminated P • precipitate 

# x • plate unscorabl. ENNG • N-ethyl-N'-n1tro-N-n1trosoguan1d1ne 4NQO • 4-N1troqu1nol1ne-l-oxide 9AA • 9-Aminoacr1d1ne 2AA • 2·Aminoanthracene BP • Benzo(a)pyrene


## 000060

### Page 55 

### PROJECT NlJ4BER: 656/2 TAB 

## LEI
TABLE OF TEST RESULTS: EXPERIMENT ,I 

# CONFIDENTIAL NAME OF TEST SUBSTANCE: (**~**

..........a ............................................................. ••• 

## ................... 

# ....

# ---­**N~8ER** OF**~EVERTANTS** (nulber of colon;••/pllt.) WITH (+) OR TEST SUBSTANCE IASE-'AIR SUiSTITUTION TYf'E FRA"ESHlFT TY'E WITHOUT (-) CONCENTRATION TA100 TA1535 1I,2uvrA- TAM TA1537 S9 "IX (USI/pl.t.)

••

__e

____s. 

# ----_ •.. 

# -- - 

# - 

# ---. 142 13 32 15 12 0 124 ( 139.3 ) 14 ( 14.3 ) 21 ( 30.7 ) 15 ( 16.3 ) 11 ( 12.0 ) 152 16 32 13 

# l'
126 34 16 10 

# l' 1.0 129 ( 125.0 ) 15 ( 16.7 ) 35 ( 34.0 ) 14 ( 15.0 ) 9 ( 10.0 ) 120 16 33 15 11 167 12 21 14 12 135 ( 144.0 ) 15 ( 14.3 ) 42 ( 32.7 ) 15 ( 13.7 ) 12 ( 11.0 ) 130 16 21 12 9 127 12 35 13 7 200 117 ( 125.0 ) 15 ( 14.3 ) 24 ( 29.7 ) 15 ( 12.7 ) 10 7.3 ) 131 16 30 10 5 126 11**~** 1" I 1000 134 ( 132.3 ) 12 ( 14.3 ) 30 ( 21.0 ) 

# , ( 12.3 ) 10 1.0 ) 

# , 137 20**~** 6 131 7 29 13 11 5000 110 ( 121.0 ) 10 ( '.7 ) 31 ( 29.0 ) 13 ( 13.7 ) 15 ( 12.7 ) 122 12 27 15 12 

# --- __ i 

# .. an NME ENN& 

## 'AA I'OSITIVE CONTROL CONCENTRATION 3.0 5.0 2.0 0.2 10.0

NOT (ug/pl.t.) REQUIRING

59 'UX' NUfllER OF 429 230 760 195 464 COLONIES"LATt 445 ( 447.3 ) 172 ( 191.7 ) 558 ( 622.0 ) 195 ( 190.3 ) 511 ( 555.7 ) 461 1M S4I 181 615 

# .­ ODOOG1

### -16­ Page S6 

## PROJECT NUMBER: 656/2 TAB L E 2 TABLE OF TEST RESULTS: EXPERIMENT 1
. (contd) 

# CONFIDENTIAl

' •• 8

__•••

___

# !'!:.~.!.~~~.~~:.:~~ 

# ..."is" 

# ,,,,,,1.: ...____.. EilE'Z""'i1'S' NUftlER OF REVERTANTS**(nu~r** of coLonies/pLate) WITH (+) OR TEST SUBSTANCE IASE-'AIR SUBSTITUTION TYPE FRAMSHIFT TY'E WITHOUT C-) CONCENTRATION TA100 TA1535 W'ZwrA- TA" TA1537 S9 "IX Cug/pLatt) 

# -.............------....... p' 

# - • • 

## .................. 

# ... 155 12 39 26 10 + 0 123 ( 130.3 ) 25 ( 17.3 ) 37 ( 36.0 ) ·29 ( 21.7 ) 8 ( 11.0 ) 113 15 32 31 15 112 16 32 26 15 + 8.0 145 ( 121.7 ) 15 ( 16.7 ) 25 ( 29.0 ) 24 ( 25.3 ) 13 ( 12.7 ) 129 19 30 26 10 133 19 35 21 13 + 40 127 ( 127.0 ) 11 ( 19.3 ) 34 ( 37.0 ) 25 ( 21.3 ) 15 ( 13.7 ) 121 22 42 17 13 128 9 29 26 12 + 200 100 ( 110.7 ) 11.0 ) 42 ( 30.7 ) 26 ( 25.3 ) 13 ( 11.3 ) 

# l' ( 104 21 24 9 

# •

. 120 10 24 22 11 + 1000 115 ( 11'.3 ) 9 ( 10.0 ) 25 ( 21.7 ) 30 ( 25.1 ) 13 ( 13.0 ) 101 . 11 22 25 15 135 13 21 35 10 + 5000 122 ( 129.0 ) 13 ( 12.3 ) 16 ( 22.7 ) 31 ( D.3 ) 15 ( 13.3 ) 130 11 29 34 15


# _ .. • 

### • '

# . 's 

## •••• • NAIll II' 2M II' II' POSITIVE CONTROL CONCEIfTRATlON 5.0 2.0 '0.0 5.0 5.0 REQUIRING (loll/pLatt)

59 lUX NUMBER OF 349 164 264 76 + COLONIES/PLATI 40'1 ( _.0 ) 167 ( 162.0 ) 

# - 2CD ( 240.3 ) 

## m ( 246.7 ) 

# " ( 91.3 ) 

# ", 1S5 236 243
••a 

## •••••• 

# " 000062

### Page 57 

## PROJECT NUMBER: 656/2 • 

## TAB L E 3 TABLE OF TEST RESULTS: EXPERIMENT 2 

# CONFIDENTIAL

# ~!:.~~.~::~.;.~~:.~~:~:J 

# .......................iiiiiiiiiiiiiiiii:Jl••• ....................................aaa NUMBER Of REVERTANTS (number of colonies/pllt.) WITN (+) OR TEST SUBSTANCE BASE'PAIR SUBSTITUTION TYPI fRAMESNIFT TYPE WITHooT (.) CONCENTRATION TA100 TA1535 ..2INrA· TA98 TA1531 59 MIX (ut/pllt.)


### ........................................................................................................................ 155 22 24 19 13 0 158 ( 157.7 ) 19 ( 19.0 ) 21 ( 23.0 ) 25 ( 23.0 ) 15 ( 14.3 ) 160 16 24 25 15 136 19 32 30 13 312.5 145 ( 141.3 ) 22 ( 17.7 ) 26 ( 27.7 ) 1Z ( 20.3 , 16 ( 15.0 ) 164 12 25 19 16 156 21 22 '1 625 145 ( 150.3 , 24 C ZO.O ) 25 C 25.3 ) 24 ( 18.3 ) 12 

# " ( 11.7 ) 150 15 29 ZO 12 155 16 34 17 12 1250 141 ( 149.7 , 20 ( 20.0 , 34 ( 32.7 , 2Z ( 21.0 , 12 C 12.0 ) 14.6 24 30 Z4 12 160 ZO 30 19 10 2500 165 ( 154.7 , 21 ( ZO.7 , 29 ( 27.7 , ZO ( 21.0 ) 12 ( 12.0 ) 139 21 24 24 14 161 19 29 15 10 5000 154 ( 154.0 ) 17 C ZO.3 ) 29 ( 26.3 ) 13 17.7 , 

# " ( 12.0 ) 147 25 21 25 15


### ........................................................................................................................ lAME lING I- E_ 41K1O 9AA POSITIVI CONTROL CONCENTRAmll 3.0 5.0 Z.O 0.2 80.0

lOT (UI/plltt) REClUIRIIG

59 lUX

.....EIt 0' 527 229 539 139 521 COlONIESIPLATE 513 ( 511.0 , Z01 ( 211.3 ) 592 ( 569.0 , 1" ( 14.6.3 ) 524 ( 516.7 ) 493 225 576 156 498


### ........................................................................................................................ 

## 000063

### -18­ Page 58 

## PROJECT NUMBER: 656/2 TAB L E 4 

## TABLE OF TEST RESULTS: EXPERIMENT 2 (contd) 

# CONFIDENTIAL

# ~~ 

# ..**~~.~.~~~.~.~~;:~~~~.:.** 

# ..........................................1 ..: ....................................... NUMBER OF REVERTANTS (I'UItler of colonie./pllte, WIT" (+, OR TEST sueSTANCE lASE-PAIR SUBSTITUTION TYPE FRAMES"IFT TYPE WI1I1QJT (-) CONCENTRAT ION TA100 TA1535 IWZWrA- TA" JA1537 S9 MIX hIt/plate)


### ....................................................................................................................... 171 26 32 40 15 + 0 160 ( 156.7 ) 21 ( 23.0 ) 26 ( 30.0 ) 26 ( 33.3 ) 12 ( 13.0 ) 139 22 32 34 12 152 22 29 39 8 + 312.5 155 ( 155.0 ) 19 ( 20.0 ) 24 ( 25.7 ) 34 ( 31.3 ) 13 ( 12.7 ) 158 19 24 21 17 160 17 2S 30 11 + 625 152 ( 153.0 ) 16 ( 19.3 ) 35 ( 32.7 ) 31 ( 31.0 ) 7 9.7 ) 147 25 38 32 11 1" 17 29 20 15 + 1250 128 ( 139.3 ) 21 ( 20.0 ) 24 ( 26.5 ) 34 ( 29.0 ) 11 ( 13.7 ) 1" 22 t 33 15 155 17 30 2S 12 + 2500 129 ( 146.7 ) 17 ( 17.0 ) 20 ( '24.0 ) 24 ( 29.7 ) 16 ( 13.7 ) 156 17 22 40 13 145 15 32 34 8 

# • 5000 17'0 ( 158.3 ) 

## l' ( 19.7 ) 24 ( 21.0 ) 35 ( 35.7 ) 14 ( 11.0 ) 160 28 2S 38 t


### ....................................................................................................................... NAME IP 2M 2M lIP IP 

# . POSITIVE CONTROL CONCENTRATION 5.0 2.0 10.0 5.0 5.0 REClUIRING (""/plate)

59 MIX'

..... 0. 379 220 164 140 104 + COLONIESIPLATI 395 ( 375.3 ) 219 ( 201.3 ) 241 ( 206.0 ) 110 ( 165.7 ) 106 ( 91.0 ) 354 116 207 177 14


### .....................................................................

# -...**-.-------~-** 

# ..

# -....

# -.. 000064

### PROJECT NtlmER: 656/2 Page 59



## A P PEN DIe E S

REVERSE MAllON ASSAY -AMES TEST- REPORT



# •

000055Page 

### 60 

## A PPEN D I X 

## I -20­ 

## PROJECT NUMBER: 656/2 FOR M I REpoRT OF RESULTS OF REVERSE-MUTATION· ASSAY IN BACTERIA [1] 

## GENERAL ITEMS

Ha
.. of tn. new en..ielL substlne. (IUPAC no.eneLltur.) 

# CONFIDENTIAL

Otner n.ft 

# • , Nol.eullr weight**~renc.** It ordinlry te.peretur.**~rt** viscous Liquid Structurll forlUle or retiOnil forlUle (or outline of ..nufectur;ng ..thad, in cas. both er. unknown). PhysicocheilicaL properti.s of the 

## '*" cn..tcaL substence. SttibiLity Nllting point Ioiltng point YlIpoUr pr••,,,r. 'ertition coefficient SOlubility ·C >100·C <760
_ MIl ( ·C) SOLuble et 50 III'IL in di_thyL IULphox1de Purity of the new ene-ieaL substene. t.sted >991 

# hg". of SOLubility vate,. lIMO Acetone Other, ( ) "tuibl. "i,cibL. "lIcibL. ..... end concent"... tion of i'PU"iti., 

### wtX



### 1. 

## ·STABILITY-
- Fill in the stability for water, other solvents, heat, light, etc. 2. 

## ·VAPOUR PRESSURE-
- F111 in the vapour pressure of the test substance at 25·C. 3. ·PARTITION COEFFICIENT-
- Fill in the value, the teaperature used and the name of solvent used for the measurement. 4. ·SOLUBILITY- • Fill in such information as water soluble, soluble in oil. 5. ·DEGREE OF SOLUBILITY- Fill in the solubility at 25·C for each solvent. 

## 000066Page 61 

## PROJECT NUMBER: 656/2 A

## P PEN D I X I 

## (contd) [2] 

## TESTER STRAINS (1) 

## Procurellent ht. Obtained Obt.;ned f \"OIl l' AUGUST 1981 BRITISH INOUSTRIAL BIOLOGICAL RESEARCH ASSOCIATION 

### (2) 

## Storage Storage T.-per.tur. COI!PO.ition lIet.rial .u.pen.ion DMSO Other. ( ) 

### '.0 al


### [3] 

## S9 MIX (1) 

## Source of S9 (Encircle the appLi cable nullber Met fiU in the Nl.v."t tntri••) 1. Mad. in-hou•• (dolt.) SUppli.r BRITISH INOUSTRIAL BIOLOGICAL RESEARCH ASSOCIATION 

# G PureN•• 03/02/94 (r8ftg-f1nc1i....tudy) 03/03/94 (_in Itudy) (dolt.) PureMled on 22/02/94 (r
......fincli... Itudy) 15/03/94 (_in Itudy) (doIte) Lot liullber AI'O.**S9/03/~/94** (r......fincl1 ... Itudy) A

1'0. 19/03/03/94 (_in Itudy) 

## (2) Storage Temperature, etc. of S9 STATEIOURNE SXR 34 

## (3) Preparation of S9 .Uf purchlsed _terial, fill in.,... to ..tllnt pcMs1bLe) SpeCi.I, Strain Sex ani_l Used lAT, SPUGUI-DAIILIY MU Induci 

# -- Adainiltration Method... MOCUIIt 1254 SINILI I.'. INJECTION ...**~**
Age (in wNkS) 1 WEEKS Adainiatered 'eriod

..,., ....t (1/1eg weight> 5 DAYS 0.5 ,/kg Weight • ZODg 

## (4) Composition of S9 Mix constituents Maunt in 1 al 19 lIix Constituenu MaI.I'rt in 1 aL 19 Nix 19 0.1 .l NADfIM

- IaOl IIgClZ '.0 .,l IMDP 4.0 IaOl 6Lucole-6-Pholphat. Delwdroaena•• IeCl 

## n.o .,l**~a** buffer pH 7.4 100.0 IaOL ",.lucose-6-pholphata 5.0 .,l Othera ( ) 

# - 00006'1

### -22­ Page 62 

### PROJECT NlJIBER: 656/2 

## AP PEN D 

## I X I (contd) 

### [4] PREPARATION OF THE SOLUTION OF THE TEST SUBSTANCE Nethod of .u.pen.ion etc. when 

### te.t .ub.tence i. difficylt to di.lOlve SOlwnt u.td
Di ..

### thyl aulpnoxide


### [5] CONDITIONS OF PRE-CULTURE Ha.. of nutrient broth and ..

### nufacturer OIOID LTD., U.K.
- NUTRIENT BROTH 10 HOURS 

### [6] MINIMUM GLUCOSE AGAR PLATE MEDIUM (Encircle the ..,plicable I'IUIiber end 

### fill in the relevent entrie.) 

# G 1. 

### MMe ;n-hou.. Agel' 

# ..... NMufacture.. Lot .......1' OIOID TEtHNICAL No. J OXOID LTD., U.K.

127'9 731" 9/91 

### Volu.e of -ear plate -.diu. 

# I 30 

### .l Menufactyrer 2. 

### I'urCMle I'rtpllrtd on (dete) I'urcM.td on (dete) Lot nuIIIbtI' 

### [7] STERILITY TESTCEM1tcle the ..,pliAble reJpOftH in 1M 

### ""l1It Mnd coLum) 

### "'terial growth 

### othel' thin thoM UHd fo.- 

### te.t Te,t iublten,e IOlution Y ..

### (~ 59 Mia Y .. 

### [8] TEST METHOD



### .(1) Test _thod (EMir,Le the ...UcabLe I'IUIiber)
1. ' .....

### inc:ublltion a.thod 

## 000068 G'late ..thod

### Page 63 

### -23­ 

## PROJECT NUMBER: 656/2 A

# PPEN 0 I X I 

## (contd) • 

## [8] TEST METHOD (contd) (2) Test condition 're-incubltion ..thad 'lett ..thad &lcttriel .uspen.ion et 0.1 .l T

••t sub'tenct solution el 0.1 et COIIPOait ion Me-pho.phat. bufftr 59 lib (in cu. of ..tebolic activation ..thad) Top agar soluUon el el el 0.5 el 0.5 el 2.0 el Othe,.a ( ) 

# - ,,..-incubation T-.,.atu,.t 

### n .. ·C .1n. Ineation T-.,.atu,.e Ti.. ·C hou,.. ]7·C 101 hou,.. 

## [9] TEST RESULTS (1) Test results should be reported on the attached for. (2) Judge.ent of the results ,.,.1tive 

### '
.......' ,
.... ,.,....> 

# I c .....t1

# Ye)

...son fo,. 1udgeMnt: No .ignificent inc,.....**i~** the f,....,.,cy of ,.eve,.tent coloni.. va recorded fo,. eny beete,.iel at,.ein uaed with any dose of Undiluted ...etint Blue X3LV in two stPI,.ete e","i-.nta eithe,. with or without ..tabolie activation. NADPH vet not lidded to the Sf co-feetort, however, MADPM it geMr.t. in-attu by the ...ition of NAD' end e.c... 6-6-, end thit it eonsi.,.... to be
...tvalent to dtrect ...itt.. of MADPM. A/"OCLor 1254 WIlt used to induet Mp,. enzyee Levels tn the I'.t Live,. 19. lI"OCLor 1254 it eonst.,.... to be equlUy effective .. the

.... of the ...tv.lent COIIIbtnatton of phenobIarl>itaL;S;6-benJofLIMIM end
..thyldloLenth,...,.. 

## (3) Referential .atters
[REMARK] -Wet.Ua" -.tt.... - ('ill tn tM vi", etc:. of the Study Dtrector on the tett ,.esulU)



### [10] OTHERS 

## 0000159

Study Director

'.at Daua ..... Titl. 

# '.11. 1'hoIIpIon, ".N.C. froe OZ/OlI94 To Z1/0]/94 51 ..t"r.**,\""",~,..,..** '

## -24­ Page 64 

## PROJECT tU4BER: 656/2 

## AP

## PEN D I X I (contd)


## KEY TO TABLE OF TEST RESULTS

NOTES: 1. When bacterial growth inhibition is found, the applicable value is marked with an asterisk. 2. The average number of colonies for each concentration is recorded in parenthesis. 3. -Number of revertants· - The observed values and average value are shown in order, beginning with the lowest concRntration of the test substance. 4. The following postfixes are used when required:­ C• contaminated P • precipitate 

# x • plate unscorable ENNG • N-ethyl-N'-nitro-N-n1trosoguan1dtne 4NQO • 4-Nitroquinoline-l-oxtde

9AA • 9-Am1noacrtd1ne 2AA • 2-Am1noanthracene

BP • Benzo(a)pyrene 

## 0000'/0__________________________________________________________________________ _ 

### -25­ Page 65 

## PROJECT NlMBER: 656/2 A P PEN 0 I X I (contd) 

### IABLE 1 

## TABLE OF TEST RESULTS: EXPERIMENT 1 

# CONFIDENTIAL

# ~E~:"~~ ..

# ~.~~:.~.::L''''''D'Dii'''''''D.m'DmDDD=l .. ...................__...:_......__....**~BER** 0' REVERTANTS**(~r** of colonie./pllte) WITH (+) OR TEST SUBSTANCE lASE-PAIR SUBSTITUTION TYPE 'RA"ESHlFT TYPE WITHOUT (-) CONCENTRATION TA100 TA1535 W'ZuvrA- TA98 TA1537 59 "IX (ug/pllte) 142 13 12 15 12 

### o 124 (139.3) 14 ( 14.3) 21 ( 30.7) 15 ( 16.3) 11 ( 12.0) 152 16 12 19 13 126 19 34 16 10 1.0 129 (125.0) 15 ( 16.7) 35 ( 34.0) 14 ( 15.0) 9 ( 10.0) 120 16 !5 15 11
167 12 21 14 12 40 135 (144.0) 15 ( 14.3) 42 ( 52.7) 15 ( 13.7) 12 ( 11.0) 130 16 21 12 9 127 12 l5 13 7 200 117 (125.0) 15 ( 14.3) 24 ( 29.7) 15 ( 12.7) 10 ( 7.3) 131 30 10 5 

# l'
126 11 ." 19 I 1000 1" (152.3) 12 ( 14.3) 30 ( 21.0) 9 ( 12.3) 10 ( 1.0) 

# , 137 20 ." 9 131 7 29 13 11 5000 110 (121.0) 10 9.7 ) 31 ( 29.0) 13 ( 13.7) 15 12.7 ) 122 12 ." 15 12 

# _. eft ••••• 

### •••• 

### • 

# .....- •• 9AA POSITIVE CONTROL. CONCENTRATION 3.0 5.0 2.0 0.2 10.0

NOT (ug/pllte)**RE~IRI~** 59 lUX HUMER 01 COlONIES/PLATE



# . 429 445 

## '" (447.3) 230 172 

## 1" (191.7) 760 

### ssa 541 (622.0) 1" 1" 111 (190.3) 464 

### sea 615 (555.7) 

## 0000'71

### ••••• Page 66 

## APPENDIX I (contd) -26­ 

## PROJECT NlItBER: 656/2 

## TAB L E 2 

## TABLE OF TEST RESULTS: EXPERIMENT 1 (contd) 

# CONFIDENTIAL

e.

# ~~~.~:r~~La=_"_=,, 

# I

. 

# - .

# ...

# - ...... ••••**~BE_** Of _EVE_TANTS (nuIOer of colonitl/plett) WITH (+) OR TEST SUBSTANCE IASE-'AII SUBSTITUTION TYPE '_AltESMIn TYPE WITHOUT (-) CONCEHnATION TA100 TA15!5 W'2wrA- TA9I TA15!1 S9 ItIX (UI/plett)

____w •• 

# ..

# __..

# _. •• • 

# .-­ 

# - 155 12 39 26 10 + 

### a 123 ( 130.3 ) 25 ( 11.3 ) 37 ( 36.0 ) 29 ( 28.1 ) 

# • ( 11.0 ) 11! 15 32 31 15 112 16 32 26 15 + 1.0 145 ( 128.7 ) 15 ( 16.1 ) ZS ( 29.0 ) 24 ( 25.3 ) 13 ( 12.1 ) 129 19 30 26 10 133 19 35 28 13 + 127 ( 121.0 ) 11 ( 19.3 ) 34 ( 11.0 ) ZS ( 23.3 ) 15 ( 13.1 ) 121 22 42 11 13 128 9 29 26 12 + 20Q 100 ( 110.1 ) 16 ( 11.0 ) 42 ( 30.1 ) 26 ( 25.3 ) 13 ( 11.3 ) 104 21 24 9 

# • 120 10 24 22 11 + 1000 115 ( 114.3 ) 9 ( 10.0 ) ZS ( ZI.1 ) 30 ( 25.1 ) 13 ( 13.0 ) 101 11 22 ZS 15 135 13 ZI 35 10 + 5000 122 ( 129.0 ) 13 ( 12.3 ) 16 ( 22.1 ) 31 ( 33.3 ) 15 ( 13.3 ) 130 11 29 34 15
•••••• I ", w_ 

# ---.. ••••• 

# ., ., ., NMI 2M POSITIVE CONTIIOL CONCENTRATION 5.0 2.0 10.0 5.0 5.0 .EQUI_IIIIG (UI/plett)

19 MIX NUME_ 0' 349 164 212 264 16 + COLONIES/flUTE 40'1 ( ••0 ) 161 ( 162.0 ) 203 ( 240.3 ) 

## m ( 246.1 ) 99 ( 91.3 ) 414 155 236 243 99 

## 000072-Z/­ Page 67 

## PROJECT NUMBER: 656/2 

## A P P E N 0 I X I (contd) 

### I 

## A B L E 3 

## TABLE OF TEST RESULTS: EXPERIMENT 2 

# CONFIDENTIAL

# ~~.~!:a~~~~.~a~~;::~~~:II"""""ii".'i""""""Ii'"•••"••

# 1.
........................................... NUMBER OF REVERTANTS Cnu.btr of colonies/pllte) WITH (+) OR TEST SUBSTANCE IASE,PAIR SUBSTITUTION TYPE FRAMESHIFT TYPE WITHooT C·) CONCENTRATION TA100 TA1535 lI>2uvrA- TA9I TA1537 59 MIX (UCI/pllte)


### ................................................................................................................

# ......**~** 155 22 24 19 13 0 158 ( 157.7 ) 19 ( 19.0 ) 21 ( 23.0 ) 25 ( 23.0 ) 15 ( 14.3 ) 160 16 24 25 15 136 19 32 30 13 312.5 145 ( 148.3 ) 22 ( 17.7 ) 26 ( 27.7 ) 12 ( 20.3 ) 16 ( 15.0 ) 164 12 Z5 19 16 156 21 22 11 11 625 145 

### « 150.3 ) 24 C 20.0 ) 25 ( 25.3 ) 24 ( 11.3 ) 12 ( 11.7 ) 150 15 Z9 20 12 155 16 34 17 12 1250 148 ( 149.7 ) 20 C 20.0 ) 34 ( 32.7 ) 22 ( 21.0 ) 12 ( 12.0 ) 146 24 30 24 12 160 20 30 19 10 2500 165 C 154.7 ) 21 ( 20.7 ) Z9 ( 27.7 ) 20 C 21.0 ) 12 ( 12.0 ) 139 21 24 24 14 161 19 Z9 15 10 5000 154 ( 154.0 ) 17 ( 20.3 ) Z9 ( 26.3 ) 13 ( 17.7 ) 

# " ( 12.0·) 147 25 21 Z5 15


### ...................................................................................................................... IIAME DIIG EIIIIG EIIG 4IIQO 9AA POSITIVI COMTROL COMeENTUTION 3.0 5.0 2.0 0.2 10.0

lOT (UCI/pllte) REGUIRING

S9 MIX NUMBER OF 527 2Z9 539 139 521 COLONIES/PLATE 513 C511.0 ) 201 ( 211.3 ) 59Z C569.0 ) 

# '" C 146.3 ) 524 ( 516.7 ) 495 

## m 576
. 156 491



# _ .............

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

# __.......... 

# --_......

# _ ...

# _.-­ 000073

## -28­ Page 68 

## PROJECT NOOER: 656/2 

## A P P E N

# PI X I 

## (contd) TAB L E 4 

## TABLE OF TEST RESULTS: EXPERIMENT 2 (contd)

# ~~ ..

# ~!:.~~~~.~e::~~~!::'ii"i"iiiii"iiiiiii'iiiii"iili'iii..::.J. •.:...

# ~~ .............. NUMIEI OF IEVEITANTS (number of colonies/plate) IIITN (+) OR TEST SUBSTANCE IASE'PAII SUBSTITUTION TYPE FltAl4ESM IFT TYPE IIITNOOT (-) CONCENTIATION TA100 TA1535 WP2wrA- TA98 TA1537 S9 MIX (UI/plat.)

......................................................................................................................at



# .. 171 26 32 40 15 0 160 ( 156.7 ) 21 ( 23.0 ) 26 ( 30.0 ) 26 ( 33.3 ) 12 ( 13.0 ) 13' 22 32 54 12



# .. 152 22 29 39 I 312.5 155 ( 155.0 ) 20.0 ) 24 ( 25.7 ) 54 ( 31.3 ) 13 ( 12.7 ) 

# l' ( 151 19 24 21 17 160 17 25 30 11 + 625 152 ( 153.0 ) 16 ( 19.3 ) 35 ( 32.7 ) 31 ( 31.0 ) 7 ( '.7 ) 147 25 31 32 11 145 17 29 20 15 + 1250 121 ( 139.3 ) 21 ( 20.0 ) 24 ( 26.5 ) 54 ( 29.0 ) 11 ( 13.7 ) 145 22 C 33 15 155 17 30 25 12 + 2500 129 ( 146.7 ) 17 ( 17.0 ) 20 ( 24.0 ) 24 ( 29.7 ) 16 ( 13.7 ) 156 17 2Z 40 13 145 15 32 34 I


# .. 5000 170 ( 151.3 ) 16 ( ".7 ) 24 ( 27.0 ) 35 ( 35.7 ) 14 ( 11.0 ) 160 21 25 31 

### c


### ....................................................................................................................... N_ IP 2M 2M IP IP POSITIVI CONTROL CONCENTRATION 5.0 2.0 10.0 5.0 5.0 IEGUIIING (ug/plaU)

59 

### .nx IIUMIEI OF 379 220 140 104


# .. '" ( COlONIES/PLATE 393 ( 375.3 ) 219 ( 201.3 ) 241 ( 204.0 ) 110 ( 165.7 ) 106 91.0 ) 3" 1. 207 177 14 

# -_......

# __._.

# __..

# _----------- •••• 

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

# .... 0000·74"0 I» OQ (I) \.Q 

# CONFIDENTIAL 0'1



### 160 

# I )lOSE-RESPONSE CURVE**~** 

### 100**~** 

### a.

........ 

### en 

### 80

# ~**~ ~**  

### 60


### 40+ 

# 20+ 0 

### 0 0 

### 0 .",.1 C,li 

### I 

# o.

0 8 .... TA100 -$9 EXPERIMENt 1

..... lA1535 -59 

### [XPERIMENT 1 I N 

## -e- WP2uvrA- - S9 EXPERIMENT 1

# I \D 

# • 

# -<>- lA98 -S9 

### EXPERIMENT 1

--- TA1537 -59 

### [XPERIMENT 1

# ~ ,." n


## ..... 

# i to ,." 

## • 

# .. :a a. 

### .0 200 1000 

### 5000 CII 

### UG/PLAT[

### ~ N

# ------------ III '"d 1)1' (JQ

...... o 

# CONFIDENTIAL
.

120
100+

&.oJ 

# 5 Go 

### 80 CA 

# " 

# § a:::

## ~'60 a::: 

### 40**~** 

### 20f 0 0 

### 0 0**~J** 

# v'J I 00 

# I ' 'loOSE-RESPONSE CURVE

n 

# - 0



# -**~** 

# .... 0.



### .... TA100 +59 EXPERIMENT 1

.... TA1535 +S9 EXPERIMENT 1 I



### ... "2uvrA- +59 EXPERIMENT 1

# I 0 W 

## • 

# -<>- lA98 

## +S9 EXPERIMENT 1
...- TA1537 

### t 59 EXPERIMENT 1

."

# ~



# .. • 

# ,., n 

### A 

# - __ 

# .... 

# i ,., :;a 

# .. 8 

### 40 eft 

### 200 1000 

### SOOO (II eft • 

### UG/PLA1£
......... N'"d, I'll ()Q

...... 

# CONFIDENTIAL R> t-' 

# r IDOSE-RESPONSE CURVE 180 

### 1
140 

### 120 

# 5 

# i**~1**

# ~
40+ 

### 20'" 0 

## I 0 0 0 

### 0 0

...] 

### <oJ 

## 8 n 

# - s: 

# -

..... lAl00 -59 EXPERIMENT 2
-.- lA15J5 -S9 EXPERIMENT 2

.... WP2uwA- - 59 EXPERIMENT 2 lA98 -59 EXPERIMENT 2 -e- 

### lA15J7 -59 EXPERIMENT 2

i:j 

### S1 :Q 

### -0. JI. 

### At.
312.5 

### 625 1250 2500 

### 5000 UG/PlA1E 

# - w 

# • •

."

# ~ n**~** -4 

# I ,.., :xl 

# .. eft**~** 

### ....... N•• "d Ill' OQ --.J 

# CONFIDENTIAL (I) N


### 180­ 160 140

# I 120+



# ..... 

# 5 

# i 100 

## Ii 80

# ~**~** 

### 60 

# 401 0 

# 1 0 0 

### 0 ",J 

### 0 

### 00 0 

# I**~OSE** -RESPONSE 

## CURVE

-II- TAl00 

## +S9 EXPERIMENT 2

..... TA1535 +S9 EXPERlUENT 2

..... WP2uwA- 

## +S9 EXPERIMENT 2

# I -<>- TA98 +59 EXPERIMENT 2 

## • -.- TA1537 +S9 EXPERlUENT 2



# - • n 

# - 0 ::::I 

# ,. A. 

# -

I W N 

## • "

# ~ ,." n -4 

# ! :t 

# !**~** 

# ! I ,." ::Ia 312.5 625 

### 1250 2500 5000 eft 

### UG/PlATE**~.** N

### • 

### • 

### . ' 

## . 

# .
. -33­ Page 73 

### PROJECT NlIIBER:**65~/2** 

## A P PEN 

## 0 I X I I I**~** R T 

### At**~** 

# 41., 'J,. Q .'. ..1 

## o .,' _.'". .:t­

..,r:. -<. 

### liE,..'"


## THE DEPARTMENT OF HEALTIf OF 1HEGOVERNMENT OF 1HE UNITED KINGDOM GOOD LABORATORY PRACI1CE 

### STATEMENT OF COMPlJANCE IN ACCORDANCE WlI1I DIRECl1VE 811320 EEC LABORATORY 

## SafePhar. Laboratories Li.ited POBox No 45 Derby DEl 2BT

DATE 

### OF INSPECI10N 31 

## January 1994 A aenera) inspecdon for compliance 

## wi. the Principles of Good 

### Laboratory Practice 

### was earned out at the above labol'litory u 

### pan ofthe UK GLP Compliance PIOIftID.lllC. At the time ofthe inspecdon no deYiad.ou were 

### found ofsufracient Dlapitude to 

### affect the validity ofstudies performed at these facilities.

# I Page 74 

### I 

# CONFIDENTIAl.

000030**Pag~** 75 I,.;ovance l.aboratorjls inC. 0.0. Box 7545 Madison. WI 53707-7545 Deliveries: 3301 Kinsman Blvd•• Madison. WI 53704 Titl: 6081241 -4471 Fax: 608/241-7227

# cov~ Sponsor: 

# I 

# ) CONFIDENTIAL
FINAL REPORT 

# lW.N301lJOJ StudyTitle: 

### Acute Oral 

### Toxicity Study

## oli.i~~ 

# ..............___.... 

# }n Rats 

## (EPA/otcn

# Guiaelines) Author: 

### StevenM. Glaza Study Completion Date: 

### March 17, 1998 . PerformiDILaboratory: 

### Covance Laboratories Ine. 3301 Kinsman Boulevard Madison, Wisconsin 53704 LaboratoryProjectIdentification: 

### Covance 71102252 Page 1of38



## 000081

### Page 76

Covance 71102252 

### COMPLIANCE STATEMENT 

# CONFIDENTIAl Acute Oral Toxicity Study oct lin Rats**(EPAJO~~C~D~G·u~la~e~ii~n~es~)------~~** 

### This study was conducted in accordance with the following Good Laboratory Practice Standards: United States Environmental Protection Agency TSCA: Good Laboratory Practice Standards, 40 CFR 792 Organisation for Economic Cooperation and Development Principles of Good Laboratory Practice, C{81)30{Final) Japanese 

### Ministry ofHealth and Welfare, Ordinance 21 Japanese 

### Ministry ofIntemalTrade and Industry Good Laboratory Practice Standards, NotificationNo. 59-85



### Date

Date



### -2­ 

## 000082Page 77

Covance 71102252 

### QUALITY ASSURANCE STATEMENT This report has been reviewed by the Quality Assurance Unit ofCovance Laboratories Inc., in accordance with the Environmental Protection Agency (EPA) Good Laboratory Practice Standards, 40 CFR 792 and the Organisation for Economic Cooperation and the Development (OECD) Principles ofGood Laboratory Practice, C(81)30(Final) and the Japanese Ministry ofHealth and Welfare (MOHW), Good Laboratory Practice Standards, Ordinance 

### 21 and the Japanese Ministry ofInternational Trade and Industry (MIT!) Good Laboratory Practice Standards, Kikyoku Notification No. 85. The following inspections were conducted and findings reported to the study director and study director management. Written status reports ofinspections and findings are issued to Covance management according to standard operating procedures. Inspection Date Reported to Dates Study Directorand From To Phase Study DirectorManagement 12/07/97 12/07/97 

### Protocol Review 12108/97 12/09/97 12109197 

### Dose Preparation 12/09/97 03/02/98 03/02198 

### Data/Report Review 03/02198



## 3-/]-,,?f Representative, Quality Assurance Unit 

### Date



### -3­ 

## 000083

### Page 78

Covance 71102252 

### STUDY IDENTIFICATION 

# CONFIDENTIAL Acute Oral Toxicity Study of lin Rats (EP

## AJO~t"C"i5p!1G~ui""d"ei"in·elllfs)----.Jj Test Material Sponsor
Sponsor's Representative Study Director
Testing Facility Study Timetable

Study Initiation Date Experimental (In-life) StartDate In-life End Date Experimental Tennination Date Study Completion Date I



### Steven M. Glaza CovanceLaboratories Inc. Telephone 608.241.7292 

### Facsimile 608.242.7936 

### Covance Laboratories Inc. 3301 

### Kinsman Boulevard Madison, WI 53704 

### December 8, 1997 

### December9, 1997 

### January 30, 1998 

### March 17, 1998 

### March 17, 1998

-4­Page- 79

Covance 71102252


### Acute Studies 

### Steven M. Glaza Study Director Manager Steven R. Sorenson Study Coordinator Jeffrey B. Hicks In-life Supervisor Rose M. Bridge Administrative Supervisor Toxicology Support 

### Kathleen R. 

### Myers Manager QualityAssurance 

### Nancy M. Centanni Manager KEY PERSONNEL LaboratoryAnimal Medicine 

### DonnaJ. Clemons, DVM Diplomate, ACLAM Supervisor Anatomical Pathology 

### Thomas E. Palmer, PhD Anatomical Pathologist Deborah L. 

### PirkeY Laurie J. Schuller Supervisors Nea:opsy



## 000085

### Page 80

Covance 7\\02252 

### CONTENTS
COMPLIANCE STATEMENT...........................................................................................2 QUALITY ASSURANCE STATEMENT ..........................................................................3 

### STIJDY IDENTIFICATION ...............................................................................................4 

### KEY PERSONNEL .............................................................................................................5 

### SUMMARy.........................................................................................................................8 OBJECTIVE .........................................................................................................................9 TEST MATERIAL ..............................................................................................................9 Identification..... t •••••••••••••••••••••••••••••a' .......................................a ••••••••••••••••••••••"' ....................

### 9 

### Purity and Stability ..........................................................................................................9

••••••••••••••••••••••••• t ........................ 

### Storage mdRetention......................................................t 

### 9 

### Safety PrecautioM...............,'" ••••• a ••••••• II I , ............

### 1 0 II •••••••• 11 •••••t "
................... ••••••••••••••••••••• 

### TEST SYSTEM ............................................................. ................................................... 1 0 Test Animal
..........................................................................,.........................................

### 1 0 

### Housing............................................................................................................................. 1 0 Animal Diet....................................................................................................................10 Animal 

### Selection and Grouping ....................................................................................1 0 Justification for Species Selection .................................................................................11 PROCEDURES .................................................................................................................11 Preparation and 

### AdministrationofTest Material .......................................................... 11 Reason for Route ofAdministration.............................................................................. 11 Observations ..................................................................................................................11 Pathology ....................................................................................................................... 12 Statistical

### Evaluation .....................................................................................................12 Location ofRaw

### Data, Records, and Final Report........................................................12 RESULTS ............................................................................................................,..,....,..,.. 12



### .Mortality ........................................................................................................................12

Body Weight.s .................................................................................... 1 ..................................

### 12 

# 000086 -6­Page 81
Covance 71102252 

### Clinical Signs................................,................................................................................. 13 Pathology ....................................................................................................................... 13 DISCUSSION.................................................................................................................... 13 SIGNATURE.....................................................................................................................14 

### REFERENCES .................................................................................................................. 

### 15 PATHOLOGY REPORT...................................................................................................16 

### TABLE 1 Mortality Summary................................................................................................... 17 2 Individual and Mean Body WeightsIBody Weight Gains (g)................................... 18 3 Individual Clinical Signs ..........................................................................................21 4 Individual PathologyComments ..............................................................................25 APPENDIX........................................................................................................................29 Protocol TP2069 ............................................................................................................30



## 00008',Page 82

Covance 71102252 

### SUMMARY 

# CONFIDENTIAL The test materiall ' 

# ,I was evaluated for its acute oral toxicity potential in rats when administered as a single gavage dose in the definitive study at levels of

### 5,000 mg/kg for males and 

### 2,000,3,000, and 

### 5,000 mg/kg for females. No mortality was observed in the five females treated at 

### 2,000 mg/kg, Clinical signs observed at the 

### 2,000 mg/kg dose level included red-stained face; liquid, blue, and/or nonformed feces; and blue-stained urogenital area, haircoat, and/or tail. All females returned to a normal appearance by Day 13 after treatment with the exception oftwo animals which exhibited a blue-stained tail through study termination. All animals treated at

### 2,000 mg/kg exhibited body weight gain during the study. No mortality was observed in the five females treated at 

### 3,000 mg/kg, Clinical signs observed atthe 

### 3,000 mg/kg dose level includedblue feces, haircoat, and tail and bluestained urogenital 

### area. All animalstreated at 

### 3,000 mg/kg exhibited body weight gain during the study. Four females treated at 

### 5,000 mg/kg died within4 days oftest material administration. Clinical signs oftoxicity observed at the 

### 5,000 mg/kg dose level includedthin appearance, hunched posture, hypoactivity, red-stained face, nonformed and/or blue feces, wet orblue-stainedurogenital area, green urine, green-stained haircoat and/or tail, and death. All surviving animalsreturned to a normal appearance by Day 

### 10 after treatment withthe exceptionof

### three males whichexhibited a green-stained tail through study termination and the remainingfemale which alsoexhibited a green-stained haircoat and tail throughto studytermination, Animals treated at 

### 5,000 mg/kg and surviving to the endofthe observationperiodexhibited body weight gain during the study. At necropsy, oneormore organs ortissues in each animal were observedto 

### be stained blue or green, which were attributed to the test material. There were no test material-related lesions observed in anyofthe animals. Sasedonthe mortality observed 

### in the study, the estimated oral LD50 values inrats were determined tobe greaterthan 

### 5,000 mg/kg for males and 4,128 mg/kg for females. 

## 000088 - 8 ­Covance 71102252 

### OBJECTIVE 

### The objective ofthis study was to assess the acute oral toxicity produced when the test material is administered by the oral route (gavage) to rats.I.2,J,4 All procedures in this study were in compliance with the Animal Welfare Act Regulations. [n the opinion ofthe Sponsor and study director, the study did not unnecessarily duplicate any previous work. The dose levels, method, frequency, and duration ofadministration utilized in this study were chosenbased on the requirements of the regulatory test guidelines. All procedural times presented in this report fall within the acceptable ranges as specified in Covance Laboratories Inc. (Covance) Standard Operating Procedure (SOP).
TESTMATERIAL 

# CONFIDENTIAl Identification 

### The test material was identified

# ai,,~

# ....______...._...... J:n.d described as a viscous, dark blue liquid. PurityandStability 

### The Sponsorassumes responsibility for purity and stability**d~terminations** 

### (including undertest conditions). Storage andRetention 

### Thetest material was 

### stored at room temperature. Areserve sample ofthe test material was 

### taken and will be

### retained ina freezer setto maintain a temperature of-20°C ±

### 10° in accordance with Covance SOP. Any unused test material will be discarded after issuance ofthe final report according to Covance SOP.

-9- 

## 000089Page 84

Covance 71102252 

### Safety Precautions 

### The test material handling procedures were according to Covance SOPs and policies.
TEST SYSTEM TestAnimal 

### Young adult albino rats ofthe Crl:CD!>(SD)BR strain were procured from Charles River Laboratories, Inc., on October 14, 1997 (Raleigh, North Carolinafacility) and December 1and 15, 1997 (Portage, Michigan facility). Housing 

### Afterreceipt, the animals were acclimated for aperiod ofat least 7days. During acclimation and throughoutthe study, the animals were separated by sex and group housed in suspended, stainless steel cages. Environmental controls for the animal room were setto maintain atemperature of18° to 

### 26°C, arelative humidity of50% ±20%, and a 12·hourlightll2·hourdark

### cycle. In cases where variations from these conditions existed, they were docwnented and consideredto have'had no adverse effectonthe study outcome.
AnimalDiet 

### The animals were providedcontinuous access to Rodent Diet#8604 (HarlanTeklad) and waterexcept for 17 to 20 hours before test material administration when the diet, butnot water, was 

### withheld. The diet is routinely analyzed by the manufacturerfor nutritional components and environmental contaminants. Samples ofthe water are periodically analyzed.**Th~** 

### wereno known contaminants in the diet orwaterat levels that would be 

### expected to 

### interfere with or affect the results ofthe study.
Animal Selection and Grouping 

### Five male and 15 female healthy, acclimatedrats, weighing from 219 to 267 gand approximately 8to 14 weeks ofage, were assigned to three 

### treatment groups. Five males

- 10·**OOOO~in**Page 85
Covance 71102252 

### were treated at 5,000 mglkg. Five females per level were treated at 2,009. 3,000, and 5,000 mglkg. The animals were identified by M:imal number and corresponding ear tag throughout the study.
Justification for Species Selection 

### Historically, rats have been used as arepresentative ofarodent species and are preferred by various regulatory agencies.
PROCEDURES

.Preparationand Administration ofTestMaterial



### The undiluted test material was administered as a single gavage dose using an average bulkdensity detenninationof1.04 g/mL 

### to detenninethe dose volume for each dose level. An

### individual dose was 

### calculatedfor each animal based on its fasted body weight
Reason for RouteofAdministration 

### Historically, the oral route has 

### been one ofthe routes USed to assess the acute toxicity of various materials.
Observations 

### Clinical observations and mortality checks were conducted at 1,2.5,and 4hours aftertest material administration and daily thereafter for 14 days. Mortality checks were conducted twice a day (morning and afternoon) for 13 days aftertest material administration and againthe morning ofDay 14. Body weights were detenninedbefore test material administration (Day 0). Additional body weights were detennined at Day 7, at tennination ofthe respective in·life phase (Day 14), orat death when survival exceeded 1day.



### ·11· 

## 000091

### Page 86

Covance 71102252 

### Pathology 

### At termination ofthe respective in-life phase for each dose level, surviving animals were euthanized. All animals, whether found dead during the study oreuthanized, were subjected to an abbreviated gross necropsy examination and any abnonnalities were recorded. Afternecropsy, the animals were discarded and no tissues were saved as there were no grossly abnormal organs ortissues.
Statistical EvaluatioD 

### Using the mortality values observed during the study, the 

### LD50 values for females was determined by acomputerprogram using a modified Behrens-Reed-Muench cumulant method. 5 

### No other statistical evaluations were required by the protocol.
Location of

### Raw Data,Records, andFinalReport 

### The raw data, records, and an original signed copy ofthe fmal report will be 

### retained in the archives ofCovance in accordance with Covance SOP. 

## RESULTS Mortality 

### A summary ofthe observed mortality for the females along with the 

### LD,o values and 95% confidence limits for the females are in

### Table I. Fourfemales treated at 5,000 mglkg were found dead within4 days oftestmaterial administration. No other mortality was observed. Basedonthe mortality observed in

### the study, the estimated oral 

### LD,o values in rats were detenninedto be greaterthan 

### 5,000 mglkg for males and 4,128 mglkg for females. Body Weights 

### Individual and meanbody weights and body weight gains are in

### Table 2. Animals sUrviving to 

### the end ofthe observation period

### exhibited body weight gain during the study.

- 12
­ 

## 000092

### Page 87

Covance 71102252 

### Clinical Signs 

### Individual clinical signs are in Table 3. Clinical signs observed at the 2,000 mglkg dose level included red-stained face; liquid, blue, and/or nonformed feces; and blue-stained haircoat, tail, and/or urogenital area. Three ofthe five animals returned to anormal appearance by Day 13 aftertest material administration. The remaining two females exhibited only ablue-stained tail through study termination. Clinical signs observed at the 3,000 mglkg dose level included blue feces, haircoat, and/or tail and blue-stained urogenital area. Clinical signs oftoxicity observed at the 5,000 mglkg dose level included thin appearance, hunchedposture, hypoactivity, red-stained face, nonformed and/or blue feces, wet orblue-stained urogenital area, green urine, green-stainedhaircoat and/or tail, and death. The surviving animals returned to anormal appearance by.Day 10 after treatment with the exception ofthree males which exhibited a green-stained tail through study tennination and the remaining female which also exhibited a green-stained haircoat and tail throughto study tennination.
PathololY 

### Individual gross**necr~psy**

### pathology findings are inTable 4. Asummary report by the study pathologistis on Page 16. Atnecropsy, one or more organs ortissues in each animal were observedto be stained blue orgreen, which were attributed to the 

### test material. There were no testmaterial-related lesions observed inany ofthe animals. 

# COtfjDENTIAL DISCUSSION 

### The acute oral toxicity

# 04 .jwas evaluated in male and female rats when administered asasingle gavage dose at levels of5,000 mglkg for males and 2,000, 3,000, and 5,000 mglkg for females. Basedon the mortality observed in the study, the estimated oral LDso values were determined to be greaterthan 

### 5,000 mglkg for mates and4,128 mglkg for females.**OOOO~3**


### - 13
­Page. 88
Covance 71 (02252 

### Clinical signs oftoxicity included thin appearance; hunched**posture~** 

### hypoactivity; redstained face; blue or green-stained**haircoat~** 

### blue, liquid, and/or nonformed**feces~** 

### blue or green-stained tail; wet orblue-stained urogenital area, green urine; and death. The only mortality observed during the study (four females treated at 5,000 mglkg) occurred within 4 days oftest material administration. Animals surviving to the end ofthe observation period exhibited body weight gain during the study. Atnecropsy, one ormore organs or tissues in each animal were observed to 

### be stained blue orgreen, which were attributedto the test material. There were no test material-related lesions observed inany ofthe animals.


### SIGNATURE

StevenM. Glaza

## :S-\1-~~ Date Study Director Acute Studies



### - 14­ 

## 00009·1

### Page 89

Covance 71102252 

## REFERENCES 1. "Acute Oral Toxicity,"

### Health Effects Test Guidelines, 40 CFR 798.1175 (1989). 2. "SingleDose Toxicity Study," 

### 1990 Guidelines/or Toxicity Studies 

## 0/Drugs Manual, First Edition, Ch. 2, pp. 9-14, YAKUJI NIPPO, LTD. (1991). 3. "Acute Oral Toxicity," 

### Organisation/orEconomic Cooperation andDevelopment Guidelines/or Testing o/Chemicals, Section4, Health Effects,Number401, Paris Cedex (February 24, 1987). 4. Kanpo Oyo No. 237, YakuhatsuNo. 306, and 62 Kikyoku No. 303 (March 31, 1987). 5. Thakur, A. 

### K. andFezio, W. 

### L."

## "AComputerProgramfor Estimating**LD~** 

### and its Confidence Limits Using a Modified Behrens-Reed-Muench Cumulant Method," 

### DrugandChemical Toxicology, 4(3):297-305(1981).

- 15
­ 

## 000095Page 91

Covance 71 102252 

### Table 1 Mortality Summary 

### Dose Level Mortality Result

(mglkg) Sex No. DiedINo. Dosed·

2,000 F 

### 0/5 3,000 F 

### 0/5 5,000 M 0/5 5,000 F 

### 4/5, Day 22

### ,31, and 41


### • Superscriptnumber indicates the numberofanimals found dead onthe indicated day. Estimated Oral L050 

### Values:

Male·>5,000 mglkg ofBody Weight Female·4,128 mg/kg ofBody Weight 95% Confidence Limits
- 3,071 to 

### 5,550 mg/kg ofBody Weight

- 17­

### Page 92
Covance 71102252 

### Table 2 Individual and Mean Body WeightsJ Body Weight Gains (g) Animal 

### Day 0 Day 7 Day 14 Number Weight Weight 

### Gain· Weight 

### Gain· Females'(2,000 mglkg) 

### C22906 260 . 289 29 302 42 C22905 251 285 34 298 47 C22903 234 261 27 271 37 C24631 245 278 33 302 57 C24627 249 291 42 306 57

Mean 

### 248 281 33 296 48 • Gain from 

### Day 0body weight.



### - 18
­ 

## 000097

### Page 93

Covance 71102252 

### Table 2 (Continued) Individual and Mean Body Weightsl Body Weight Gains (g) 

### Animal 

### Day 0 Day 7 Day 14 Number Weight Weight 

### Gain· Weight 

### Gain· Females (3,000 mgfkg) 

### C24634 249 288 39 301 52 C24637 240 258 18 286 46 C24628 267 306 39 321 54' C24639 246 277 31 294 48 C24640 239 272 33 277 38

Mean 

### 248 280 32 296 48 • Gain from Day 0body weight.



### - 19­ 

## 000098Page 94

Covance 11102252 

### Table2 (Continued) Individual and Mean Body Weights/ Body Weight Gains (g) Animal 

### Day 0 Day 7 Day 14 Number Weight Weight 

### Gain· Weight 

### Gain· Males (5,000 mglkg) 

### C22841 C22842 C22843 C22844 C22845 219 239 228 231 236 297 315 253 306 311 78 76 25 75 75 351 389 330 372 370 132 150 102 141 134 Mean 

### 231 296 66 362 132 Females (5,000 mglkg) 

### C22016 C22017 C22013 C22014 C22015 252 248 244 250 235 266 (224)1 (21IY' (226)1 (177t 

### 14 273 21

Mean 

### 246 266 14 273 21 • Gain from 

### Day 0body weight.



### - 20· 

## 000099

### Page 95

Covance 71102252 Table 3 Individual Clinical Sllft. Animal Number Observauion 1.0 Hour 2.5 4.0 I­ 2t 3 4 5 6**Da~** 7 8 9 10 II 12 13 14 Femaln (1.000 mllkl) C22906 Normal appcll.!'llrlCC ./ ./ ./ Liquid feces ./ ./ Blue feces ./ ./ Red-stained faec ./ Blue-stained haircoat 

### · ./ ./ ./ ./ ./ Blue-stained tail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ C2290S Normal appcll.!'llrlCC ./ Liquid feces ./ ./ Blue feces ./ ./ Blue-stained haireoat 

### · ./ ./ ./ ./ ./ Blue-stainedtail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ C22903 Normal appell'llllCe ./ ./ Nontbrmed feces ./ ./ Blue feces ./ ./ Blue-stained ./

urogenital area Blue-stained haircoa 

# - ./ ./ ./ ./ Blue-stained tail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ C24631 Normal appearance ./ ./ Liquid feces ./ Blue feces ./ Blue-stainedbaircoat 

# - ./ ./ ./ ./ Blue-stained tail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ C24627 Normal appell'llllCe ./ ./ Red-stainedtaco ./ ./ ./ Blue-stainedbaircoat 

## · ./ ./ ./ ./ Blue-stainedtail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ Condition existed• Condition notevident.



# - Pan paper under ellebidblue f'eces. blue stainJ, III'Id nontbnned feces. t Pan paper under ellebidblue stains.**ooo~.:)o**

### Page 96

Covance 71102252 TableJ (Continued) Individual Clinical Sign. Animal Hour**Da~** Number Observation Lot 2.S 4.0 2 3· 4· S· 6 7 8 9 10 II 12 13 14 Females (3.000 m&lkc) C24634 Normal appearance Blue haircoat Blue-stained

urogenital area Blue feces Blue tail .I .I .I .I .I .I .I .I .I .I
.I .I
.I .I
.I .I
.I .I
.I .I
.I .I
.I .I
.I .I .I .I .I C24637 Normal appearance Blue-stained

urogenital area Blue feces Blue ha.ircoat Blue tail .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I C24628 Normal appearance BIue-stained

urogenital area Blue feces Blue haircoat Blue tail .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I C24639 Normal appearance Blue-stained

urogenital area Blue haircoar Blue feces Blue !iii .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I. .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I C24640 Normal appeIfIIICC Blue-stained

uroaenital area Blue haircoar Blue tail .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I .I Conditionexisted. Condition notevident. t Blue haircoatand tail observed, lrll'llfem:d from paves to hairofanimals while dosing. BIue stains and blue feces observed on pan pIpII'.



### - 22­ 

## GOO::L01Page. 97

Covance 71 102252 Table 3 (Continued) Individual Clinical SigDI Anima! Hour Day Number Observation 1.0 2.S 4.0 2 3" 4" S" 6" 7 8 9 10 II 12 13 14 Miles (S.OOO mlfkl) C2284l Nonna! appel/'lllCC Blue-stained

urogenital arel Green urine Green haircoat Green tail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ C22842 Nonfonned feces Blue feces Blue-stained

urogenital arel Green haircoll Green tail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ C22843 Nonna! appel/'lllCC Nonfonned feces Blue feces Blue-stained

urogenital area Green urine Thin appel/'lllCC Green haircoat Green tail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./
./ ./ ./

./ ./

./ ./ ./ ./ ./ ./ ./ ''/ C22844 Nonna! appelll'lllCC Nonformcd feces Blue feces Blue-stained

urogenitalarel Green-stained tail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./. ./ ./ ./ ./ ./ ./ ./

C22845 Nonna! appcanncc Nonfonned feces Blue feces Blue-stained

urogenital area Green-stained tail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./

./ Condition existed. Condition notevident. Blue-stainedpili paperobserved undercqeofanimals C2284I. C22842. and C22843. 

### "**OOO~t02** 

### - 23­Pag.e 98

Covance 71102252 Table l (Continued) Individual Cllalcal Siga. Animal Number Observation 1.0 Hour 2.S 4.0 2 3­ 4­ 5­ 6­**Da~** 7 8 9 10 11 12 13 14 Female. (5.000 m&lkl) C22016

C22017

C22013

C22014

C220lS Nonformed feces Blue feces Blue-stained

urogenital area Green haircoat Green tail Wet urogenital area Blue-stained

urogenital are Red-stained face Thin appearance Hunched posture Found dead Normal appcanmce Nonformcd feces Blue feces Wet urogenital area Blue-stained

urogenital area Red-stained face Found dead Wet urogenital area Blue-stained

urogenital area Red-stained face Nonformcd feces Hypoacdvity Thin appclll1lllCC Found dead (after

clinical observation) Wet urogenital area Blue-staincd

urogenital area Red-stained face Thill appcmnce Green haircc* Green tall Found dead ./ ./

./ ./

./

./ ./

./ ./ ./ ./

./

./ ./

./ ./

./ ./ ./

./ ./

./ ./

./ ./

./ ./ ./ ./ ./

./ ./ ./ ./ ./

./ ./ ./ ./ ./ ./

./ ./ ./ ./

./

./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./

./

./ ./ ./ ./ ./ ./ ./ ./

./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./

./ 

### • Conditionexisted. Condition notevidenl Pan paper under cage had blue-stained spots.



### - 24­ 

## 000103Page. 

### 99
Covance 71102252 

### Table 4 Individual Pathology Comments Dose Level: 2,000 mglkg ofBody Weight 

### Animal Test Day Number Sex Died Sacrificed Necropsy Observation 14 

### All internal and external lymph nodes are green. The ovaries are diffusely green. 14 

### The mesenteric lymph nodes are green. The ovaries are dark green. 14 

### The mesenteric lymph nodes are green. 14 

### The mesenteric lymph nodes are green. The ovaries are dark green. 14 

### .The mesenteric lymph nodes are green. The ovaries are dark green. C22906
C22905
C22903 C24631

C24627 

### F
F
F F


### F



### - Not applicable.

- 25­ 

## U00:104Page 100

Covance 7\\02252 

### Table 4 (Continued) Individual Pathology Comments Dose Level: 3,000 mglkg ofBody Weight Animal 

### Test Day Number Sex Died Sacrificed Necropsy Observation 

### 14 The tail and the mesenteric lymphnodes are green. 

### 14 The tail and thejejunum, ileum, and mesenteric lymph nodes are green. 

### 14 The tail and thejejunum,ileum, and mesenteric lymphnodes are -green. 

### 14 The tail and the mesenteric lymphnodes are green. 

### 14 The tail andthemesenteric lymphnodes are green. 

### C24634 C24637
C24628
C24639 C24640 F F


### F
F F

- Not applicable.



### -26­ 

## 000105Page 101
Covance 7110:2252 

### Table 4 (Continued) Individual Pathology Comments Dose Level: 5,000 mglkg ofBody Weight Animal Test Day Number Sex Died Sacrificed Necropsy Observation C2284I M 

### 14 The tail is stained blue, the mesenteric lymph nodes are diffusely blue. C22842 M 

### 14 The tail is stained blue, the mesenteric lymph nodes are diffusely blue. C22843 M 

### 14 The tail is stained blue. The jejunum, mesenteric lymph nodes, mandibular lymph nodes, both adrenals, and both tracheobronchial lymph nodes are diffusely blue. C22844 M 

### 14 The tail is stained 

### blue, the mesenteric lymph nodes are diffusely blue. C22845 M 

### 14 The mesenteric lymph nodes are diffusely blue.

- Not applicable.



### - 27· 

## 0001.06Page 102

Covance 7t 102252 

### Table4 (Continued) Individual Pathology Comments Dose Level: 5,000 mglkg ofBody Weight 

### Animal Test Day Number Sex Died Sacrificed Necropsy Observation C22016 F

C22017 

### F 2



### C22013 

### F 3



### C22014 

### F 2

C2201S 

### F 4



### - Not applicable. 14 The haircoat and tail are stained blue. The entire gastrointestinal tract, mandibular, mesenteric lymphnodes, adrenals, and ovaries are blue. The entire haircoat and gastrointestinal tract are stained blue. The entire haircoat and gastrointestinal tract are stained blue. The entire haircoat and gastrointestinal tract are stained blue. The haircoat, stomach, colon, and rectum is stainedblue. The stomachcontents are darkblue with a thick semifluid. The small intestine, pancreas, mesenteric lymph node, cecum, right ovary, and right hom ofthe uterus are cannibalized.



### - 28­ 

## U00:10',Page 103

Covance 71102252 

### APPENDIX Protocol TP2069

- 29­ 

## 000108Page 104

Covance 71102252

# cov~

__ P.O••• 7...,

..<ICII_""'--1ft ':mI1-7141 

# "" _.0I'tI
.... SIIMClI 

## ""'*
.... ::taO, 1(...._ ._...

............_Im
.. Tel: 10112<4'''471 'ar. "/141 ·n:rl' Spoillei': 

# I I CONFIDENTIAL PROTOCOLTP2069

Acute Oral Toxicity Study

# of**==~** 

# ___jmR.III (EP 

## AI01:.co GUidfiIiDILi)
Dati: 

# December" 1997**~** 

### 30­Page 105

Covance 71102252

Study Acute Oral Toxicity Study inRats (EPAlOECD Guidelines) rurpoM To assess the acute oral toxicity producedwhen the test materia.I is admiDistcred by the oral route (gavap)to rats. SPOuor

Study Dincter StevellM. Glaza Covance Laboratories lac. Telephone 601.241.7292 TestiDl

### ,aciIItJ CoV8IICCLabontories lac. 3301 KiDsmaa Boulevard MadiIoa,WI 53704

,...,.....Sbldy

### n........ Experim.entaI StartDUe ExperimeDtaJ. TermiDalioa DIIII FinaJ ReportDIte 

# CONFIDENTIAL

WeekofDecemberIt 1997 Week ofFebruIry 2, 1991 Week ofFebruIry 2, 1991



# ooo:tlifl

# I Page 106

Covance 71102252

TPl069

Pap3 Replatol')'CompllaDce This studywill be conducted in accordance with the followina Good Laboratory Practice RegulalioDIIStandards:

] Conduct ISaNonreauJated Study ] 21 CFR 58 (FDA) 

### [ J 40 CFR 160 (EPA-FIFRA) [Xl 40 CFR792 (EPA-TSCA) [Xl C(81)30(Final) (OECD) [ ] 59 NobSmNo. 3850 (J
.......MAFF) [Xl 0rdinaDc:e 21 (JapaneaeMOHW) [Xl Notification No. 59-15 (Japanese MI11) AIliaIilCan
..d U
..St.
...... All procedures illthisprotocol are illcompliaaccwith the Animal Welfare Act Regulations. Iathe OpUUOIlofthe Spoasor IDII studyd.Uector. the studydoes not unnecessarilyduplicate any previouI

### wort. QuUtyA.u....... The

### protocoL. studyconduct, IDIIthefiaaI report willbe audited bythe QualityAssurance Unit inaecordance with CoVIIDCC Laboratories IDe. (Cownce) StaadardOperatiaa Procedures (SOPS)aDd poticia. TeltMaterW 

# CONFiDENTIAL 

# I

Purity
..d St....,. The SpoMOl' assumes respoasibility for purity IDII stabilitydeterminatioas (iac1udiDa UDdertest c:oqdidoDs).



### ·32·**ooo~t:iL**Page 107

Covance 71 102252

TP2069



### Pace" Stoneeeoaditiou Room temperature RacncSa.p" Ifthis study is more thaD four weeks inexperimental dmation, reserve samples ofeach batchIIotofthe test material wi,1 be taken and stored atCovance, in a freezer setto maintain atemperature of-20"C ±lOO. The reserve samples will be maintaiDed as outlined inthe Record Retent.ioa section. Dispelido.ofTestMaterial AnyWlused testmaterial will be discarded afterissuance ofthe final report. unless otberwUc directed by SponIOl'. SaletJ Precaadou AsrequDecl by Covaoce SOPS aDd policies Abaerpdo. Thepurpose ofthis study is DOtto detenniDe absorpdoa.

Stnia adSell.. Crl;COo(SO)BR.., Charles RivetLaboratories IDe. 

### Ace atI.......fT..__t Youoalldult WeiPtat1ll1da
... ofT
...... 200 to 300.



### ·33
- 

# OOOl:12Page. 108

Covance 71102252

TP2069 Pqo' Number••dSa. Smales aDd Sfemales for the initial dose level Smales and/or Sfemales for lilY additional dose levels (ifrequired) Ideadftcatlu Individual numbered ear tag BlIIbudl'7 Bo..... Separa.ted bysex aDd II'OUP howled in suspeoded. S1aiD.Iesssteel caps Diet R.odcDt Diet#1604 (HarlanTeklad) adIlblttmt**~cept** far 17 to 20 boutsbefore testmate.rW administratiOll. The diet is routinelymalyzedbythe maraufactuler fornutritiooal componcIdSand eawiroameatal contamialatl. 

### w..... Adlibitwlrt&om lID , ..romaDe system. Samples ofdie waterare periodically malyadfor specifiedmicroorpaismsandeaviroDmeratal cootJunirumts Co.......... There are 110 !mowncontammarrts illthe dietorwater Itlevels tbIItwouldbe expected iat.aferewith this study. Ea'riro._t Enviroamcutal controlsforthe IIlimaIroom will be IIItolDIintain • tempc:latInofIS- to26OC••relative humidityofSCM

### ±ZO%. ad. 12-110111' liibfl12-bourdarkcycle.



### -34­ 

## 0001.1.3Page 109

Covance 71102252

TP2069 ·P.6 Selectioa .fTestADiJuII Based ODhealthaDd body weight according to Covance SOPs. An adequate numberofex1rlanimals will be purchased so that no animal inobviouslypoor health is placed ODtest. Justifteatioa f.rSpeciaSeIeetio. Historic:alIy, rats have beeD used as representatives of.rodent species andare preferred byvar10Ul regulatory aaCDCiOl. Treatllld.Proced..... DoMLmII A sinaJe dose of5.000 

### maIkI ofbody weight will be admioi.stendto &ve males and five females. The dole level, method, 1iequeocy.andt.bo duratioaof administrationare cboseD buedoat.borequiremadI ofthen=guIatory test &uidcUaes. ItDOtestmaterial-relatedmortality isproduced atthislevel, DO furI:ber 

### testinc will be required. IflD)' mortality oc:curs atthe5,000

### m&IkI dolelevel, additioaal dole Icveb maybe added at thedirectioaofthe studydirector illorderto meetthe objectiwlloft.bo study. DonAd
....iItn.. All animals will n:ceivethe samecooc:entratioooftcstmaterial per dole level. 1'be testmaterial will be doledundiluted. usiqt.bobulkdeasity todctamiDe the dole volume. llldividualdolawillbe calcu.lafed buedupoat.bo animal's fasted body weiahttakabefore test material admiDistratioaand admiaisterecl by

## aaV9- The animals will have food witbbeld. for 17to20 hours befoIe testmaterial admiDistratioa.

..... few RIa
....f Ad.......,..
.. Historic:aIIy. the oral route bas baa ODeofthe routes used toassess the acute toxicityofvarious matcriaIJ.



### •3S- 

## 000114:Page. 110

Covance 71102252

TP2069

PIp1 ObtcrnltoaofA.baall CIiIIicaI ObJerv.dou Atapproximately 1.2..5, and 4hours after test mataialadmiDistration and daily thereafter for atleast 13 days for clinical sipsandtwice daily (UD. andp.m.) for mortality (Lm.mortality ooI.y on Day 14). ObservatioDl may be exta.Ided wbeD directed bythe studydi.rec:tor. BodyWapa Before experimentalinitiltioD, at 7and 14 days aftertestmat.erW administntiOD, orat dcadJ. (wbeasurvival exceeds 1 day). 

# '.CIao..., . All animals. wbctherdyiqdurinathe study. sacrific:ed ill• moribUDdc:oaditioa. or euthanizedatits respective expcrimeDtaI tc:rmwtioa(Day 14).wiD be subjected to anabbnMated IJ'OSI DCCIOpS)' exarniDalioaand allabaormaJitieawiD berecorded.. All tissueswith 1csi0QlwiD bec:oUcctedand preserwei illpbospIIa1e-buffaed formalia forpossible t\dure bistoloakal eYIluatioau determDIeclbytile Spouor (tobe IIddressed byameDdment). StadltlcalKv........ Wheo applicable. LDSt values wiD bedetermiDed

### usiD&• modifiedBelueDl-Jteed.. MueDCb cumuJant method (Covaace StatisticaLibray). No ad.stadstical eva.lumODlIrereqllinld. Rep." AfiDal.n:port

### iDc.ludiD&thoJe items listedbelowwiD be submitted. Oescriptioaoftile testmaterial Desc:ripdoa oftile test system Procedarea DataofexpcrimemIJ initiatioaIIIId terminatioa Tabulatioaofmonalitydata byleXand dose Iewl Desc:riptiQl'l ofany

### tOxic effects



### - 36-**OOO~t:t5**Page 111

Covance 7t t02252

TP2069 Pap' Tabulatioa ofmeaa body lWiahts by leXaDd dose level LO"calculations for each sex with95% confidence iatervals (wbc:a appJ.ic:able) Gross pathology fiDdinp'poss patbolOCY reports Histopathology fincliftp (ifapplicable) Record Retea... All raw data, tissues, documentation, records.protocol. protoc:ollDWldments aDdreserve samples (ifapplicable), aDd the fiDai tq)OI'tgcoeratedu a result ofthis studywill be archived ia the storage facility ofCovance for aperiod ofteD yanfoUowiq**si~** of the final report. Tenyem after signjqofthe fioalreport. allofthe aCoremc:atioaed materiab wiD be sent tothe SpoMOI'. aDd aretumfee will be cIuqed. T'be SpoIIIOI'may electtohavethe maaeriaIs retaiaed intheCovance _hivesfor an additiODll periodof time.forwhich Covance will cbarpa

### storaaefee. Ifthe SpotIIIOl'c.boosea tohaw Covancc c:IispoIe ofthe materials,adisposal fee will becbarpd. All raw'"stored OG magnetic mediawill be retaiDed byCcmace. T'be foUowiq supportiDarecordswillbemaiDcdatCovmcebutwin DOt be archived withthe studydata. Animal receipclacclimatioanx:ordI Waf«analysis IeCOIda Animalroom and humidityrecorda RefripraroraDd freezer recorda Instrumcatc:ab1ndoaaDd maj,.,..,...,recordI



### ·37
­ 

## 000'1'" ,-" _ •.L'\:)

### Page 112

Covance 7t 102252

TP2069

Pace 9 PROTOCOLAPPR ' OVAL 

## /7,/'ir7 ,

n;:::_--1:\l-t...• ' 

### ·00 studyDirector Acute Studies Covaoce' ..... ........ntories Inc.
I]. (,

### J., , Date I (1110m2-pI')



### ·38· 

## OOO:t1.7

# I Page 113 

# I CONFIDENTIAL

. •



### J.



# . 

# . ...**OOO~t:1~~**":ovatlce LaD()raIO(le" 1m::. P.O. SOl.**7~5** Madison. WI 53707.7545 Oeliver'e": 3301 I<insman Blvd
.. Madison. WI 53704 Tel: S08l241·4471 Fax:**~08l241.** 7227 Page 114

# cov~
Sponsor: 

# I**~** 

# I CONFIDENTIAL FINAL REPORT StudyTide: Primary 

### Dermal 

### Irritation Study 

### o·iJ_...............

# ~_IIIIIII_..-j·J

# .. in 

### Rabbits (EP

### AlO~D GUldehnes) Author. 

### Steven M. Glaza Study Completion Date: January 

### 22. 1998 

### PerforminlLaboratory: 

### Covance Laboratories Inc. 3301 

### Kinsman Boulevard Madison, Wisconsin 53704 

### Laboratory Project Identincation: 

### Covance 71102253 

### Page 1of29



## 0001.:19

# CONFIDENTIAL Page 115
CO'iance 71102253 

### COMPLIANCE STATEMENT 

### Primary Dermal Irritation Study

## {'~'fIII..a~~~~ _____ ._

.. iillj_in Rabbits (EP**A/O~6** 

### GUtdehnes) This study was conducted accordance with the following Good Laboratory Practice Standards: United States Environmental Protection Agency TSCA: Good Laboratory Practice Standards, 40 CFR 792 Organisation for Economic Cooperation and Development Principles ofGood Laboratory Practice, C(SI)30(Final) Japanese Ministry ofInternational Trade and**Indu~try** 

### Good Laboratory Practice Standards, Notification No. 59-SS Japanese Ministry ofHealth and Welfare, Ordinance 21

Study Director Acute Studies CovanceLaboratories Inc. Date

Date



# -z1,/711

000:1.20Page 116
Covance 7t 1022S3 

### QUALITY ASSURANCE STATEMENT This report has been reviewed by the Quality Assurance Unit ofCovance Laboratories Inc., in accordance with the Environmental Protection Agency (EPA) Good Laboratory Practice Standards. 40 CFR 192, the Organisation for Economic Cooperation and Development (OECD) Principles ofGood Laboratory Practice, C(81)30(Final), the Japanese Ministry ofIntemational Trade and Industry (MITI) Good Laboratory Practice Standards, Kikyoku**Notifi~ation** 

### No. 85, and the Japanese Ministry ofHealth and Welfare (MOHW), Good Laboratory Practice Standards, Ordinance 21. The following inspections were conducted and findings reported to the study director and study director management. Written status reports ofinspections and findings are issued to Covance management according to standard operating procedures. Inspection Date Reported.to Dates Study Director and From To Phase Study DirectorManagement 12107/97 12107/97 

### Protocol Review 12108/97 12/11197 12111/97 

### Dose Preparation 12111197 01119/98 01119/98 

### DatalReport Review 01/19/98



### Date

-3­Page 117
Covance 71102253 

### STUDY IDENTIFICATION 

### Primary Dermal Irritation Study

# 01\ lin Rabbits (EP

### AJo.J~"c"'6"'d"'u-la"'e·h-ne-s") ----... Test Material Sponsor
Sponsor'5 

### Representative Study Director
Testing Facility Study Timetable

Study Initiation Date Experimental (In-life) Start Date In-life End Date Experimental TerminationDate Study Completion Date . Steven M. Glaza Covance Laboratories Inc. Telephone 608.241.7292 

### Facsimile 608.242.7936 

### Covance Laboratories Inc. 3301 

### Kinsman Boulevard Madison, WI 53704 

### December 8, 1997 

### December 11, 1997 

### December 14, 1997 

### December 14. 1997 

### January 22, 1998

-4­ 

## 000:122Page 118
Covance 71102253 

### KEY PERSONNEL Acute Studies 

### Steven M. Glaza Study Director Steven R. Sorenson Study Coordinator Jeffrey B. Hicks In-life Supervisor Rose M. Bridge Administrative Supervisor Toxicology Support 

### Kathy Myers Manager Calvin L. 

### Horton Supervisor Quality Assurance 

### Nancy M. Centanni Manager LaboratoryAnimal Medicine 

### Donna 1. 

### Clemons, DVM Diplomate, ACLAM Supervisor

-5­**OOO~** 

### 1 "'-'3**/~**Page 119
Covance 71102253 

### CONTENTS COMPLIANCE STATEMENT ....................................................................................... 2 

### QUALITY ASSURANCE STATEMENT ...................................................................... 3 

### STUDY IDENTIFICATION........................................................................................... 4 

### KEY PERSONNEL......................................................................................................... 5 

### StJrv1l\1AR

### Y................................................................................................"'" ............... 8 

### OBJECTIVE ................................................................................................................... 8 

### TEST MATERIAL.......................................................................................................... 8 

### Identification................................................................................................................ 8 

### Purity and Stability.................................................. .................................................... 8 

### Storage and Retention..................................................................................................9 

### Safety Precautions ....................................................................................................... 9 

### TEST SySTEM .............................................................................................................. 9 

### Test Animal ................................................................................................................. 9 

### Housing.......................................................................................................................9 

### Animal Diet ................................................................................................................. 9 

### Animal Selection and Preparation ofExposure Area................................................... 10 

### Justification for Species Selection .............................................................................. 10 

### PROCEDURES............................................................................................................. 

### 10 Preparation ofTest Material ...................................................................................... 10 

### Treatment.................................................................................................................. 10 

### Reasonfor Route ofAdministration........................................................................... 10 

### Observations.............................................................................................................. 11 Termination............................................................................................................... 1 1 

### Statistical Evaluation ................................................................................................. 11 Location ofRaw 

### Data, Records, and Final Report...................................................... 1 1 

### RESULTSIDISCUSSION ............................................................................................. 12 SIGNATURE................................................................................................................ 

### 12

- 6­**0001.:~4:**Page 120

Covance 71l02253 

### REFERENCES.................................................................................................... ..13 

### TABLE Primary Dermal Irritation Scoring Scale (Draize Technique) ................................ 14 1 

### Individual Dermal Irritation Scores ............................................................. . . . . . . . ... 15 

### 2 Individual Dermal Irritation Index Scores.............................................................. 16 

### 3 

### Average Primary Dermal Irritation Scores............................................................. 17 4 

### Individual Body Weights (g) ................................................................................,I7 

### APPENDIX......................................................................,......................................'..... 18 Protocol Deviation....................................................,.......................,.......,..,..,.......... I

### 9 Protocol TP3014 ,...........................................................................................,..,...... 20



## 000125Pag-e 121 

# CONFIDENTIAL
Covanc:e il102253 

### SUMMA,RY 

### The primary dermal irritation potential ot ; ,

# tas evaluated in rabbits under 4-hour semioccluded conditions. The test material produced only avery slight erythema reaction in two animals. No other dermal irritation was observed, All 

### irritation cleared by the 48 hour observation. The primary dermal irritation index is 02 (considered to be slightly irritating). I


### OBJECTIVE



### ,The objective ofthis study was to assess the relative level ofprimary skin'irritation ofa test material on rabbits under semioccluded conditions.2.3 

### All procedures used in this study were in compliance with the Animal Welfare Act Regulations. In the opinion ofthe Sponsor and study director. the study did not unnecessarily duplicate any previous work; The dose, method, frequency. and duration of administration utilized in 

### this study were chosen based on the requirements ofthe regulatory test guidelines. All 

### procedural times presented in 

### this report fall within the acceptable ranges as specified in Covance Laboratories Inc. (Covance) Standard Operating Procedure (SOP).
TEST MATERIAL 

# CONFIDENTIAL Identification The test material was identified

# 4'
. Fddescribed as a viscous, dark-blue liquid. ...---------­ 

### Purity and Stability The Sponsor assumes responsibility for purity and stability determinations (including under**~est** 

### conditions).

·8­**OOO~t~~6**

### Page 122
Co\'ance 7llO2253 

### Storage and Retention 

### The test material was stored at room temperature. Any 

### unused test material will be discarded after issuance ofthe final report according to Covance SOP. Safety Precautions 

### The test material handling procedures were according to Covance SOPs and policies.
TEST SYSTEM Test Animal 

### Adult albino rabbits ofthe Hra:(NZW)SPF strain were procured from Covance Research Products Inc., Kalamazoo, Michigan on October 29, 1997. Housing 

### After receipt, the animals were acclimated for a period ofat least 7 days. During acclimation and throughout the study, the animals were individually housed in-suspended stainless steel cages with the exception ofone female that was found out ofits cage on Day 2. Environmental controls for the animal room were set to maintain atemperature of 16° to 22°C, a relative humidity of50% :1:20%, and a 12-hourlightll2-hourdark cycle. In cases where variations from these conditions existed, they were documented and considered to have had no adverse effect on the study outcome.
Animal 

### Diet The animals were provided access to water adlibitum 

### and a measured amount ofHigh Fiber Rabbit Diet#7015 (Harlan Teldad). The diet is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Samples ofthe water are periodically analyzed. There were no known contaminants in the diet orwater at levels that would be expected to interfere with or affect the results ofthe study.

-9­**OOO'l~)'''''** 

# -**~-** (

### Page 123
Covance 711022.53 

### Animal Selection and Preparation ofExposure Area 

### Three male and three female healthy, acclimated rabbits, weighing from 2,610 to 2,806 g and approximately 19 weeks ofage, were selected at random and identified by animal number and corresponding ear tag. On the day before treatment, the back and/or flanks of each animal were clipped free ofhair to obtain one unblemished skin site.
Justification for Species Selection 

### Historically, the New Zealand White albino rabbit has been the animal ofchoice for evaluating the effect ofchemicals on the skin.
PROCEDURES Preparation ofTest Material 

### The test material was administered as received. The pH ofthe test material was determined to be 7.5.


### Treatment 

### The undiluted test material was applied to the intact skin site on each animal's back (approximate exposure area of6.2S 

### cml) in 

### the amount of0.5 

### mL. Each area of application was covered with an 8-ply 2.S-cm

### x2.S-cm 

### gauze patch secured with paper tape, loosely overwrapped with SaranWrap·, and secured with Elastoplast'" tape to provide a semiocclusive dressing. At the end ofthe 4-hour exposure period, the patches were removed and the test sites washed using liquid Ivorr- soap mixed with water, rinsed with water, and dried with disposable papertowels.
Reason for Route ofAdministration 

### Historically, the dermal route has beenthe route ofchoice based onthe method ofDraize. 4



### - 10­

### Page 124

Covance 71lO2253 

### Obsen'ations 

### Thirty minutes after removal ofthe test material, the degree oferythema and edema at each test site was read according to the Draize technique (recorded as the 4-hour score). Subsequent examinations were made at 24, 48, and 72 hours. The untreated skin ofeach animal was used for comparison. Animals were weighed before test material administration.
Termination 

### At termination ofthe in-life phase, all animals were euthanized and discarded.
Statistical Evaluation 

### No statistical evaluations were required by the**protoc~l.** 

### Location ofRaw 

### Data, Records, and Final Report 

### The raw data, records, and an original signed copy ofthe final reportwill

### be retained in the archives ofCovance for aperiod often years following signing ofthe final report. Ten years after signing ofthe 

### final report, 

### all ofthe aforementioned materials will be sent to the Sponsor.

- 11"­**OOO~t29**

### Page 125 

# CONFIDENTIAL Covance 71102253 

### RESULTSfDlSCUSSION 

### [ndividual dermal irritation scores are presented in Table 1, with the individual dermal irritation index scores in Table 2. 

### Average primary dermal irritation scores and individual body weights are in Tables 3 

### and 4, respectively. Application of . 

# +0 the skin ofrabbits under 4-hour semioccluded conditions resulted in only avery slight erythema reaction in two animals. No other dermal irritation was observed. 

### All irritation cleared by the 48 hour observation. The primary dermal irritation index (the average ofthe individual 

### animal index scores) is found in Table 2 

### and determined to be 0.2 

### (considered to be slightly irritating).
SIGNATURE

Date Steven M. 

### Glaza Study Director Acute Studies

- 12
­ 

## 0001.30

### Page 126

Co..-ance 71102253 

## REFERENCES 1. Seabaugh, V. M. and Vocci, F., "Dermal Irritation, Addendum 3 on Data Reporting," 

### Pesticide Assessment Guidelines, Subdivision F(January 1988). 2. "Primary Dermal Irritation," 

### Health Effects Test Guidelines, 40 CFR 798.4470 (1989). 3. "Acute Dermal Irritation/Corrosion," 

### Organisationfor Economic Cooperation and Development Guidelines for Testing of Chemicals, Section4, Health Effects, Number 404, Paris Cedex: (July 

### 17, 1992). 4. Draize,1. H., "Primary Irritation ofthe Skin," In: 

### Appraisalof the Safety of Chemicals in Foods, Drugs, and

### Cosmetics
- Dermal Toxicity, Association ofFood and Drug Officials ofthe U.S., pp. 46-47 (1959).



## 0001.3:1.

### Page 127
Co\'ance 71[02253 

### Primary Dermal Irritation Scoring Scale (Draize Technique) 

### (1) Erythema and Eschar Formation No erythema Very slight erythema (barely perceptible) Well-defined erythema Moderate to severe erythema Severe erythema (beet redness) to slight eschar

formation (injuries in depth) 

## o 1 2 

### 3 

### Highest possible erythema score 4 

### (2) Edema Formation No edema Very slight edema (barely perceptible) Slight edema (edges are well defined by definite raising) Moderate edema (raised approximately 1mm) Severe edema (raised approximately 1mm and extending

beyond area ofexposure) 

# o I 2 3


### Highest possible edema score 4



### - 14
­ 

## 000:13ZPage 128

Covance 711022.:53 

### Table 1 Individual Dermal Irritation Scores 

### Animal Hour Number 

### 4 24 48 72 Erythema 

### F65363 1 I 

### 0 0 

### F65364 1 

### 1 0 0 

### F65388 0 0 0 0 

### F65365 0 0 0 0 

### F65387 0 0 0 0 

### F65389 0 0 0 0 Edema 

### F65363 0 0 0 0 

### F65364 0 0 0 0 

### F65388 0 0 0 0 

### F65365 0 0 0 0 

### F65387 0 0 0 0 

### F65389 0 0 0 0



### • 15 • 

## 000:.t.33

### Page 129

Covance 71102253 

### Table 2 Individual Dermal Irritation Index Scores Animal Index Number Scores· F65363 2 / 4 = 

### F65364 2 /4 = 

### F65388 

# o /4 = 

### F65365 

# o /4 

## = F65387 

# o /4 = 

### F65389 0/4 = 

### POll" 1.00 / 6 = 

### 0.50 0.50 0.00 0.00 0.00 0.00 0.2
• The individual index scores are the total ofthe erythema and edema scores at 4,24,48, 

### and 72 hours divided by the number ofobservation periods (4). "'. The primary dennal irritation index (POll) is the sum. ofthe individual index scores, divided bythe number ofanimals (6), and rounded to the nearest tenth.



### • 16·CO\'ance 711022;3 

### Table J 

### Average Primary Dermal Irritation Scores 

### Observation Period (Hour) Average Score* 4 

### 24 48 72 0.3 

### OJ 0.0 0.0 

### * The average primary dermal irritation score is the total dermal irritation score for all 

### the animals (erythema and edema) divided by the number oftest sites (6) at each observation period.

Table 4 Individual Body Weights (g) Animal 

### Number Sex 

### Predose 

### F6S363 M 2,786 

### F6S364 M 2,724 

### F6S388 F 2,730 

### F6S36S M 2,806 

### F6S387 F 

### 2,6S1 F6S389 F 2,610

- 17 - 

## 000:135Page 131
CO\-ance 71102253 

### APPENDlX 

### Protocol Deviation

Protocol TP30 14



## 000:1,36

### Page 132

Covance 71102253 

### Protocol Deviation 

### Protocol Actual Procedure Page 5, .'\nimals, Husbandry, Housing. One animal was found out ofits cage on Individually, in suspended, stainless steel Day 2. cages This deviation is not considered to have had an adverse effect on the outcome ofthe study.

- 19 ­ 

## 00013'(1

### Page 133

CO"ance 71102253

## cov~ po. 10. 7U. 'HI OI'IIlM"l1IT 511MC11 C-'" 1I••lIi..... WI.e....I" 53707·7U. P.ek....: 3301 KI"....." I.ul....... r••: 10112",'.71 F ••: 10112.1·7227 Spouor: 

# I 

# I CONFIDENTIAL
PROTOCOL 11'3014 Stllcly TItle: PrimIryDermailrritatiOD Study

### off~,....,.~~ 

# ....___**~ln** Rabbits**(EPAlOE~b** 

### GW&GDeI)

DecemberIt 1m
Perfe..... Labentory: Covmce Labontories Inc. 3301 K.insmm 80uIevInl MadiJoa, W'18COIIIia 53704

Covmce 711022S3



### - 20­**OOO~t38**

### Page 134

Covance 7[1112253

TP3014 Pile 2 Study Primary DcnnaJ Irritation Study in Rabbits (EPAlOECD Guidelines) PUrpoH To assess the relative level ofprimary skin irritation of.testmaterial onrabbits under semioccluded conditions SOODlOr

SooDlOr t , ReDreseatatiYe

Study Director StevenM. Olaza Covance Laboratories lac. Telephone 601.241.7292 Taba

### 'adUtJ COVUICCLaboratories IDe. 3301 K.insnumBouIC'YIl'd Madisoa, WI S3104 Pnpo... StudyT.....b.. Experimental StiltDale ExperimentalTenn.i.DalioD Dace Final Report Date 

# CONFIDENTIAL

WeekofDecembcr8.1m WeekofDecember29. 

### 1m WeekofFebruary 9, 1998



### - 21
­ 

## 000139**p~~** 135

11'3014 Pile) Replatery COlDpu.ace This study will be conducted in accordance with the followina Good Laboratory Practice RegulatiomiStandards: 

### 1 Conductas a Nonregulated Study 

### 1 21 CFR 58 (FDA) ] 40 CFR 160 (EPA-FIFRA) [X] 40 CFR 792 (EPA-TSCA) [X] C(81)30 (Final)(OECD) 

### ( 159 NohSan No. 38'0(Japanese MAFF) [X] 0rdinaDce 21 (Japanese MOHW) [X] NotificationNo. 59..1'(Japanese MITI) ADbDatC.n.adUN Statemeat All procedures inthis protocol are incompliance with

### the Animal Welfare Act Regulations. In the opinion ofthe Sponsorand study director, the study does not unneceaarilyduplic:ate lIlY pnMous 

### wort. Quallt1 AuU'lUlOt The protocol. study conduct,and thetiDal reportwill be

# _ted bytheQuality Assuraace Unit inaccordaocc with COVaDCC Laboratories loc. (Covance) Standard Operating Procedures (SOPs) aad policies. TiltMaterial 

# CONFIDENTIAL
Pbyslcal DeIcrtpdIa Viscous,dark. blueliquid Parityaad

## sea....,. The·SpoaIGI'assumes responsibility forpurity determinations (incJudiqIIIKIertest conditions).



### - 22­ 

## OOO:1.:tO

### Page 136
Covancc 7110225}

mOl4

Pace 4 Sto....e Co.did... Room temperature Reserve Sa.p'" Ifthis StUdy is more thanfour weeks in experimental duntioa, reserve samples ofeach batchllot ofthe testmaterial will be taken aDd stored atCovaace. illa .fm:zer setto maintaiD atemperatureof·20·C ±Io-. The reserve samples wiD be maintaiDed u outlined in the Record Retention section. Dispoaidoa Any unused testmaterial wiD be discarded afterissuance ofthe final report, unless otherwise directed bySponsor. Safety rrecaatloaa As required byCovance SOPs andpoIiciel Absorpdoa The purpose ofthis study isnot to determU1e absorption.

S
..... Rabbit Stnlllaad

### s..rc. Hra:(NZW)SPF. Covance Raan:hProductsInc. 

### AI- at1Dida.. ofTra.....t Adult 

## w

..... t atlaida".fTra.....t 2.0 

### m3.5 

### q



### - 23­ 

# 000141

### Page 137

Co\ance**711012~3**

TP3014 Pag" Number ••d Sex 6 ofmy sex Ideatifleatioa Individual numbered car

### taa HUlb.adry HousiDI individually. in stainless steel cases Diet Ameasured amount ofLaboratory HighFiber RabbitDiet 1#7015 (Harlan Teldad). The diet is routinely analyzedby the manufacturerfor nutritional components andenvironmental contaminaDts. 

## w

..... A.dlibitum from an automatic system. Samplesofthe waterare periodica.Uy analyzed for specified microorpDisms IDdenvironmental contami.tw:lts. COD
.......... There are no known contaminants in the dietorwater atlevels thatwould be expectedtointerfere withthis study. Eaviroll_t Environmeptal controls for the animalroom will be setto maintain a temperature of 16·to**2l~,** I ndative humidity ofSO% :t;20%, and a 12-hour Iightl12-bourdarkcycle. Acdiaadoa At lout7 days



## 000:1/12Page 138
Co,'ance 71102253

TP3014 Pqe6 SelecdoD ofTatADjma. Basedon health and body weight according to Covaac:e SOPs. An adequate numberofextraanimals will bepun:ha.scd so that no animal in obviously poor health is placedon test. JU.tiflcadOD for Species Selecdo. Historically. the New Zealind White albino rabbithas been the animal ofchoice for evaluatiqthe effcctofcbcm.icals on the skin. TnatllleDt Procedures PnpandoaofE.lpoIunA.ra Onthe day before test material administration, remove the hairfrom the back and., ifne«sSaI'Y (to obtain unblcm.isbed skin), the flanks ofCICh animal with an electric clipper. The tratmeDtsites will be inspected for int.ert'erini laioas, irritation. or defects that would precludethe use ofanyofthe animals. The animals will be clipped at needed tbroupoutthe study. DoN Admutntioa Before administrationofthe testmaterial the pH ofthe material will be determined (ifpossible). The test material will be applied tothe intacl testarea (approximately 6.25·anz ) on eachrabbit,in tbe amountof0.5 mL. The dose lewl. metbod. frequency. and duratioD ofadminisaVioD Mechosen batedonthe rec:tuiremerdS of the rcpdatorytestguidelines. The test material will be applied undiluted. The area ofapplication will becoveredwith IIIa-ply 2.'-cmx 2.5-cm puzepat.cb secured with papertapeand overwrappcd with 

## wrap- and 

### El&!RoJILut-tape to provide a scmiocclusive drcssiq. The rabbits will DOt be col1arcddurina the 4­ hour applicationperiod.

..... forReate ofAd....tndo. Historically,the dermal route has beeDthe route ofchoicebated Oilthe method of Drai&



### - 2S

## 000:143Page 139

Covancc 71101153

TP10l4 Pqe1 Remov.1 .fTestM.terial After 4 bours ofexposure, the patches and testmaterial will be removed as thoroughly aspossible using waterand/or an appropriate substance without irritating the skin.

ReadlacofDel"lllallrritatioa Within 30-60minutes after removing patches. tbedqreeoferythemaaDd edema will be evaluatedaccordiq tothe Dra.ize technique(AttacbmeDt 1) aDd recordedas the
....hour score. The untreated skinofeacb animal will serve as itsown conttol. Sub"equentradinpwill be taken atapproximately 24. 41, aDd

## n hours afterpatch removal. Ifirritation is presentat the n·hourexamination, additional observations will be madeatapproximately 96 hours and at Days 7. 14, and21. oruntil irritatioll has clCSRd. Basedon thelevel ofirritationobserved atany ofthese time points. the study may be tcrmi.Dated atthe direetiODofthe studydirector. BodyW..... Before testmaterial admiDistraIioD only. P.t..., Anyanimals dyiqorsacrific:cd in • moribuDd conditionduriDa the study will be subjected to an abbreviated polSnecropsy examination andall abnormalities will be recoaded. After 1ICClOPIY. theanimals will be discarded and DO tissues will be saved. Atte.rminatioaoftheexperimental phase. surviviqanimals will be sacrificed aDddiscarded. StadaUcal

## z..... tIOII No statiIcical evaluar:ioas are requiRd.



### - 26­ 

## 000:1<14Page 140

TPlOt4 Pale' Report A final report includina those items listed below will be submitted: Description ofthe testmaterial Descriptionofthe test system Procedures Dates ofexperimental initiation and termination Tabulation ofinitationdata Record Reteatto. All raw data. documentation. records, protocol, protocol amendments and reserve samples (ifapplicable), and the final report aenentecl as aresultoftbis study will be archived in the storage facility ofCovancc for a periodoftenyears followiDa sipingof the fmal report. Tenyears aftersipinaoftile final report, all ofthe aforementioned materials will be sent totile SPODlOl', and a fee will be cbarpd. The Sponsormay electtohave the materialsretained intile COVllDCe archives for anadditional period of time,for which Covance will cbarae astorage fee. Ifthe sponsorchooses tohave Covance dispose ofthe

### matcrials. adisposal fOIl will be charpd. All raw data storedon magnetic mediawillbe retained by Cov.oce. The foUowiDa supportiDarecords will be retainedatCovance butwillnot be archived with the study data: Animal receiptllCCtimationreconIa Waf«analysis records Animal room tempel"IhRandhumidityreconIa RefrileratorIDd treea:a:r tem.pcraNrereconIa In.strumentcalibradonand maintelWlCereconIa



### - 27­ 

## 0001/15Page 141

Covance i [ [02253

TP10l4

Page 9 PROTOCOL APPROVAL

Steven M. Ollza 

# JJ...:.. (Z,

## 1fC) Date 

# CONFlOENllAl

1

# "'t.-\-'" Study Directm AcuteStudies Covaoce Laboratories Inc.

Date (71102253.pr)

### Page 142
CO\ance 71102253

TP3014 P.e 10 Act.cb....t I
Pri..aryDenaallrritadoaSeoriaC Scale (DraizeTecbaique) Erytbema adEscharFormado. No erythcl1'la ..•.

_ ••••:............................................................................ 0 Very slight erythcI1'Ia(barely perc:eptible) .................................................................1 WeU-dcfincd erytbema
.......................................................................................2 Moderate to severe erythana
.....................................................................................3 Severe erythema (beet redneu)to sliKbteschar formation (injuries in dcpcb) ......................................................................................4 Highestpossible crytbenIa sc;ore....................................................................4 Edema Forma" No edema .......................................................................................0 Very slightedaDa ....................................................................................... 1 SlightedaDa (qesare well defiDed by Severe edema(raised approximately 1mIDIDd definite raisiDI)
.......................................................................................2 Moderate edaDa(railedapproximately 1mID) .........................................................3 exteDdinl beyond ....**of~)** ...................................................................:......4 Hia.bestpossible edeIua sc;ore ........................................................................4



### - 29- 

## 00014'7

# I Page 143 

# I 

# CONFIDENTIAl .

t



# . t'

### Page 144



### PAGE 1 OF 32PAGES
CONFIDENTIAL 

# CONFIDENTIAL 

# ( 

# 1 ACUTE TOXICITY TO DAPHNIA MAGNA 

### SPL PROJECT NUMBER: 656/011



### AUTHORS: P M WmON 

## AJ BARTLm 

# CONFIDENTIAl

STUDY SPONSOR: ISSUED BY: Safepharm Laboratories Limited 

### P.O. Box No. 

### 45 DERBY DEl 2BT U.K. Telephone: (01332) 792896 Facsimile: (01332) 799018

## 000:149

### Page 145



### -2­ SPl PROJECT NUMBER: 656/011
QUALITY ASSURANCE REPORT

The routine inspection of short term studies at Safepharm is carried out as a continuous process designed to encompass all major phases of each study type 

### once per month. Inspection findings are reported to Management/Study Directors on the day of inspection in each case. Dates of relevant monthly inspections are as follows: 06, 08, 13, 14, 27 January 1998 This report has been audited by Safepharm Quafity Assurance Unit. It is considered to be an accurate account of the data generated and ofthe procedures followed. 

### Date of Report Audit: 24 February 1998 

# . 1**~** 

## HAY 1991 DATE. ................................................ 

## J R Pateman For Safepharm Quality Assurance Unit



## 000:150Page 146



### -3­ SPL PROJECT NUMBER: 656/011
GLP COMPLIANCE STATEMENT \, the undersigned, hereby declare that the objectives laid down in the protocol were achieved and as nothing occurred to adversely affect the quality or integrity of the study, I consider the data generated to be valid. This report fully and accurately reflects the procedures used and data generated. The work described was performed in compliance with UK GLP standards' (Schedule 1, Good laboratory Practice Regulations 1997 (SI 1997/654». These Regulations are in accordance with GLP standards published as OECD Monograph No. 45 (OCDElGD(92}32); and are in conformity with, and implement, the requirements of Directives 87/181EEC and 881320/EEC. These international standards are acceptable to the United States Environmental Protection Agency and Food and Drug Administration, and fulfil the requirements of 40 CFR Part 160, 40 CFR Part 792 and 21 CFR Part 58 (as amended)
.



# .............!ll...J.l
............................................ Date: ...........

# ..**1.J.~.Al .l~~~**  .................. P M Wetton BSc Study Director
I, t nder my supervision and accurately reflect the data obtained. 

## ". 1

## 2 HAY 1998

......••......•......•................................................. Date: .•...................•............................... A 

### J

## B~rtlett CChem MRSC Head of Analytical Chemistry



## 0001.S1

### Page 147



### SPL PROJECT NUMBER: 656/011 CONTENTS PAGE QUALITY ASSURANCE REPORT 2 GLP COMPLIANCE STATEMENT 3 SUMMARY 6 1. INTRODUCTION 7 2. TEST MATERIAL AND EXPERIMENTAL PREPARATION 7 2.1 Description, Identification and Storage Conditions 7 2.2 Experimental Preparation 8 

### 3. M£rHODS 8 3.1 Test Species 

### 8 3.2 Test Water 

### 9 3.3 Procedure 

### 9 3.3.1 Range-finding study 

### 9 3.3.2 Definitive study 10 

### 4. ARCHIVES 11 

### 5. RESULTS 12 5.1 Range-finding Study 12 5.2 Definitive Study 12 5.2.1 Immobilisation data 12 5.2.2 Physico-chemical measurements 13 5.2.3 Verification oftest concentrations 13 

### 6. CONCLUSION 13 

### 7. REFERENCE 13 

### TABLES 1 Cumulative Immobilisation Data in the Range-finding Study 14 2 Cumulative Immobilisation Data in the Definitive Study 15 

### FtGURES 1 Concentration-Response Curve After 24 Hours 16 

### 2 Concentration-Response Curve After 48 Hours 

### 17 

## 000:152

### Page 148



### -5­ 

### SPl PROJECT NUMBER: 656/011 

### PAGE APPENDICES 18 

### Physico-chemical Measurements 19 

### II Verification of Test Concentrations 

### 20 III Reconstituted Water 31 IV Statement of GlP Compliance in Accordance with Directive 

### 88/320/EEC 

### 32



## 000153Page' 149



### -6­ 

### SPL PROJECT NUMBER: 656/011 

### SUMMARY 

# CONFIDENTIAL STUDY SPONSOR STUDY TITLE ACUTE TOXICITY TO DAPHNIA 

### MAGNA TEST MATERIAL 

# :[

__________ , CONFIDENTIAL Methods A study was performed to assess the acute toxicity of the test material to Daphnia 

### magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 202, "Daphnia sp, 

### Acute Imfnobilisation Test and Reproduction Testn 

### referenced as Method C.2 of Commission Directive 

### 92169/EEC (which constitutes Annex V of Council Directive 67/548/EEq. 

### Procedures Following a preliminary range-finding study, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous dispersion of the test material at concentrations of 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2,.5.6 and 10 mgtl for 48 hours under static test conditions. The number of immobilised Daphnia 

### were recorded after 24 and 48 hours. 

### Results The 48-Hour EC

### so for the test material to Daphnia 

### magna based on nominal test concentrationswas 1.1 mgtl with 95'0 confidence limits of 0.92 - 1.3 mgt!. The No Observed Effect Concentration was 0.56 mgtl. Analysis of the test solutions at 0 and 48 hours showed the measured test concentrations to be in excess ofthe required 80% of nominal test concentrations and so the results are based on nominal test concentrations only. 

## 000:154Page 150



### -7­ 

# CONFIDENTIAl SPL PROJECT NUMBER: 656/011 

# I I ACUTE TOXICITY TO DAPHNIA MAGNA 

# CONFIDENTIAL 1. INTRODUCTION This report contains a description ofthe methods used and results obtained during a study to investigate the acute toxicity

# 01-. )to 

### Daphnia magna. The method (Safepharm Laboratories Standard Test Method 900.02) 

### followed the recommendations of the OECD Guidelines for Testing of Chemicals (1984) 

### No 202 

### "Daphnia sp, Acute Immobilisation Test and Reproduction Test" referenced as Method C.2 of Commission Directive 

### 92169/EEC (which constitutes Annex V of Council Directive 

### 67IS48/EEC). Daphnia magna is a freshwater invertebrate representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems. The range-finding study was conducted between 2 January 1998 and 4 January 1998 and the definitive study between 2 February 1998 and 4 February 1998. 

### 2. -rEST MATERIAL AND EXPERIMENTAL PREPARATION 

### 2.1 Description, Identification and Stora e Conditions 

# CONFIDENTIAL Sponsor's identification Description extremely dark b ue opaque VISCOUS 

### liquid Date received 24 

### November 1997 Storage conditions room temperature Data relating to the identity, purity and stability of the test material are the responsibility of the Sponsor.



## 000:155Page 151

-8­ 

### SPL PROJECT NUMBER: 656/011 

### 2.2 Experimental Preparation For the purpose ofthe definitive study the test material was prepared by a direct dispersion in water. An amount of test**materi~1** 

### (100 m8> was dispersed in reconstituted water and the volume adjusted to 

### 1 litre to give a 100 mgtl aqueous stock solution. A dilution was made from this stock solution to give the test concentration of 10 mgtl from which dilutions were made to prepare the remainder of the test series of 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2 and 5.6 mgtl. Given that the pre-study stability analysis performed indicated the test material to 

### be unstable in light, the above preparations were carried out using minimal light conditions. The concentration and stability of the test material in the test solutions were verified by chemical analysis at 0 and 48 hours (see Appendix 11). 

### 3. METHODS 

### 3.1 Test Species The test was carried out using 1st instar Daphnia magna. Daphnia magna 

### were maintained in a laboratory culture originating from a strain supplied by the Institut National de Recherche Chimique Appliquee (lRCHA), France. Adult Daphnia 

### were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at 21 •C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a suspension of mixed algae (predominantly Chlorella spp.). 

### Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated 24 hours prior to the initiation of the test, the young daphnids produced ovemight were then removed for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study. 

## OH01S6

### Page 152



### -9­ 

### SPL PROJECT NUMBER: 656/011 

### 3.2 Test Water The reconstituted water used for both the range-finding and definitive studies was the same as that used to maintain the stock animals. The reconstituted water is defined in Appendix III. 

### 3.3 Procedure 3.3.1 Range-finding study The test concentrations to 

### be used in the definitive study were determined by a preliminary range-finding study. In the range-finding study 

### Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mgtl. The test material was dispersed directly in water. An amount oftest material (100 mg) was dispersed in reconstituted water and the volume adjusted to 1 litre to give a 100 mgtl test concentration. Serial dilutions were made from this test concentration to give the remainder of the test series. For each concentration 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21 • C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of48 hours. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised 

### Daphnia magna were recorded. The control group was maintained under identical conditions but not exposed to the test material.**OOO~t.S'7**

### Page 153



### -10­ 

### SPL PROJECT NUMBER: 656/011 

### 3.3.2 Definitive study Based on the results ofthe range-finding study the following test concentrations were assigned to the definitive study 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6 and 1

### 00 mgt!. 

### 3.3.2.1 Preparation of the test material For the purpose of the definitive study the required amount of test material was added to each test vessel using the method described in Section 2.2. 

### 3.3.2.2 Exposure conditions For the definitive study 250 ml glass jars containing approximately 250 ml of test solution were used. At the start of the study 10 daphnids were placed in each test and control vessel at random, in the prepared test solutions. Duplicate test vessels were used for each test and control group. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21·C. The test vessels were shielded from light given that the pre-study stability analysis (see Appendix 11), 

### showed the test material to 

### be unstable in light. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. The control group was maintained under identical conditions but not exposed to the test material. The test solutions were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia 

### were considered to be immobilised ifthey were unable to swim for approximately 15 seconds after gentle agitation. 

### 3.3.2.3 Physico-chemical measurements Water temperature was recorded daily throughout the study. Dissolved oxygen concentrations and pH were recorded at the start and termination of the study
. 

# . 000:158Page 154

-11­ 

### SPL PROJECT NUMBER: 656/011


### 3.3.2.4 Verification of test concentrations Water samples were taken from the control and all test groups (replicates 

### Rl
- R2 pooled) at 0 and 48 hours for quantitative analysis. The method of analysis, stability,
.recovery and test solution analyses are described in Appendix II. 

### 3.3.2.5 Evaluation of data The EC

### so values and associated confidence limits were calculated by the moving average method of Thompson (1947). 

### 4. ARCHIVES Unless instructed otherwise by the Sponsor, all original data and the final report will 

### be retained in the Safepharm archives for five years, after which instructions will be sought as to further retention or disposal.



## 0001.59Page 155



### -12­ 

### SPl PROJECT NUMBER: 656/011 5. RESULTS 5.1 Range-finding Study Cumulative immobilisation data from the exposure of Daphnia magna 

### to the test material during the range-finding study are given in Table 1. The results showed no immobilisation at the test concentrations of 0.10 mgtl. However, immobilisation was observed at 1.0, 10 and 100 mgtl. Based on this information test concentrations of 0.10, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2, 5.6 and 10 mg/l were selected for the definitive study. The study was carried out with the test vessels shielded from light given that pre-study stability analysis (see Appendix II) 

### showed the test material 

# ro be unstable in light conditions. 

### 5.2 Definitive Study 

### 5.2.1 Immobilisation data Cumulative immobilisation data from the exposure of Daphnia magna 

### to

, kUririg the definitive study are given in Table 2. The relationship between percentage immobilisation and concentration at 24 and 48 hours are given in Figures 1 and 2. Analysis of the immobilisation data by the moving average method of Thompson (1947) based on the nominal test concentrations gave the following results:Time (h) EC

### so (mgtl) 950f0 Confidence Limits (mgtl) 24 48 1.3 1.1 

### 1.1 

# - 1.5 0.92 - 1.3 The No Observed Effect Concentration after 24 and 48 hours exposure was 0.56 mgl!. The No Observed Effect Concentration is based upon zero immobilisation at this concentration. 

## 0001bOPage 156



### 13­ SPL PROJECT NUMBER: 656/011


### 5.2.2 Physico-chemical measurements The results of the physico-chemical measurements are given in Appendix 

### I. Temperature was maintained at 21 • C throughout the study, wh iIe there were no treatment related differences for oxygen concentration or pH. 

### 5.2.3 Verification of test concentrations Analysis of the test preparations at 0 hours (see Appendi,x II) 

### showed the measured test concentrations to be within 20% of nominal values with the exception ofthe 0.10, 0.18, 0.32, 0.56 and 1.0 mg/l test groups which showed measured concentrations of 139, 127, 145, 136 and 123% of nominal respectively. These high measured values are considered to be due to sampling/analytical variation given that the measured concentrations at 48 hours were all near nominal. It was therefore considered justifiable to calculate the EC

### so values based on nominal test concentrations only. Shielding the test vessels from direct light maintained the test concentrations near nominal as pre-study stability analysis (see Appendix II) 

### showed the test material to be unstable in light. 6. CONCLUSION The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC

## so value of 1.1 mg/I with 95% confidence limits of0.92 - 1.3 mg/l. The No Observed Effect Concentration at 48 hours was 0.56 mg/I. 7. REFERENCE Thompson, W R 

### (194n Use of Moving Averages and Interpolation to Estimate Median-Effective Dose 

### Bact Reviews, 11, 115 - 145. 

## 000:16.1Pag.e 157 

# CONF\OENTlAl SPL PROJECT NUMBER: 656/011



# , Ii ACUTE TOXICITY TO DAPHNIA MAGNA

### ~----------------~TABLE1 

### CUMULATIVE IMMOBILISATION DATA IN THE RANGE-FINDING STUDY Nominal Concentration (mgt!) i 

## I 'Cumulative Immobilised 

### Daphnia (Initial Population: 10 per Replicate) 24 hours 48 hours Control 0 0 0.10 0 0 1.0 

### 6 8 10 10 10 100 10 10



## 0001.62

### Page 158 

# CONFIDENTIAL SPL PROJECT NUMBER: 656/011


# , 1ACUTE TOXICITY TO DAPHNIA MAGNA

### ~--------------~TABLE2 CUMULATIVE IMMOBILISATION DATA IN THE DEFINITIVE STUDY 

## I Cumulative Immobilised 

### Daphnia Nominal (Initial Population: 

### 10 per Replicate) Concentration 

### 24 hours 

### 48 hours (mgt!) R, R2 Total 

### Ofo R, R2 Total 

### % Control 

### 0 0 0 0 0 0 0 0 0.10 0 0 0 0 0 0 0 0 0.18 0 0 0 0 0 0 0 0 0.32 0 0 0 0 0 0 0 0 0.56 0 0 0 0 0 0 0 0 1.0 4 5 9 

### 45 5 6 11 

### 55 1.8 6 7 

### 13 65 8 8 16 80 3.2 10 10 20 100 10 10 20 100 5.6 10 10 20 100 10 10 20 100 10 10 10 20 100 10 10 20 100 R, - R2 - Replicates 1 and 

### 2**000:1~:i3**

# I Page 159 

# CONFlDENTlAL -16­ 

### SPl PROJECT NUMBER: 656/011 

# I: ACUTE TOXICITY TO DAPHNIA MAGNA

### ~--------------~F'IGUREl CONCENTRATION-RESPONSE CURVE AFTER 24 HOURS
99.91**~-------------o---o---<:)**

91.8 

# J



# I 30 If.
10

1 .. ­

0.010---0---0----(>---..1.------------' 0.1 1 10 COIlCiilUlon ( mgII)


## 000164: 0.1Page 160

-17­ 

# CONFIDENTIAl SPL PROJECT NUMBER: 656/011


# , t ACUTE TOXICITY TO DAPHNIA MAGNA

## ~----------------~FIGURE2 CONCENTRATION-RESPONSE CURVE AFTER 48 HOURS 

### 99.• ,....--------------0---0---9

1 <...<
0.1 

### 0.016---0---0--<>--..1....-----------:**~1** 1 

### 10 

## CcncIi*.,(mgII)


## 000165Page 161

-18­ 

### SPL PROJECT NUMBER: 656/011

APPENDICES



## 000:166

### Page. 162 "



### -19­ 

# CONFIDENTIAL SPL PROJECT NUMBER: 656/011 

# I IJ ACUTE TOXICITY TO DAPHNIA MAGNA



### ....

### -------~A~'PENDIX I PHYSIC()'cHEMICAL MEASUREMENTS

I 

## o hours 

### 24 hours 

### 48 hours Nominal I Concentration 

### I (mgtl) pH mg 

## 0/1 T'C T'C pH mg 

## 0/1 T'C Control Rl 

### 7.9 8.3 21.0 21.0 

### 7.9 8.0 21.0' R2 

### 7.9 8.3 21.0 21.0 

### 7.9 8.1 21.0 0.10 Rl 

### 7.9 8.3 21.0 21.0 

### 7.9 8.1 21.0 R2 

### 7.9 8.3 21.0 I 21.0 An 

### 8.0 21.0 

## ......... 0.18 Rl 

### 7.9 8.3 I 

### 21.0 21.0 8.0 8.0 21.0 . R2 A'l 

### 1.0 21.0 

# I 7.9 8.1 21.0 0.32 Rl 'l 

### 21.0 .21.0 

### 7.9 8.1 21.0 R2 

### 7.9 8.3 21.0 21.0 

### 7.9 7.9 21.0 0.56 Rl 

### 7.9 8.3 21.0 21.0 

### 7.9 8.0 21.0 R2 

### 7.9 8.3 21.0 21.0 

### 7.9 8.0 21.0 1.0 Rl 

### 7.9 8.3 21.0 21.0 

### 7.9 8.0 21.0 R2 

### 7.9 8.3 21.0 21.0 

### 7.9 8.1 21.0 1.8 Rl 

### 7.9 8.3 21.0 21.0 

### 7.9 8.1 21.0 R2 

### 7.9 8.3 21.0 21.0 

### 7.9 8.0 21.0 3.2 Rl 

### 7.9 8.3 21.0 21.0 8.0 8.0 21.0 R2 

### 7.9 8.3 21.0 21.0 

### 7.9 7.9 21. 5.6 Rl 

### 8.3 .0 21.0 

### 7.9 8.0 

# ......... R2 

### 8. 

## ..... ... 

### 21.0 

### 7.9 8.0 21.0 10 R, 

### 8.3 21.0 21.0 

### 7.9 7.9 21.0 R2 

### 7.9 8.3 i 

### 21.0 21.0 

### 7.9 8.0 21.0 Rl and R2 - Replicates 1 and 

### 2. 

## 000167

### Page 

### 163

-20­ 

# CONFIDENTIAL SPL PROJECT NUMBER: 656/011 

# I
........________**,~I** ACUTE TOXICITY TO DAPHNIA MAGNA 

### APPENDIX II VERIFICATION OF TEST CONCENTRATIONS 1. METHOD OF ANALYSIS 1.1 Summary The test material concentration in the test samples was determined spectrophotometrically. 1.2 Samples A 

### volume of test sample was passed through an isolute C'8(EO cartridge which had been pre<onditioned with methanol (10 ml) followed by water (10 mI). The test material was eluted from the cartridge with methanol:5M sodium**hy~roxide** 

### (49.5:0.5 v/v) to give a final theoretical test material concentration of 10 or 20 mgt!. 1.3 Standards Standard solutions of test material were prepared in methanol:5M sodium hydroxide (49.5:0.5 v/v) at nominal concentrations 

# of 10 and 20 mgtl. 1.4 Procedure The standard and sample solutions were analysed spectrophotometrically using the following conditions: Spectrophotometer Perkin-Elmer Lambda 2 Wavelength Amax at - 629 nm Cell path length 1 cm Reference medium methanol:5M sodium hydroxide (49.5:0.5 v/v)



## 000:168

### Page 164 

# CONFIDENTIAl SPL PROJECT NUMBER: 656/011 

# I )ACUTE TOXICITY TO DAPHNIA MAGNA 

### APPENDIX II (continued) VERIFICATION OF TEST CONCENTRATIONS 2. VALIDATION 2.1 Linearity A range of standard solutions was prepared covering 0 to approximately 200% of the higher working concentration and analysed. The detector response was determined to be linear up to approximately 40 mgt\. The results are presented graphically on page 22. 2.2 Recoveries A range of test samples was prepared in bottles, accurately fortified at known concentrations of test material, and analysed.

Fortification Recoveries (mgtl) (mgtl) (Ofo) . 

### Mean ('0) 

### 0.0988 0.127 128
98 0.316 0.291 92 0.988 0.883 89 3.16 2.63 83 9.88 9.49 96 The method has 

### been considered to 

### be sufficiently acurate for the purposes of this study. The test sample results have not been corrected for recovery. 2.3 Limit of Quantitation The limit of quantitation has been assessed down to 0.0098 mgtl. 

## 0001G9Page 165 

# CONFIDENTIAL lV1lN3Q\:JNOJ -22­ SPL PROJECT NUMBER: 656/011 

# , IACUTE TOXICITY TO DAPHNIA MAGNA



### .........

## -----~A~P~PE~N~D~IX·II (continued) VERIFICATION OF TEST CONCENTRATIONS LINEARITY OF DETECTOR RESPONSE

50.
40.
30.
20.
10. 

# 0._ 

# 0._
UIID 11._ 

# I'.. H._ •._ 11._ A 0.0.• a-LIm 

### ,-1,..



### o

..**~----~------~----~------~**

0.0 10.0 20.0 30.0 40.0 

### CONCEN"l'RAnONcmtm



## 000:1';'0Page 166



### -23­ 

# CONFIDEN11AI. SPL PROJECT NUMBER: 656/011 

# I I ACUTE TOXICITY TO DAPHNIA MACNA



### ...

### ------A!I!P!I!!P!I!!E!I!IIN!I!!!D~IX II (continued) VERIFICATION OF TEST CONCENTRATIONS 3. STABILITY 

### A range of pre-study test samples was prepared in bottles and analysed initially and then after storage at ambient temperature in light and dark conditions for approximately 48 hours (equivalent to the study exposure period).

Nominal concentration (mglL) 0.10 0.32 1.0 3.2 10 Concentration found initially (mglL) 0.129 0.318 0.914 2.70 8.73 Concentration found after storage in light conditions (mg/L) 0.0751 0.182 0.628 2.03 6.37 Expressed as a % of the initial concentration 58 57 69 75 73 Concentration found after storage in dark conditions (mg/L) 0.147 0.354 1.01 3.32 10.2 Expressed as a % of the initial concentration 114 111 110 123 117 

### The test samples have been shown to be unstable in light conditions.



## 000:1.'; .i

### Page 167 

# CONFIDENTIAL -24­ SPL PROJECT NUMBER: 656/011 

# :**~:** ACUTE TOXICITY TO DAPHNIA MAGNA 

# I

....

### ------A~P!I!!P~E~N!I!D~IX II (continued) VERIFICATION OF TEST CONCENTRATIONS 4. RESULTS Samples 

## I Nominal concentration (mgt!) Concentration found (mgt!) Expressed as a 

### % of the nominal concentration 

# o hours Control 0.10 0.18 0.32 0.56 1.0 1.8 3.2 5.6 10 <LOQ 0.139 0.229 0.463 0.763 1.23 2.13 3.70 6.04 10.4 

# - 139 127 145 136 123 118 116 108 104 48 hours Control 0.10 0.18 0.32 0.56 1.0 1.8 3.2 5.6 10 <LOQ 0.101 0.197 0.331 0.628 1.03 1.91 2.98 5.52 10.4 

# - 101 109 104 112 103 106

93 99 104 LOQ - Limit of quantitation



## 0001''72

# 1 Page 168



# e.a**.,....--------------~** 

### 8.G724 

### 8.li18

1\ 

### 8.8322 8.8U16 

# CONFIDENTIAL -25- SPL PROJECT NUMBER: 656/011 

# I: ACUTE TOXICITY TO DAPHNIA MAGNA



### - ....-----IIII!IAl!!lp!'!PE!I!INII!I!O!l!IX II (continued) 

### TYPICAL SPECTRA
.



# • Standard 10 mill 8.151 .,..-------------------, 

### 8.118 8.• A 

## '.854 

# 8.m -i.811 

# +----r-----r---.,...-----,-----t 2.tI 

# • Standard 20 mill


## 000173Page. 169 

# CONFIDENTIAl. -26­ SPL PROJECT NUMBER: 656/011



# , .1: ACUTE TOXICITY TO DAPHNIA MACNA

## '--------~A~PP!I!!E!I!INII!I!D~IX II (continued)


### 8.S 

### 8.m4 1.8518

A 

### I.W 8.81.**~.81.**

# •
8.S 8.8724 8.8518



# " '.8322 

### ' ••186**~.11GI**

# • TYPICAL SPECTRA



# ... see 

# • Control



# ...• Test Sample 0.10 

## millPage 170 

# CONFIDENTIAl SPL PROJECT NUMBER: 656/011


# , Z ACUTE TOXICITY TO DAPHNIA MAGNA



### ....

## -----~AII!!IP!!I!!PE!!I!INI!I!D~IX II 

### (continued) TYPICAL SPECTRA 

### e.8938 e.8724 8,8518

A 

### e.8312 8.el"

....el.. 

## zee 

# • Test Sample 0.18 

### in&ll 

# • 

# • SM 

# e.2S8 --------------------, e.11I e

.• A 

# e.a 

# e.m

....el • .j.ll---..----r-----r------,------i 

## zee 

# • Test Sample 0.32 

### m&ll


## 0001.'/5

# I Page 171 

# CONFIDENTIAL -28­ 

### SPL PROJECT 'NUMBER: 

### 656/011 

# :: ACUTE TOXICITY TO DAPHNIA MAGNA

# L.------A~P~P!!'!!E~N~D~IX II (continued) 

### TYPICAL SPECTRA 8.8938 .,...-----------------, 

### 8.8?24 

### 8.8518

A 

### 8.8322 8.81.

### -e.el~.U1---r--- 

# • Test Sample 0.56-

### mg/I 

# •**..,.----S8t.,..----r---~** 

# e.15I.....----------------, e.l18 

# e

.• A 

# e.est e.1Z2**~'**



# •**..,.----S8t.,..----,...--~** 

# •**-e.e~w.---r---** Test Sample 1.0 

### mgli


## OOO:1'-;t:i

# • • Page 172 

# CONFIDENTIAL -29- SPL PROJECT NUMBER: 656/011 

# I i ACUTE TOXICITY TO DAPHNIA MACNA

## 4-------A~P~P~E~N~D~IX II 

### (continued) TYPICAL SPECTRA 

# e.158 ------------------, e.U8 

# e

.• A 

## e.854

.. 

## e.122 

# ....e1l .j..II..--..,...-----,r-----r-----r----; 

## zee

e.s e.11I
e.• A 

## '.854 e.122



# .....1. Z8I 

# • Test Sample 1.8

## mill



# • see Test Sample 3.2

## mill
0001.'7'7

# • 

# •• 

# I Page. 173



### ·30- . 

# CONFIDENTIAL SPL PROJECT NUMBER: 656/011 

# ; ACUTE TOXICITY TO DAPHNIA MAGNA



### - ....-----A-P
..

## P~EN~D~IX II 

### (continued)

TYPICAL SPECTRA 

# '.158

••US 

# e

.• A 

# '.851 '.m

.....11 

# • !

Test Sample 

### 5.6 mgll 

# '.158

••UI 

# e

.• A 

# e.851 e.m

....111 

# • 

# • 

# •• Test Sample 10 

### mgII

### Page 174 

# CONFIDENTIAL -31- SPL PROJECT NUMBER: 656/011



# , I: ACUTE TOXICITY TO DAPHNIA MAGNA



### ...

### --------~A~PPENDIX IV RECONSTITUTED WATER i) 

### Stock Solutions a) CaCI 2

### ·2H2

### O 11.76 

### gil 

## b) MgS04

### ·7H 2

### O 4.93 gil c) NaHCO) 2.59 gil d) KCI 0.23 gil ii) 

### Preparation An aliquot (25 ml) ofeach of solutions 

### a-d wa'S added to each litre (final volume) ofdeionised water with a conductivity of 

## <5 

### pS

## cm-1 

### and pH equal to 7.8 

### ± 0.2, adjusted (if necessary) with NaOH or HC!. The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value. The reconstituted water has an approximate theoretical total hardness of 250 mgll as CaCO).**UOO1.'./~**

### Page 175



### -32­ SPL PROJECT NUMBER: 656/011



### THEDEPARTMENT OF HEALTHOF THE GOVERNMENT OFTHE UNITED KINGDOM GOOD LABORATORY PRACTICE STA**TE.~E.vr** OFCOMPLlANCI IN ACCORDANCI WITH DIRECTIVE 111320 IIC LABORATORY TEST TYPE SaCePhar. Laboratortes Ltd. Analytical Cha.lstry P.O. Box No. 4.5 Envlron.antal Tox. Derby DEl 28T Environ.ental Fate Hutaaanictty Phys/Cha•. tests ToxlcololY

DATE OF INSPECTION 22 January 1996 A Ill'Iem jQspllCtion fot compliance with the Principles of Good Laboratory Praclice was carried OIIt II the above liboratory IS pan of the UK GLP Compliance Prorramme. At the time of the inspection no dcviitiollJ were found of suMaent malDitude to affect the validity offtOD-clinicai siudia performed at these facilities.

# @It(~ ,/1

# ~

# 1,( D.F.

# ~oorc '(tI.{' Director UK GLP**~onilorinl** Allchoril), 

# (JI018d