ft 

~EPA \\ Jl~t1 

UHl1pktcd 


'~lld thl' 

101111 to 

Enter the total number of pages 

in the Premanufacture Notice 

Ires 10-3 I -96.! 

• 	 You must provide all information requested in this form to the extent that it is known to or reasonably ascertainable by you. Make reasonable estimates if you 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 by calling 202·554.1404, or faxing 202·554·5603). 

If a 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~

CDDT 

" I I 

Company Sanitized 

PREMANUFACTURE 

NOTICE 

FOR NEW 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 lNFORMA nON 

You must provide the currently correct Chemical Abstracts (CAl Name ofthe new 

chemical substance, even if you claim the identity as confidentiaL You may authorize 

another person to submit chemical identity information for you, but your submission will 

not be complete and the review will not begin until EPA receives this information. A 

letter in support of your submission should reference your TS user fee identification 

number, You must submit an original and two copies of this notice including all test data. 

If you 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, or use operations to be described in Part II, 

sections A and B of this notice, reproduce the sections as needed. 

Part III - LIST OF A TT ACHMENTS 

Attach additional sheets if there 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 evaluating the new 

substance. On page II of this 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 of consent orders or Significant New Use 

Rules. Except in the case of exemption 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 itsel f prohibit the submitter from later deviating from the information (except 

chemical identity) reported in the form. 

CONFIDENTIALITY CLAIMS 

You may claim any information in this notice as confidential. To assert a claim on 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. If you claim information in the notices as confidential, 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) if any information in this notice is claimed as confidential. 

TEST DATA AND OTHER DATA 

You are required to submit all test data in your possession or control and to provide 

a description of all other data known to or reasonably ascertainable by you, if these 

data are related to the health and environmental effects on the manufacture, 

processing, distribution in commerce, use, or disposal of the new chemical 

substance. Standard literature citations may be submitted for data in the open 

scientific literature. Complete test data (written in English), not summaries of data. 

must be submitted if they do not appear in the open literature. You should clearly 

identitY whether test data is on the substance or on an analog. Also, the chemical 

composition of the tested material should be characterized. Following are 

examples of test data and other data. Data should be submitted according to the 

requirements of §720.50 of the 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 

or control oftne submitter 

A physical and chemical properties worksheet is located on the last page of this form. 

TYPE OF NOnCE (Check Only One) 

[8J 

D 

D 

D 

D 

D 

D 

PMN (Premanufacture Notice) 


INTERMEDIATE PMN (submitted in sequence with final product PMN) 


SNUN (Significant New Use Notice) 

TMEA (Test Marketing Exemption Application) 

-T'1

LVE (Low Volume Exemption)@40 CFR 723.5~1) nQ 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 A CONSOLlDA TED PMN? Yes 

# of chemicals 


(Prenotice Communication # required, enter # on page 3) 


EPA FORM 7710·25 (Rev. 5·95) 	 Replaces previous editions of EPA Fonn 7710·25. 00000:1 
Public reporting burden for this collection of information 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 of this collection of information, including suggestions for 

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

20460; and to the Office of Management and Budget, Paperwork Reduction Act (2070-00! 2), Washington, D.C. 20503. 

CERTIFICATION 

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

1. The company named in Part I, section A, subsection la ofthis notice form intends to manufacture or import for a 

commercial purpose, other than in small quantities solely for research and development, the substance identified in Part I, 

Section B. 

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

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

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

Additional Certification Statements: 

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

statement that applies: 

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

o The Company named in Part I, Section A has remitted the fee of$1 000 for an Intermediate PMN (defmed @ 40 CFR 

700.43) in accordance with 40 CFR 700.45(b), or 

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

in accordance with 40 CFR 700.45(b). 

If you are submitting a low volume exemption (LVE) application in accordance with 40 CFR 72 3 .50( c)( 1) or a Low release 

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

statements: 

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

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

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

o The new chemical substance for which the notice is submitted meets all applicable exemption conditions. 

Ifthis application is for an LVE in accordance with 40 CFR 723.50(c)(I), the manufacturer intends to commence 

manufacture of the exempted substance for commercial purposes within 1 year of the date of the expiration of the 30 day 

review period. 

Confidential 

x 

Signature of agent - (ifapplicable) Date 

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

Confidential 

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. If you are submitting this notice as part of a joint submission, 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 of authorized official 

Company 

Mailing address (number and street) 

City. State, ZIP Code 

[f YOll have had a prenotice connmmication (PC) concerning this notice 

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

4. If YOll previously submitted an exemption application for the cbemical 

sllbslance covered by this notice, enter tbe exemption number assigned by 

EP A. [f you previously submitted a PMN for this subSlance enter the 

PMN number assigned by EPA (i.e. withdrawn or incompiel£). II 

for the chemical subslance covered by this notice. enter the notice nllmber 

assigned by EP A. .. 

Telephone Area Code 

Telephone 

Mark (Xl ~ 

if none 

Mark (Xl ~ 

ifnone 

Mark (Xl ~ 

if none 

0 

0 

0 

X 

X 

X 

\. Manufacture 2. Import 6. Type of Notice - Mark (X) 3. Both 

o Only Only 0 Binding Option 0 Binding Option 

Mark Mark 

EPA FORM 7710-25 (Rev. 5-95) Page 3 
I . 

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 of the reaction or process. (3) Indicate the 
 x 

range of composition and the typical composition (where appropriate). (4) Provide a correct representative or partial chemical structure 

diagram, as complete as can be known, if one can be reasonably ascertained. 

f. 

Complete either item I 

Class I or 2 

a. Class of substance - Mark (X) D Class I or 2 Class 2 x 

rg] 	Mark (X) this box if you attach a continuation sheet. (See Page 15) 

EPA FORM 7710-25 (Rev. 5-95) 	 Page4a 
If another person will submit chemical identity information for you (for either Item I or 2). mark (X) the box at the right. 

of that in a continuation sheet. 

1. Class 1 or 2 chemical substances (for definitions of class I and class 2 substances, see the Instructions Manual) 

a. Class of substance Mark (X) I D Class I or 2 D Class 2 

b. Chemical name (Currently correct Chemical Abstracts (CA) Name that is consistent with TSCA Inventory listings for similar substances. 

For Class I substances a CA Index Name must be provided. For Class 2 substances either a CA Index Name or CA 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 or obtain the specified chemical identity information reported in this notice: (check one). 

o Method I (CAS Inventory Expert Service - a copy of the 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 (if a 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 of the reaction or process. (3) Indicate the 

range of composition and the typical composition (where appropriate). (4) Provide a correct representative or partial chemical structure 

diagram, as complete as can be known, if one can be reasonably ascertained. 

Contidential 

x 

x 

x 

attach a continuation sheet. (See 16) 

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

000005 
Class I or 2 chemical substances (for definitions 

a. Class of substance· Mark (X) 1 

b. Chem1cal name (Currently correct 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 either a 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 of the 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 	

d. 	 Mo substance)

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 their respective CAS Registry Numbers. (2) Describe the nature of the reaction or process. (3) Indicate the 

range of composition and the typical composition (where appropriate). (4) Provide a correct representative or partial chemical structure 

diagram, as complete as can be known, if one can be reasonably ascertained. 

f. 

attach a continuation sheet. 

Page 4c EPA FORM 7710·25 (Rev. 5·95) 

000006 


x 
substance based on 

If another person will submit chemical identity information for you (for either Item I or 2), mark (X) the box at the right. 

the of that in a continuation sheet. 

I, Class I or 2 chemical substances (for delinitions of class I and class 2 substances, see tlie Instructions Manual) 

a, Class of substance - 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 of the 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 (if a number already exists for the substance) 

e, 1 substance, a complete and correct 

precursor substances with their respective CAS Registry Numbers, (2) Describe the nature of the reaction or process. (3) Indicate the 

range of composition and the typical composition (where appropriate). (4) Provide a correct representative or partial chemical structure 

diagram, as complete as can be known. if one can be reasonably ascertained. 

Conndential 

x 

x 

CAS# 

structure 2 substance - (I) List the immediate 

x 

this box if you attach a continuation sheet. 

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

00000'( 
Section B - CHEMICAL substance based on IDENTITY 

Complete enher item 1 (Class 1 or 2 substances) or 2 (Polymers) as appropriate, Complete all other items, 


If another person will submit chemical identity information for you (for either Item I or 2), mark (Xl the box at the right 
 Contildenti the and 	 in a sheet. dential 

I, Class 1 or 2 chemical substances (for definitions of I and class 2 substances. see the Instructions Manual) 

a, Class of substance - Mark (X) I Class 1 or 2 0 Class 2 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 of the 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 of the reaction or process. (3) Indicate the 

range of composition and the typical composition (where appropriate). (4) Provide a correct representative or partial chemical structure 

diagram. as complete as can be known, if one can be reasonably ascertained. 

f 

[8J Mark (X) this box if you attach a continuation sheet. (See Page 19) 

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

000008 

x 
Confidential 

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

d. 

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

below 1,000 absolute molecular weight of that composition. 

Describe the methods of measurement 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 (if a number exists) of each monomer or other reactant used in the manufacture of 

the polymer. 

(2) Mark (Xl this column if entry in column (I) is confidential. 

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

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

description on the TSCA Chemical Substance Inventory. 

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

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

commercial purposes. 

% % 

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 or partial chemical sttucture diagram, as complete as can be known, if one can be reasonably 

D 

Page 5a 

000009 
X 

Contidential 

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

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

below 1,000 absolute molecular weight of that composition. 

Describe the methods of measurement 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. 

o 

Mark 

obtained from CAS Inventory Expert Service must be submitted as 

as attachment to this 

X 

Typical 

% 

% 

% 

% 

% 

X 

% 

X % X 

% 

% 

% 

% 

d. The currently correct Chemical Abstracts (CAl name for the polymer that is consistent with TSCA Inventory listings for similar polymers. 

e. Provide a correct representative or partial chemical structure diagram, as complete as can 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 claim as confidential 

(I) Provide the specitic chemical name and CAS Registry Number (if a number exists) of each monomer or other reactant used in the manufacture of 

the polymer. 

(2) Mark (X) this column if entry in column (I) is contidentiaL 

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

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

description on the TSCA Chemical Substance Inventory. 

(5) Mark (X) this column ifentries in columns (3) and (4) are confidential. 

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

commercial purposes. 

attach a continuation sheet. 

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

00001.0 
Contidential 

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

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

below 1,000 absolute molecular weight of that composition. 

Describe the methods of measurement 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 claim as confidential 

(I) Provide the specific chemical name and CAS Registry Number (if a number exists) of each monomer or other reactant used in the manufacture of 

the pol ymer. 

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

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

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

description on the TSCA Chemical Substance Inventory. 

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

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

commercial purposes. 

Mark 

method you 

X 

x 

(CAS Inventory Expert Service - a copy of the identification report 

obtained from CAS Inventory Expert Service must be submitted as 

as attachment to this 

% 

% X 

% X 

% 

% 

% 

(check one). 

currently correct name for the polymer that is consistent with TSCA Inventory listings for similar polymers. 

% 

% X 

% X 

% 

% 

% 

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

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

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

000011 
Part I - GENERAL INFORMATION ­Continued 

Section 8 - CHEMICAL IDENTITY INFORMATION - Continued 

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

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

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

below 1,000 absolute molecular weight of that composition, 

Describe the methods of measurement 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 separate cont1dentiality claims for monomer or other reactant 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 (if a number exists) of each monomer or other reactant used in the manufacture of 

(2) -

(3) 

(4 ) 

the polymer, 

Mark (X) this column if entry in column (1) is confidential. 

Indicate the typical weight percent of each monomer or other reactant in the polymer. 

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

description on the TSCA Chemical Substance Inventory. 

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

(6) Indicate the maximum weight percent of each monomer or other reactant that may be present as a residual in the polymer as manufactured 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 or obtain the specified chemical identity information reported in this notice (check one). 

D Method I (CAS Inventory Expert Service - a copy of the identification report D Method 2 (other source) 

obtained from CAS Inventory Expert Service must be submitted as 

as 

The currently correct Chemical Abstracts (CA) name for the polymer that is with TSCA Invenf,flrv 

Prov' a correct or partial chemical structure diagram, as complete as can be known, if one can be reasonably ascertained. 

X 

00001.2 

Contidential 

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

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

below 1,000 absolute molecular weight of that composition. 

Describe the methods of measurement 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 other reactant 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 (if a number exists) of each monomer or other reactant used in the manufacture of 

the poIymer. 

(2) Mark (X) this column if entry in column (I) is confidential. 

(3) Indicate the typical weight percent of each monomer or other reactant in the polymer, 

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

description on the TSCA Chemical Substance Inventory. 

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

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

commercial purposes, 

Mark this column if 

% % 

X % X % X 

X % X % X 

% % 

% % 

% 

reported in this notice (check one). 

Method 2 (other source) 

known, if one can be reasonably ascertained. 

X 

obtained from CAS Inventory Expert Service must be submitted as 

as attachment 

d. The currently correct Chemical Abstracts (CA) name for the polymer that is consistent with TSCA Inventory 

e. Provide a correct representative or partial 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 if available. [f there are unidentified impurities, enter "unidentified." 

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

6. 	 Generic chemical name- If you claim chemical identify as w"'".....ltial, you must provide a generic name for your substance that reveals 

the specific chemical identity of the 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 box if you 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 if available. 

""rMIll'! CAS RegisyY Number ~JI'(I)~~' 

(2) ;;~j

X 

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

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

000014: 

0 
Part I - GENERAL INFORMATION -- Continued 

Section C - PRODUCTION, IMPORT, AND USE INFORMATION: 

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

l. Production volume - Estimate the maximum production volume during the first 12 months of production. Also estimate the maximum 

production volume for any consecutive 12-month period during the first three years of production. Estimates should be on 100% new chemical 

substance basis. For a Low Volume Exemption application, if you 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. If granted, you are bound to this volume 

Maximum first 12-month production (kg/yr) Maximum 12-month production (kglyr) Confi· Binding 

(100% new chemical substance basis) (100% new chemical substance basis) dential Option 

Mark (x) 

X 

2. Use Information - You must make separate confidentiality claims for the description of the category of use, the percent of production volume 

devoted to each category, the formulation 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 of use of the new chemical substance by function and application .. 

(2) -­ Mark (X) this column if entry column (1) is confidential business information (CBI). 

(3) -­ Indicate your willingness to have the information provided in column (I) binding. 

(4) -­ Estimate the percent of total production for the first three years devoted to each category of use. 

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

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

commercial purposes at sites under your control associated with each category of use. 

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

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

your willingness to have the use type provided in (8) binding. 

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

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

Option tion% Form­ (8) 

(by function and application i.e, a dispersive 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 

% -

% % 

% % 

% % 

% % 

% % 

• If 

In 

you 

addition 

have 

include 

identified 

estimates 

a "consumer" 

of the concentration 

use, please provide 

of the 

on 

new 

a continuation 

chemical substance 

% 

sheet a 

as 

detailed 

expected 

description 

in 

% 

consumer 

of the 

products 

use(s) of 

and 

this 

describe 

chemical 

the 

substance 

chemical 

in 

reactions 

consumer 

by 

products, 

which 

H

this 

substance loses its identity in the consumer product. 

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

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

use Instructions Manual for examples of generic use descriptions, 

description Colorant for aqueous ink applications 

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

3. Hazard Information - Include in the notice a copy of reasonable facsimile of any hazard warning statement, label, material safety data sheet, or other r= 

information which will be provided to any person who is reasonably likely to be exposed to this substance regarding protective equipment or practices 

for the safe handing, transport, use, or disposal of the new substance. List in part III hazard information you include, 

[gJ Mark (X) this box if you attach hazard information, (See pages 25-29) 

EPA FORM 7710-25 (Rev, 5-95) Page 7 

000015 
x 

Complete section A for each type of manufacture, 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. Confiof the site at which the will occur. dential 

Site address (number and street) 

City, County, State, ZIP 

If the same operation will occur at more than one site, enter the number of sites. Identify the 


additional sites on a continuation sheet, and if any of the sites have significantly different 


production rates or operations, 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) 	 Diagram the major unit operation steps and chemical conversions. Include interim storage and transport containers (specify- e.g. 5 gallon pails, 55 gallon 

drum, rail car, tank truck. etc.). 

(2) 	 Provide the identity, the approximate weight (by kg/day or kgibatch on a 100010 new chemical substance basis), and entry point of all starting materials and 

feedstocks (including reactants, solvents, catalysts, etc.), and of all products, recycle streams, and wastes. Include cleaning chemicals (note frequency if not 

used daily or per batch.). 

(3) 	 Identify by number the points of release, including small or intermittent releases, to the environment of the new chemical substance. 

See Process Description on Page 30 

Mark (X) this box if you attach a continuation sheet. (See page 30) 

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 of worker activity, physical form of the new chemical substance, 

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

at the time of exposure. 

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

(8) -- Estimate the maximum number of workers involved in each activity for all sites combined. 

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

eI0) and (II) -­ Estimate the maximum duration of the activity for any worker in hours per day and days per year. 

(12) -­ Mark (X) this column if entries 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 if you attach a continuation sheet 

3. Environmental Release and Disposal -­ You must make separate contldentiality claims for the release number and the amount of the new chemical substance 

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

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

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

(3) Mark (X) this column if entries 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 or off-site land or incineration. POTW. or other (specify» to 

which the new substance will be released from that release point 

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

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

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

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

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

(7) Identify the destination(s) of releases to water. Please supply NPDES (National Pollutant Discharge Elimination System) numbers for direct discharges or 

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

Release Amount of new 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 

(7) Mark (X) the o POTW provide 

H

name(s) below: CBI o Navigable o Other - Specify provide NPDES # CBI ,-­ destination(s) of waterway 

releases to water. 

Mark (X) this box if you 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 or bracket the specific information that you claim as confidential. 

In this section you may provide information not reported elsewhere in this form regarding your efforts to reduce 

or minimize potential risks associated with activities surrounding manufacturing, processing, use and disposal 

ofthe PMN substance. Please include new information pertinent to pollution prevention, including source 

reduction, recycling activities and safer processes or products available due to the new chemical substance. 

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

modification, process and procedure modification, product reformulation, raw materials substitution, and/or 

inventory control. Recycling refers to the reclamation of useful chemical components from wastes that would 

otherwise be treated or released as air emissions or water discharges, or land disposal. Descriptions of pollution 

prevention, source reduction and recycling should emphasize potential risk reduction subsequent to compliance 

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

information to assess overall net reductions in toxicity or environmental releases and exposures, not the shifting 

ofrisks to other environmental media or non-environmental areas (e.g., occupational or consumer exposure). In 

addition, information on the relative cost or performance characteristics ofthe PMN substance to potential 

alternatives may be provided. 

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

substance. See Instructions Manual and Pollution Prevention Guidance manual for guidance and 

examples. 

Describe the expected net benefits, such as (1) an overall reduction in risk to human health or the environment; (2) a reduction in the 

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

reduction in potential toxicity or human exposure and/or environmental release; (5) an increase in product performance, a decrease in 

the cost ofproduction and/or improved operation efficiency of the new chemical substance in comparison to existing chemical 

substances used in similar application; or (6) the extent to which the new chemical substance may be a substitute for an existing 

substance that poses a greater overall risk to human health or the environment. 

CONF\DEN1\~1 ~ 

I'2J Mark (X) this box if you attach a continuation sheet. (See Study Reports 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 of the fonn and test data and other data (including physical/chemical properties and 

structure/activity infonnation), and optional infonnation after this page. Clearly identify the attachment and the section of the fonn to 

which it relates, if appropriate. Number consecutively the pages ofthe attachments. In the column below, enter the inclusive page 

numbers of each attachment. 

Mark (X) 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 of the notice fonn a sanitized 

version of any attachment in which vou claim information as confidential. 

Attachment name Attachment Conlipage number(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 if you attach a continuation sheet. Enter the attachment name and number. 

EPA FORM 7710-25 (Rev. 5-95) Page 12 


000019 
PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET 

To assist EPA's review of physical and chemical properties data, please complete the following worksheet for data you provide and 

include it in the notice. Identify the property measured, the page of the notice on which the property appears, the value of the property, 

the units in which the property is measured (as necessary), and whether or not the property is claimed as confidential. The physical 

state of the neat substance should be provided. These measured properties should be for the neat (100% pure) chemical substance. 

Properties that are measured for mixtures or formulations should be so noted (% PMN substance in --->. You are not required to 

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

information is properly protected. You should submit this worksheet as a supplement to your submission of test data. This worksheet 

is not a substitute for submission of test data. 

Property 

(a) 

Mark 

(X) tf 

provided 

Page 

number 

(b) 

Value 

(c) 

lMeasured 0 

Estimate 

(M or E) 

Confidenti.1 

Mark (X) 

(d) 

Physical state of neat 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) torr pressure °C 

Spectra X 

Dissociation constant 

Particle size distribution 

Octanol / water partition coefficient 

Herny's Law constant 

Volatilization from water 

Volatilization from 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 CONFJ DENTIAL 

PAGE 14 


000021 
Page l5 

Cffi Inventory Expert 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. OhIo 43110-0334 
Page 16 

ern Inventory Expert Service 


A division of the American 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. Ohio 43110.0334 000023 
Page 17 

ern Inventory Expert Service 


A division of the American 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. Ohio 43210-0334 
Page 18 

ern Inventory Expert Service 


A division of the American 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. Ohio 43210.0334 000025 
Page 19 

Inventory Expert 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]]4 
Page 20 

Qnent 0IIte 1011912000 
 CONFIDENTIAL 10f5 

000027 

Page 21 


CONFIDENTIAL 


000028 

Page 22 


000029 

Page 23 

Page ')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 

Company 

CAS Number: 

Identification 

Blend A'..

Milliken 

P.O. Box 

Chemical 

817 
 MILLIKEN

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 

000032 
Page 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" 1111111111111 111I 

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 111111 II I111111111111111 II 1111111111111111111 

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. 

000041 

2 

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_ 

000044 

5 

Pag~ 39 

SAMPLE NUMBER: 70600017 PAGE

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 

00004:6 

Page 41 

CONFIDENTIAL 


.CONFIDENTIAL 


IREVERSE ~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 IIItabol1 sing , systell. 

4. I 'Ica~sed no reduction in the growth of the 

bacterial lawn at any dose level either with or without metabolic 

act 1 vat i on. ~ -.. - -----]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. 000051 
Page 46 PROJECT UBER: 656/2

CONFIDENTIAL I.......--..... ··~-· ~-·......
 ,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 

Batch number : I >:~. _on .._-­ I

Purity 

Date Received 28 February 1994 

Description dark purple viscous _liquid 

Container clear gl ass 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. A solvent treatlent group was used as the negatfve control and 

the positfve control aaterfals were as follows: 

000053 
Page 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 5 pg/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 E and 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. 

Jest 	Material 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 101 ten. 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, 2 00 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 

TAl 00 103.0 111.0 113.5 103.5 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 

sat i sfactory. 

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._______________1was 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. 

000059 

Page 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 

COLON IES"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 

COlON IESIPLATE 513 ( 511.0 , Z01 ( 211.3 ) 592 ( 569.0 , 1" ( 14.6.3 ) 524 ( 516.7 ) 

493 225 576 156 498 

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


000063 
Page 58 -18­

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 (-) CONCENTRATION 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 

+ 	 COLON IESIPLATI 395 ( 375.3 ) 219 ( 201.3 ) 241 ( 206.0 ) 110 ( 165.7 ) 106 ( 91.0 ) 

354 116 207 177 14 

..................................................................... 
-...-.-------~-..-....-.. 000064 
Page 59 PROJECT NtlmER: 656/2

A P PEN DIe E S 


REVERSE MAllON ASSAY -AMES TEST- REPORT 

• 


000055 

Page 60 

A P PEN 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 

Ioi l tng 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. 

000066 
Page 61 PROJECT NUMBER: 656/2 

A P PEN D I X I (contd) 

[2] TESTER STRAINS 

(1) Procurellent 

Obt.;ned f \"OIl ht. Obtained

BRITISH INOUSTRIAL BIOLOGICAL RESEARCH ASSOCIATION l' AUGUST 1981

(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 appL i cable nullber Met fi U 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) 

A1'0. 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 

Weight • ZODg 0.5 ,/kg

(4) Composition of S9 Mix 


constituents Maunt in 1 al 19 lIix Consti tuenu MaI.I'rt in 1 aL 19 Nix 

19 0.1 .l NADfIM - IaOl 

IIgClZ '.0 .,l IMDP 4.0 IaOl 

6 Lucole-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 


SOlwnt u.td Nethod of .u.pen.ion etc. when te.t .ub.tence i. difficylt to di.lOlve

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.) 

G1. 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 

G'late ..thod 000068
Page 63 -23­

PROJECT NUMBER: 656/2

APPEN 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 

COIIPOa i t 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
.....t1Ye) 

...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 

A P 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 ~~~.~:r~~La=_"_=,, I .- e. ....-...... •••• ~BE_ Of _EVE_TANTS (nuIOer of colonitl/plett) 

WITH (+) OR TEST SUBSTANCE IASE-'AII SUBSTITUTION TYPE '_AltESMIn TYPE 

WITHOUT (-) CONC EHnATION 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 C 511.0 ) 201 ( 211.3 ) 59Z C 569.0 ) '" C 146.3 ) 524 ( 516.7 ) 

_ 495 m 576 . 156 491 .............--_._--.---...__.......... --_......_..._.-­

000073 

-28­

Page 68 PROJECT NOOER: 656/2

A P P E N P I 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 I FT 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 

...... CONFIDENTIAL . o

120 

100+ 

&.oJ 

5 

Go 80 

CA " §

a::: 

~'60 a::: 

40 

~ 

20f 

0 

0 

0 

0 

~J v'J 

I 

0 

0 

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 

." 

~ 

.. 	 • 

,., 


A

n

- __ 	 .... i

,., 

:;a

.. 8 40 	 200 	 eft 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 -0. JI. 	 :Q At. 

312.5 	 625 1250 2500 5000 

UG/PlA1E 

-

w• 

• 

." 

~ n 

~ 

-4 

I

,.., 


:xl

.. 


eft 

~ ....... 


N 
•• 

"d 

Ill' 

OQ 

CONFIDENTIAL --.J

(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- TAl 00 +S9 EXPERIMENT 2 


..... TA1535 +S9 EXPERlUENT 2 


..... WP2uwA-+S9 EXPERIMENT 2 I 


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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 1HE GOVERNMENT 


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 of the UK GLP Compliance PIOIftID.lllC. 

At the time of the inspecdon no deYiad.ou were found of sufracient 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 

Study Title: 

Acute Oral Toxicity Study oli.i~~..............___....}n Rats 


(EPA/otcn Guiaelines) 

Author: 

Steven M. Glaza 

Study Completion Date: 

March 17, 1998 . 

PerformiDI Laboratory: 

Covance Laboratories Ine. 

3301 Kinsman Boulevard 

Madison, Wisconsin 53704 

Laboratory Project Identification: 


Covance 71102252 


Page 1 of38 

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 ofIntemal Trade and Industry Good Laboratory Practice 

Standards, Notification No. 59-85 

Date 

Date 

-2­ 000082 
Page 77 

Covance 71102252 

QUALITY ASSURANCE STATEMENT 

This report has been reviewed by the Quality Assurance Unit of Covance 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 of Good Laboratory Practice, C(81 )30(Final) and the 

Japanese Ministry of Health and Welfare (MOHW), Good Laboratory Practice Standards, 

Ordinance 21 and the Japanese Ministry of International 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 of inspections and findings are issued to Covance 

management according to standard operating procedures. 

Inspection Date Reported to 

Dates Study Director and 

From To Phase Study Director Management 

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 

(EPAJO~t"C"i5p!1G~ui""d"ei"in·elllfs)----.Jj 

T est Material 

Sponsor 

Sponsor's Representative 

Study Director 

Testing Facility 

Study Timetable 

Study Initiation Date 

Experimental (In-life) Start Date 

In-life End Date 

Experimental Tennination Date 

Study Completion Date 

I 


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 9, 1997 

January 30, 1998 

March 17, 1998 

March 17, 1998 

-4­
Page- 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 

Quality Assurance 

Nancy M. Centanni 

Manager 

KEY PERSONNEL 

Laboratory Animal Medicine 

Donna J. 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 


Storage md Retention ...................................................... t •••••••••••••••••••••••••t ........................
9 


Safety PrecautioM ............... , '" II •••••••• 11 ••••• t ••••• a ••••••• " II ................... I ••••••••••••••••••••• 
, ............ 1 0 


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 Administration of Test Material .......................................................... 11 


Reason for Route of Administration .............................................................................. 11 


Observations .................................................................................................................. 11 


Pathology .......................................................................................................................12 


Statistical Evaluation ..................................................................................................... 12 


Location of Raw Data, Records, and Final Report ........................................................ 12 


RESULTS ............................................................................................................ , .., .... , ..,.. 12 


. Mortality ........................................................................................................................ 12 


Body Weight.s .................................................................................... 1 .................................. 12 


-6­ 000086
Page 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 Pathology Comments ..............................................................................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 at the 3,000 mg/kg dose level included blue feces, haircoat, and tail and bluestained urogenital area. All animals treated at 3,000 mg/kg exhibited body weight gain 

during the study. 

Four females treated at 5,000 mg/kg died within 4 days oftest material administration. 

Clinical signs oftoxicity observed at the 5,000 mg/kg dose level included thin 

appearance, hunched posture, hypo activity , red-stained face, nonformed and/or blue 

feces, wet or blue-stained urogenital area, green urine, green-stained haircoat and/or tail, 

and death. All surviving animals returned to a normal appearance by Day 10 after 

treatment with the exception ofthree males which exhibited a green-stained tail through 

study termination and the remaining female which also exhibited a green-stained haircoat 

and tail through to study termination, Animals treated at 5,000 mg/kg and surviving to 

the end ofthe observation period exhibited body weight gain during the study. 

At necropsy, one or more organs or tissues in each animal were observed to be stained 

blue or green, which were attributed to the test material. There were no test 

material-related lesions observed in any ofthe animals. 

Sased on the mortality observed in the study, the estimated oral LD50 values in rats were 

determined to be greater than 5,000 mg/kg for males and 4,128 mg/kg for females. 

- 8 ­ 000088
Covance 71102252 

OBJECTIVE 


The objective of this 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 of administration utilized in this study were chosen based 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). 

TEST MATERIAL CONFIDENTIAl 

Identification 

The test material was identified ai,,~....______...._ ......J:n.d described as a 

viscous, dark blue liquid. 

Purity and Stability 

The Sponsor assumes responsibility for purity and stability d~terminations (including 

under test conditions). 

Storage and Retention 

The test material was stored at room temperature. A reserve sample ofthe test material 

was taken and will be retained in a freezer set to maintain a temperature of -20°C ±10° in 

accordance with Covance SOP. Any unused test material will be discarded after issuance 

of the final report according to Covance SOP. 

-9- 000089 

Page 84 

Covance 71102252 

Safety Precautions 


The test material handling procedures were according to Covance SOPs and policies. 


TEST SYSTEM 

Test Animal 

Young adult albino rats ofthe Crl:CD!>(SD)BR strain were procured from Charles River 

Laboratories, Inc., on October 14, 1997 (Raleigh, North Carolina facility) and 

December 1 and 15, 1997 (Portage, Michigan facility). 

Housing 

After receipt, the animals were acclimated for a period of at least 7 days. During 

acclimation and throughout the study, the animals were separated by sex and group 

housed in suspended, stainless steel cages. Environmental controls for the animal room 

were set to maintain a temperature of 18° to 26°C, a relative humidity of 50% ±20%, and 

a 12·hour lightll2·hour dark cycle. In cases where variations from these conditions 

existed, they were docwnented and considered to have' had no adverse effect on the study 

outcome. 

Animal Diet 

The animals were provided continuous access to Rodent Diet #8604 (Harlan Teklad) and 

water except for 17 to 20 hours before test material administration when the diet, but not 

water, was withheld. The diet is routinely analyzed by the manufacturer for nutritional 

components and environmental contaminants. Samples of the water are periodically 

analyzed. Th~ were no known contaminants in the diet or water at levels that would be 

expected to interfere with or affect the results ofthe study. 

Animal Selection and Grouping 

Five male and 15 female healthy, acclimated rats, weighing from 219 to 267 g and 

approximately 8 to 14 weeks of age, 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 a representative ofa rodent species and are preferred 

by various regulatory agencies. 

PROCEDURES 

. Preparation and Administration ofTest Material 

The undiluted test material was administered as a single gavage dose using an average 

bulk density detennination of 1.04 g/mL to detennine the dose volume for each dose 

level. An individual dose was calculated for each animal based on its fasted body weight 

Reason for Route of Administration 

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 4 hours after test 

material administration and daily thereafter for 14 days. Mortality checks were 

conducted twice a day (morning and afternoon) for 13 days after test material 

administration and again the morning ofDay 14. 

Body weights were detennined before test material administration (Day 0). Additional 


body weights were detennined at Day 7, at tennination of the respective in·life phase 


(Day 14), or at death when survival exceeded 1 day. 


·11· 

000091 
Page 86 

Covance 71102252 

Pathology 

At termination of the respective in-life phase for each dose level, surviving animals were 

euthanized. All animals, whether found dead during the study or euthanized, were 

subjected to an abbreviated gross necropsy examination and any abnonnalities were 

recorded. After necropsy, the animals were discarded and no tissues were saved as there 

were no grossly abnormal organs or tissues. 

Statistical EvaluatioD 

Using the mortality values observed during the study, the LD50 values for females was 

determined by a computer program using a modified Behrens-Reed-Muench cumulant 

method.5 No other statistical evaluations were required by the protocol. 

Location of Raw Data, Records, and Final Report 

The raw data, records, and an original signed copy of the 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. Four females treated at 5,000 mglkg 

were found dead within 4 days oftest material administration. No other mortality was 

observed. Based on the mortality observed in the study, the estimated oral LD,o values in 

rats were detennined to be greater than 5,000 mglkg for males and 4,128 mglkg for 

females. 

Body Weights 

Individual and mean body weights and body weight gains are in Table 2. Animals 

sUrviving to the end of the 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 of the five animals returned to a normal 

appearance by Day 13 after test material administration. The remaining two females 

exhibited only a blue-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 of toxicity observed at the 5,000 mglkg dose level included thin 

appearance, hunched posture, hypoactivity, red-stained face, nonformed and/or blue 

feces, wet or blue-stained urogenital area, green urine, green-stained haircoat and/or tail, 

and death. The surviving animals returned to a normal appearance by.Day 10 after 

treatment with the exception of three males which exhibited a green-stained tail through 

study tennination and the remaining female which also exhibited a green-stained haircoat 

and tail through to study tennination. 

PathololY 

Individual gross necr~psy pathology findings are in Table 4. A summary report by the 

study pathologist is on Page 16. At necropsy, one or more organs or tissues in each 

animal were observed to be stained blue or green, which were attributed to the test 

material. There were no test material-related lesions observed in any of the animals. 

DISCUSSION COtfjDENTIAL

The acute oral toxicity 04 .jwas evaluated in male and 

female rats when administered asa single gavage dose at levels of 5,000 mglkg for males 

and 2,000, 3,000, and 5,000 mglkg for females. Based on the mortality observed in the 

study, the estimated oral LDso values were determined to be greater than 5,000 mglkg for 

mates and 4,128 mglkg for females. 

OOOO~3 - 13 ­
Page. 88 

Covance 71 (02252 

Clinical signs of toxicity 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 or blue-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 of the observation 

period exhibited body weight gain during the study. At necropsy, one or more organs or 

tissues in each animal were observed to be stained blue or green, which were attributed to 

the test material. There were no test material-related lesions observed in any of the 

animals. 

SIGNATURE 

Steven M. 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. 	 "Single Dose 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/or Economic Cooperation and Development 

Guidelines/or Testing o/Chemicals, Section 4, Health Effects, Number 401, Paris 

Cedex (February 24, 1987). 

4. 	 Kanpo Oyo No. 237, Yakuhatsu No. 306, and 62 Kikyoku No. 303 (March 31, 

1987). 

5. 	 Thakur, A. K. and Fezio, W. L." "AComputer Program for Estimating LD~ and its 

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

Drug and Chemical Toxicology, 4(3):297-305(1981). 

- 15 ­ 000095 
Page 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 

• 	 Superscript number indicates the number of animals found dead 

on the indicated day. 

Estimated Oral L050 Values: 

Male· >5,000 mglkg of Body Weight 

Female· 4,128 mg/kg of Body Weight 

95% Confidence Limits - 3,071 to 5,550 mg/kg of Body 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 0 body 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 0 body weight. 

- 19­ 000098 
Page 94 

Covance 11102252 

Table 2 (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 0 body 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-stained tail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ 

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-stained baircoat - ./ ./ ./ ./ 

Blue-stained tail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ 

C24627 Normal appell'llllCe ./ ./ 

Red-stained taco ./ ./ ./ 

Blue-stained baircoat · ./ ./ ./ ./ 

Blue-stained tail ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ ./ 

./ Condition existed • 

Condition not evident. 

- Pan paper under elle bid blue f'eces. blue stainJ, III'Id nontbnned feces. 

t Pan paper under elle bid blue stains. 

ooo~.:)o 
Page 96 

Covance 71102252 

Table J (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 

BI ue-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 Condition existed . 

Condition not evident. 

t Blue haircoat and tail observed, lrll'llfem:d from paves to hair of animals while dosing. 

BI ue stains and blue feces observed on pan pIpII'. 

- 22­ GOO::L01 
Page. 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 not evident. 

Blue-stained pili paper observed under cqe of animals C2284I. C22842. and C22843. " 

OOO~t02 - 23­
Pag.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 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

./ 

• 

Condition existed. 

Condition not evidenl 

Pan paper under cage had blue-stained spots . 

- 24­ 000103 
Page. 99 

Covance 71102252 

Table 4 


Individual Pathology Comments 


Dose Level: 2,000 mglkg of Body 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

U00:104 
Page 100 

Covance 7\\ 02252 

Table 4 (Continued) 


Individual Pathology Comments 


Dose Level: 3,000 mglkg of Body Weight 


Animal Test Day 

Number Sex Died Sacrificed Necropsy Observation 

14 The tail and the mesenteric 

lymph nodes are green. 

14 The tail and the jejunum, ileum, 

and mesenteric lymph nodes are 

green. 

14 The tail and the jejunum, ileum, 

and mesenteric lymph nodes are 

-green. 

14 The tail and the mesenteric 

lymph nodes are green. 

14 The tail and the mesenteric 

lymph nodes are green. 

C24634 


C24637 


C24628 


C24639 


C24640 


F 

F 

F 

F 

F 

- Not applicable. 

- 26­ 000105 
Page 101 

Covance 7110:2252 

Table 4 (Continued) 


Individual Pathology Comments 


Dose Level: 5,000 mglkg of Body Weight 


Animal Test Day 

Number Sex Died Sacrificed Necropsy Observation 

C2284 I 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· 

0001.06 
Page 102 

Covance 7t 102252 

Table 4 (Continued) 


Individual Pathology Comments 


Dose Level: 5,000 mglkg of Body 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 

lymph nodes, 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 stained blue. The 

stomach contents are dark blue 

with a thick semifluid. The 

small intestine, pancreas, 

mesenteric lymph node, cecum, 

right ovary, and right hom of the 

uterus are cannibalized. 

- 28­ U00:10', 
Page 103 

Covance 71102252 

APPENDIX 


Protocol TP2069 

- 29­

000108 
Page 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 

PROTOCOL TP2069 CONFIDENTIAL

Acute Oral Toxicity Study of ==~___jm R.III 

(EPAI01:.co GUidfiIiDILi) 

Dati: 

December" 1997 

~ 30­
Page 105 

Covance 71102252 

Study 


Acute Oral Toxicity Study in Rats (EPAlOECD Guidelines) 


rurpoM 


To assess the acute oral toxicity produced when the test materia.I is admiDistcred by the 


oral route (gavap) to rats. 


SPOuor 

Study Dincter 

Stevell M. Glaza 

Covance Laboratories lac. 

Telephone 601.241.7292 

TestiDl ,aciIItJ 

CoV8IICC Labontories lac. 

3301 KiDsmaa Boulevard 

MadiIoa, WI 53704 

,...,..... Sbldy n........ 

Experim.entaI Start DUe 

ExperimeDtaJ. TermiDalioa DIIII 

FinaJ Report DIte 

CONFIDENTIAL 

Week ofDecember It 1997 

Week ofFebruIry 2, 1991 

Week ofFebruIry 2, 1991 

ooo:tlifl 

I 

Page 106 

Covance 71102252 

TPl069 


Pap 3 


Replatol')' CompllaDce 

This study will be conducted in accordance with the followina Good Laboratory Practice 

RegulalioDIIStandards: 

] Conduct IS a NonreauJated Study 

] 21 CFR 58 (FDA) 

[ J 40 CFR 160 (EPA-FIFRA) 

[Xl 40 CFR 792 (EPA-TSCA) 

[Xl C(81)30 (Final) (OECD) 

[ ] 59 NobSm No. 3850 (J....... MAFF) 

[Xl 0rdinaDc:e 21 (Japaneae MOHW) 

[Xl Notification No. 59-15 (Japanese MI11) 

AIliaIil Can ..d U .. St....... 


All procedures ill this protocol are ill compliaacc with the Animal Welfare Act 


Regulations. Ia the OpUUOIl ofthe Spoasor IDII study d.Uector. the study does not 


unnecessarily duplicate any previouI wort. 


QuUtyA.u....... 


The protocoL. study conduct, IDII the fiaaI report will be audited by the Quality Assurance 


Unit in aecordance with CoVIIDCC Laboratories IDe. (Cownce) Staadard Operatiaa 


Procedures (SOPS) aDd poticia. 


TeltMaterW 

CONFiDENTIAL I 

Purity ..d St....,. 


The SpoMOl' assumes respoasibility for purity IDII stability determinatioas (iac1udiDa 


UDder test c:oqdidoDs). 


·32· ooo~t:iL 
Page 107 

Covance 71 102252 

TP2069 

Pace" 

Stonee eoaditiou 

Room temperature 

RacncSa.p" 

If this study is more thaD four weeks in experimental dmation, reserve samples ofeach 

batchIIot of the test material wi,1 be taken and stored at Covance, in a freezer set to 

maintain a temperature of-20"C ±lOO. The reserve samples will be maintaiDed as 

outlined in the Record Retent.ioa section. 

Dispelido. ofTest Material 

Any Wlused test material will be discarded after issuance ofthe final report. unless 

otberwUc directed by SponIOl'. 

SaletJ Precaadou 


As requDecl by Covaoce SOPS aDd policies 


Abaerpdo. 


The purpose ofthis study is DOt to detenniDe absorpdoa. 


Stnia ad Sell.. 


Crl;COo(SO)BR.., Charles Rivet Laboratories IDe. 


Ace at I.......fT..__t 

Youoalldult 

WeiPt at 1ll1da...ofT...... 

200 to 300. 

·33 - OOOl:12 

Page. 108 

Covance 71102252 

TP2069 

Pqo' 

Number ••dSa. 


S males aDd Sfemales for the initial dose level 


S males and/or S females for lilY additional dose levels (ifrequired) 


Ideadftcatlu 


Individual numbered ear tag 


BlIIbudl'7 

Bo..... 


Separa.ted by sex aDd II'OUP howled in suspeoded. S1aiD.Iess steel caps 


Diet 

R.odcDt Diet #1604 (Harlan Teklad) ad Ilblttmt ~cept far 17 to 20 bouts before 

test mate.rW administratiOll. The diet is routinely malyzed by the maraufactuler 

for nutritiooal componcIdS and eawiroameatal contamialatl. 

w ..... 

Adlibitwlrt &om lID , ..romaDe system. Samples ofdie water are periodically 


malyad for specified microorpaisms and eaviroDmeratal cootJunirumts 


Co.......... 


There are 110 !mown contammarrts ill the diet or water Itlevels tbIIt would be 


expected iat.afere with this study. 


Ea'riro._t 


Enviroamcutal controls for the IIlimaI room will be III to lDIintain • 


tempc:latIn of IS- to 26OC•• relative humidity ofSCM ±ZO%. ad. 12-110111' 


liibfl12-bour dark cycle. 


-34­

0001.1.3 
Page 109 

Covance 71102252 

TP2069 

·P.6 

Selectioa .fTest ADiJuII 

Based OD health aDd body weight according to Covance SOPs. An adequate 

number of ex1rl animals will be purchased so that no animal in obviously poor 

health is placed OD test. 

Justifteatioa f.r Specia SeIeetio. 


Historic:alIy, rats have beeD used as representatives of. rodent species and are 


preferred by var10Ul regulatory aaCDCiOl. 


Treatllld. Proced..... 

DoMLmII 

A sinaJe dose of 5.000 maIkI ofbody weight will be admioi.stend to &ve males 

and five females. The dole level, method, 1iequeocy. and t.bo duratioa of 

administration are cboseD bued oa t.bo requiremadI ofthe n=guIatory test 

&uidcUaes. ItDO test material-related mortality is produced at this level, DO furI:ber 

testinc will be required. IflD)' mortality oc:curs at the 5,000 m&IkI dole level, 

additioaal dole Icveb may be added at the directioa ofthe study director ill order to 

meet the objectiwll oft.bo study. 

Don Ad....iItn.. 

All animals will n:ceive the same cooc:entratioo oftcst material per dole level. 1'be 

test material will be doled undiluted. usiq t.bo bulk deasity to dctamiDe the dole 

volume. llldividualdola will be calcu.lafed bued upoa t.bo animal's fasted body 

weiaht taka before test material admiDistratioa and admiaisterecl by aaV9- The 

animals will have food witbbeld. for 17 to 20 hours befoIe test material 

admiDistratioa. 

.....few RIa....f Ad.......,.... 


Historic:aIIy. the oral route bas baa ODe ofthe routes used to assess the acute 


toxicity ofvarious matcriaIJ. 


• 3S- 000114: 

Page. 110 

Covance 71102252 

TP2069 

PIp 1 

Obtcrnltoa of A.baall 

CIiIIicaI ObJerv.dou 

At approximately 1.2..5, and 4 hours after test mataial admiDistration and daily 

thereafter for at least 13 days for clinical sips and twice daily (UD. and p.m.) for 

mortality (Lm. mortality ooI.y on Day 14). ObservatioDl may be exta.Ided wbeD 

directed by the study di.rec:tor. 

BodyWapa 


Before experimental initiltioD, at 7 and 14 days after test mat.erW administntiOD, 


or at dcadJ. (wbea survival exceeds 1day). 


'.CIao..., . All animals. wbcther dyiq durina the study. sacrific:ed ill • moribUDd c:oaditioa. or 

euthanized at its respective expcrimeDtaI tc:rmwtioa (Day 14). wiD be subjected to 

an abbnMated IJ'OSI DCCIOpS)' exarniDalioa and all abaormaJitiea wiD be recorded.. 

All tissues with 1csi0Ql wiD be c:oUccted and preser wei ill pbospIIa1e-buffaed 

formalia for possible t\dure bistoloakal eYIluatioa u determDIecl by tile Spouor 

(to be IIddressed by ameDdment). 

Stadltlcal Kv........ 

Wheo applicable. LDSt values wiD be determiDed usiD& • modified BelueDl-Jteed.. 

MueDCb cumuJant method (Covaace Statistica Libray). No ad. stadstical 

eva.lumODlIre reqllinld. 

Rep." 


A fiDal.n:port iDc.ludiD& thoJe items listed below wiD be submitted. 


Oescriptioa oftile test material 


Desc:ripdoa oftile test system 


Procedarea 


Data ofexpcrimemIJ initiatioa IIIId terminatioa 


Tabulatioa ofmonality data by leX and dose Iewl 


Desc:riptiQl'l ofany tOxic effects 


- 36- OOO~t:t5 
Page 111 

Covance 7t t 02252 

TP2069 

Pap' 

Tabulatioa of meaa body lWiahts by leX aDd dose level 

LO" calculations for each sex with 95% confidence iatervals (wbc:a appJ.ic:able) 

Gross pathology fiDdinp'poss patbolOCY reports 

Histopathology fincliftp (if applicable) 

Record Retea ... 

All raw data, tissues, documentation, records. protocol. protoc:ollDWldments aDd reserve 

samples (ifapplicable), aDd the fiDai tq)OI't gcoerated u a result ofthis study will be 

archived ia the storage facility ofCovance for a period ofteD yan foUowiq si~ of 

the final report. Ten yem after signjq ofthe fioal report. all ofthe aCoremc:atioaed 

materiab wiD be sent to the SpoMOI'. aDd a retum fee will be cIuqed. T'be SpoIIIOI' may 

elect to have the maaeriaIs retaiaed in the Covance _hives for an additiODll period of 

time. for which Covance will cbarp a storaae fee. Ifthe SpotIIIOl' c.boosea to haw 

Covancc c:IispoIe ofthe materials, a disposal fee will be cbarpd. All raw'" stored OG 

magnetic media will be retaiDed by Ccmace. 

T'be foUowiq supportiDa records will be maiDcd at Covmce but win DOt be archived 

with the study data. 

Animal receipclacclimatioa nx:ordI 

Waf« analysis IeCOIda 

Animal room and humidity recorda 

Refripraror aDd freezer recorda 

Instrumcat c:ab1ndoa aDd maj,.,..,..., recordI 

·37 ­ 000'1'" _ • .L '\:) ,-"
Page 112 

Covance 7 t 102252 

TP2069 

Pace 9 

PROTOCOLAPPR OVAL '

/7,/'ir, 

7

n;:::_--1:\l-t...•' ·00 

study Director 

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 

FINAL REPORT CONFIDENTIAL

Study Tide: 

Primary Dermal Irritation Study o·iJ_...............~_IIIIIII_..-j·J..in Rabbits 

(EPAlO~D GUldehnes) 

Author. 

Steven M. Glaza 

Study Completion Date: 

January 22. 1998 

Performinl Laboratory: 

Covance Laboratories Inc. 

3301 Kinsman Boulevard 

Madison, Wisconsin 53704 

Laboratory Project Identincation: 

Covance 71102253 

Page 1 of29 

0001.:19 
CONFIDENTIAL Page 

115 

CO'iance 71102253 

COMPLIANCE STATEMENT 

Primary Dermal Irritation Study {'~'fIII..a~~~~_____._..iillj_in Rabbits 

(EPA/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 of Good 

Laboratory Practice, C(SI )30(Final) 

Japanese Ministry of International Trade and Indu~try Good Laboratory Practice 

Standards, Notification No. 59-SS 

Japanese Ministry of Health and Welfare, Ordinance 21 

Study Director 

Acute Studies 

Covance Laboratories Inc. 

Date 

Date 

-z1,/711 


000:1.20 

Page 116 

Covance 7t 1022S3 

QUALITY ASSURANCE STATEMENT 


This report has been reviewed by the Quality Assurance Unit of Covance 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 of Health 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 of inspections and findings are issued to Covance 

management according to standard operating procedures. 

Inspection Date Reported. to 


Dates Study Director and 


From To Phase Study Director Management 


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 

-3­
Page 117 

Covance 71102253 

STUDY IDENTIFICATION 

Primary Dermal Irritation Study 01\ lin Rabbits 

(EPAJo.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 Termination Date 

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

000:122 
Page 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 

Laboratory Animal 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\1ARY ................................................................................................ " '" ............... 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 of Exposure Area.......................... ......................... 10 


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


PROCEDURES .................................................................................................... ......... 10 


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


Treatment ............................... ................................................................................... 10 


Reason for Route of Administration ........................................................................... 10 


Observations.............................................................................................................. 11 


Termination ................................. .............................................................................. 11 


Statistical Evaluation ................................................................................................. 11 


Location ofRaw Data, Records, and Final Report...................................................... 11 


RESULTSIDISCUSSION ............................................................................................. 12 


SIGNATURE ................................................................................................................ 12 


- 6­ 0001.:~4: 
Page 120 

Covance 71l 0225 3 


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 


000125 

Pag-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 a very 

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 of this study was to assess the relative level of primary skin' irritation of a 

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 of the 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 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). 


TEST MATERIAL CONFIDENTIAL 

Identification 

The test material was identified 4' .Fd described 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 of the 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 of the 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 of at 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 of its cage on 

Day 2. Environmental controls for the animal room were set to maintain a temperature of 

16° to 22°C, a relative humidity of 50% :1:20%, and a 12-hour lightll2-hour dark 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 ad libitum and a measured amount of High 

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 or water 

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 of Exposure Area 

Three male and three female healthy, acclimated rabbits, weighing from 2,610 to 2,806 g 

and approximately 19 weeks of age, 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 of hair to obtain one unblemished skin site. 

Justification for Species Selection 

Historically, the New Zealand White albino rabbit has been the animal of choice for 

evaluating the effect of chemicals on the skin. 

PROCEDURES 

Preparation of Test Material 

The test material was administered as received. The pH of the 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 x 2.S-cm gauze patch secured with paper 

tape, loosely overwrapped with Saran Wrap·, and secured with Elastoplast'" tape to 

provide a semiocclusive dressing. 

At the end of the 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 paper towels. 

Reason for Route of Administration 

Historically, the dermal route has been the route of choice based on the method of Draize.4 

- 10­
Page 124 

Covance 71lO2253 

Obsen'ations 

Thirty minutes after removal of the test material, the degree of erythema 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 of each 

animal was used for comparison. 

Animals were weighed before test material administration. 

Termination 


At termination of the in-life phase, all animals were euthanized and discarded. 


Statistical Evaluation 


No statistical evaluations were required by the protoc~l. 

Location of Raw Data, Records, and Final Report 


The raw data, records, and an original signed copy of the final report will be retained in 


the archives ofCovance for a period of ten 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 of rabbits under 4-hour 

semioccluded conditions resulted in only a very 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 

Steven M. Glaza Date

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," Organisation for Economic Cooperation and 

Development Guidelines for Testing ofChemicals, Section 4, Health Effects, 

Number 404, Paris Cedex: (July 17, 1992). 

4. 	 Draize,1. H., "Primary Irritation ofthe Skin," In: Appraisal ofthe Safety of 

Chemicals in Foods, Drugs, and Cosmetics - Dermal Toxicity, Association of Food 

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 1 mm) 

Severe edema (raised approximately 1 mm and extending 

beyond area of exposure) 

o 

I 

2 

3 

Highest possible edema score 4 

- 14 ­

000:13Z 

Page 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 of the 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. of the 

individual index scores, divided 

by the number of animals (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 -	 000:135 

Page 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 of its cage on 

Individually, in suspended, stainless steel Day 2. 

cages 

This deviation is not considered to have had an adverse effect on the outcome of the 

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: 

PrimIry DermailrritatiOD Study off~,....,.~~....___~ln Rabbits 

(EPAlOE~b GW&GDeI) 

December It 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 of primary skin irritation of. test material on rabbits under 


semioccluded conditions 


SOODlOr 

SooDlOrt , ReDreseatatiYe 

Study Director 

Steven M. Olaza 

Covance Laboratories lac. 

Telephone 601.241.7292 

Taba 'adUtJ 

COVUICC Laboratories IDe. 

3301 K.insnum BouIC'YIl'd 

Madisoa, WI S3104 

Pnpo... Study T .....b.. 

Experimental Stilt Dale 

Experimental Tenn.i.DalioD Dace 

Final Report Date 

CONFIDENTIAL 

WeekofDecembcr8.1m 

Week ofDecember 29. 1m 

Week of February 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: 

1Conduct as a Nonregulated Study 


121 CFR 58 (FDA) 


] 40 CFR 160 (EPA-FIFRA) 


[X] 40 CFR 792 (EPA-TSCA) 

[X] C(81 )30 (Final) (OECD) 

( 1 59 NohSan No. 38'0 (Japanese MAFF) 

[X] 0rdinaDce 21 (Japanese MOHW) 

[X] Notification No. 59..1' (Japanese MITI) 

ADbDat C.n .ad UN Statemeat 

All procedures in this protocol are in compliance with the Animal Welfare Act 

Regulations. In the opinion ofthe Sponsor and study director, the study does not 

unneceaarily duplic:ate lIlY pnMous wort. 

Quallt1 AuU'lUlOt 

The protocol. study conduct, and the tiDal report will be _ted by the Quality Assuraace 

Unit in accordaocc with COVaDCC Laboratories loc. (Covance) Standard Operating 

Procedures (SOPs) aad policies. 

Tilt Material 

CONFIDENTIAL 

Pbyslcal DeIcrtpdIa 

Viscous, dark. blue liquid 

Parity aad sea....,. 


The·SpoaIGI' assumes responsibility for purity determinations (incJudiq IIIKIer test 


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 than four weeks in experimental duntioa, reserve samples ofeach 

batchllot ofthe test material will be taken aDd stored at Covaace. ill a .fm:zer set to 

maintaiD a temperature of·20·C ±Io-. The reserve samples wiD be maintaiDed u 

outlined in the Record Retention section. 

Dispoaidoa 

Any unused test material wiD be discarded after issuance of the final report, unless 

otherwise directed by Sponsor. 

Safety rrecaatloaa 

As required by Covance SOPs and poIiciel 

Absorpdoa 


The purpose of this study is not to determU1e absorption. 


S..... 

Rabbit 

Stnlll aad s..rc. 


Hra:(NZW)SPF. Covance Raan:h Products Inc. 


AI- at 1Dida..ofTra.....t 


Adult 


w ..... t at laida" .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 

A measured amount ofLaboratory High Fiber Rabbit Diet 1#7015 (Harlan 


Teldad). The diet is routinely analyzed by the manufacturer for nutritional 


components and environmental contaminaDts. 


w ..... 

A.d libitum from an automatic system. Samples ofthe water are periodica.Uy 

analyzed for specified microorpDisms IDd environmental contami.tw:lts. 

COD.......... 


There are no known contaminants in the diet or water at levels that would be 

expected to interfere with this study. 

Eaviroll_t 

Environmeptal controls for the animal room will be set to maintain a 

temperature of 16· to 2l~, I ndative humidity of SO% :t;20%, and a 12-hour 

Iightl12-bour dark cycle. 

Acdiaadoa 

At lout 7 days 

000:1/12 

Page 138 

Co,'ance 71102253 

TP3014 


Pqe6 


SelecdoD of Tat ADjma. 


Based on health and body weight according to Covaac:e SOPs. An adequate 


number ofextra animals will be pun:ha.scd so that no animal in obviously poor 


health is placed on test. 


JU.tiflcadOD for Species Selecdo. 


Historically. the New Zealind White albino rabbit has been the animal ofchoice 


for evaluatiq the effcct ofcbcm.icals on the skin. 


TnatllleDt Procedures 

Pnpandoa of E.lpoIun A.ra 


On the day before test material administration, remove the hair from the back and., 


if ne«sSaI'Y (to obtain unblcm.isbed skin), the flanks ofCICh animal with an electric 


clipper. The tratmeDt sites will be inspected for int.ert'erini laioas, irritation. or 


defects that would preclude the use of any of the animals. The animals will be 


clipped at needed tbroupout the study. 


DoN Admutntioa 


Before administration ofthe test material the pH ofthe material will be determined 


(if possible). The test material will be applied to the intacl test area (approximately 


6.25·anz) on each rabbit, in tbe amount of0.5 mL. The dose lewl. metbod. 


frequency. and duratioD ofadminisaVioD Me chosen bated on the rec:tuiremerdS of 


the rcpdatory test guidelines. The test material will be applied undiluted. The area 


of application will be covered with III a-ply 2.'-cm x 2.5-cm puze pat.cb secured 


with paper tape and overwrappcd with wrap- and El&!RoJILut-tape to 


provide a scmiocclusive drcssiq. The rabbits will DOt be col1arcd durina the 4­

hour application period. 


.....for Reate of Ad....tndo. 


Historically, the dermal route has beeD the route ofchoice bated Oil the method of 


Drai& 


- 2S000:143 
Page 139 

Covancc 71101153 

TP10l4 


Pqe1 


Remov.1 .fTest M.terial 

After 4 bours of exposure, the patches and test material will be removed as 

thoroughly as possible using water and/or an appropriate substance without 

irritating the skin. 

Readlac of Del"lllallrritatioa 

Within 30-60 minutes after removing patches. tbe dqree oferythema aDd edema 

will be evaluated accordiq to the Dra.ize technique (AttacbmeDt 1) aDd recorded as 

the ....hour score. The untreated skin of eacb animal will serve as its own conttol. 

Sub"equent radinp will be taken at approximately 24. 41, aDd n hours after patch 

removal. If irritation is present at the n·hour examination, additional observations 

will be made at approximately 96 hours and at Days 7. 14, and 21. or until irritatioll 

has clCSRd. Based on the level of irritation observed at any of these time points. 

the study may be tcrmi.Dated at the direetiOD of the study director. 

BodyW..... 


Before test material admiDistraIioD only. 


P.t..., 

Any animals dyiq or sacrific:cd in • moribuDd condition duriDa the study will be 

subjected to an abbreviated polS necropsy examination and all abnormalities will 

be recoaded. After 1ICClOPIY. the animals will be discarded and DO tissues will be 

saved. At te.rminatioa of the experimental phase. surviviq animals will be 

sacrificed aDd discarded. 

StadaUcal z. .... tIOII 


No statiIcical evaluar:ioas are requiRd. 


- 26­ 000:1<14 
Page 140 

TPlOt4 


Pale' 


Report 

A final report includina those items listed below will be submitted: 

Description of the test material 

Description of the test system 

Procedures 

Dates of experimental initiation and termination 

Tabulation of initation data 

Record Reteatto. 

All raw data. documentation. records, protocol, protocol amendments and reserve 

samples (if applicable), and the final report aenentecl as a result oftbis study will be 

archived in the storage facility ofCovancc for a period often years followiDa siping of 

the fmal report. Ten years after sipina of tile final report, all of the aforementioned 

materials will be sent to tile SPODlOl', and a fee will be cbarpd. The Sponsor may 

elect to have the materials retained in tile COVllDCe archives for an additional period of 

time, for which Covance will cbarae a storage fee. If the sponsor chooses to have 

Covance dispose ofthe matcrials. a disposal fOIl will be charpd. All raw data stored on 

magnetic media will be retained by Cov.oce. 

The foUowiDa supportiDa records will be retained at Covance but will not be archived 

with the study data: 

Animal receiptllCCtimation reconIa 

Waf« analysis records 

Animal room tempel"IhR and humidity reconIa 

Refrilerator IDd treea:a:r tem.pcraNre reconIa 

In.strument calibradon and maintelWlCe reconIa 

- 27­

0001/15 
Page 141 

Covance i [ [02253 

TP10l4 

Page 9 

PROTOCOL APPROVAL 

Steven M. Ollza 

JJ...:.. (Z, 1fC) Date

CONFlOENllAl 

1"'t.-\-'" 

Study Directm 

Acute Studies 

Covaoce Laboratories Inc. 

Date 

(71102253.pr) 
Page 142 

CO\ance 71102253 


TP3014 

P.e 10 


Act.cb....t I 


Pri..ary Denaallrritadoa SeoriaC Scale 

(Draize Tecbaique) 

Erytbema ad Eschar Formado. 

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 sliKbt eschar 


formation (injuries in dcpcb) ......................................................................................4 


Highest possible crytbenIa sc;ore....................................................................4 


Edema Forma" 

No edema ....................................................................................... 0 


Very slight edaDa .......................................................................................1 


Slight edaDa (qes are well defiDed by 


Severe edema (raised approximately 1 mID IDd 


definite raisiDI) .......................................................................................2 


Moderate edaDa (railed approximately 1 mID) .........................................................3 


exteDdinl beyond .... of~)................................................................... : ...... 4 


Hia.best possible edeIua sc;ore ........................................................................4 


- 29- 00014'7 

I Page 143 I CONFIDENTIAl . 

t 

. 


t' 
Page 144 

PAGE 1 OF 32 	PAGES 

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 of the procedures followed. 

Date of Report Audit: 

24 February 1998 

. 1~ HAY 1991 DATE. ............................................... . 

J R Pateman 

For Safepharm Quality Assurance Unit 

000:150 

Page 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. 

". 12 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 of test 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 

000153 

Page' 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 ECso for the test material to Daphnia magna based on nominal test 

concentrations was 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 of the required 80% of nominal test concentrations and 

so the results are based on nominal test concentrations only. 

000:154 
Page 150 

CONFIDENTIAl -7­

SPL PROJECT NUMBER: 656/011 

I I 

ACUTE TOXICITY TO DAPHNIA MAGNA CONFIDENTIAL 

1. INTRODUCTION 

This report contains a description of the 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 

Descri ption 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. 

000:155 
Page 151 

-8­

SPL PROJECT NUMBER: 656/011 

2.2 Experimental Preparation 

For the purpose of the 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 of test 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 of 48 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 of the 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 100 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 if they 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 . . 000:158 
Page 154 

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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 ECso 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. 

0001.59 

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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) ECso (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. 

0001bO 
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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 mai ntained at 21 • C throughout the study, whiIe 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 of the 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 ECso 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 ECso value of 1.1 mg/I with 

95% confidence limits of 0.92 - 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. 

000:16.1 
Pag.e 157 

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, 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 

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CONFIDENTIAL 

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, 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 

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CONFlDENTlAL -16­

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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: 


0.1 
Page 160 

CONFIDENTIAl -17­

SPL PROJECT NUMBER: 656/011 

, tACUTE 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) 


000165 
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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 Nominal I o hours 24 hours 48 hours

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 
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CONFIDENTIAL -20­

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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. 


0001G9 
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, 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'RAnON cmtm 

000:1';'0 

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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 

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: 

~: 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 

000173 

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CONFIDENTIAl. -26­

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, .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 mill 
Page 170 

CONFIDENTIAl SPL PROJECT NUMBER: 656/011 

, ZACUTE 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) CaCI2·2H2O 11.76 gil 


b) MgS04·7H2O 4.93 gil 


c) NaHCO) 2.59 gil 


d) KCI 0.23 gil 


ii) 	 Preparation 

An aliquot (25 ml) of each of solutions a-d wa'S added to each litre (final volume) 

of deionised 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 

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT 


OF THE UNITED KINGDOM 


GOOD LABORATORY PRACTICE 

STATE.~E.vr OF COMPLlANCI 

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 of ftOD-clinicai siudia performed at these facilities. 

@It(~ ,/1~ 1,( D.F. ~oorc '(tI.{' Director 

UK GLP ~onilorinl Allchoril), 

(JI018d