SANITIZED VERSION 


FormA 

U.S. ENVIRONMENTAL PROTECTION AGENCY AGENCY USE ONLY 

PREMANUFACTURE 

NOTICE 

Date of receipt 

CES 

03 OCT 27 Aii 6: 11 

Company Sanitized 

: ,

Ih,I, 

,'. i III nIt II II Ii ;l li

elll!;;r til!;; lUlal lIumoer 01 pages 

in the Premanufacture Notice 

INS1'RUCTIONS 

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 ifyou do 

not have actual data. 

Before you complete this form, you should read the "Instructions Manual for Premanufacture Notification" (the Instructions Manual is available from the Toxic 

Substances Control Act (TSCA) Information Service by calling 202-554-1404, or faxing 202-554-5603). 

• 	 Ifa user fee has been remitted for this notice (40 CFR 700.45), indicate in the boxes above the TS-user fee identification number you have generated. Remember, 

your user fee 10 number must also appear on your corresponding fee remittance, which is sent to EPA, HQ Accounting Operations Branch (PM·226), P.O. 

360399M, Pittsburgh, PA 15251-6399, Attn. TSCA User fee. 

Part I-GENERAL INFORMATION 

You must provide the currently correct Cbemical Abstracts (CA) Name ofthe new 

chemical substance, even ifyou claim tbe 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 ofyour submission should reference your TS user fee identification 

number. You must submit an original and two copies ofthis notice including all test data. 

Ifyou claimed any information as confidential, a single sanitized copy must also be 

submitted. 

Part II - HUMAN EXPOSURE AND ENVIRONMENTAL 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 Ill-LIST OF ATTACHMENTS 

Attach additional sheets ifthere is not enough space to answer a question fully. Label 

each continuation sheet with the corresponding section heading. In Part III, list these 

attachments, any test data or other data and any optional information included in the 

notice. 

OPTIONAL INFORMATION 

You may include any information that you want EPA to consider in evaluating the new 

substance. On page II ofthis form, space has been provided for you to described 

pollution prevention and recycling information you may have regarding the new 

substance. 

So-called "binding" boxes are included throughout this form for you to indicate your 

willingness 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 

that routinely accompany the development ofconsent orders or Significant New Use 

Rules. Except in the case of exemption applications (such as TMEA, LVE, 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 

by itself prohibit the submitter from later deviating from the infonnation (except 

chemical identity) reported in the form. 

CONFIDENTIALITY CLAIMS 

You may claim any information in this notice as confidential. To assert a claim on the 

form, mark (X) the confidential box next to the infonnation that you claim as 

confidential. To assert a claim in an attachment, circle or bracket the information you 

claim as confidential. If vou claim information in the notices as confidential you must 

also provide a sanitized version ofthe notice (including attachments), For additional 

instructions on claiming information as confidential, read the Instructions Manual. 

Mark (x) if any infonnation 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 ofall 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 ofdata, 

must be submitted [fthey do not appear in the open literature. You should clearly 

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

composition 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 ofthe Premanufacture Notification Rule (40 CFR Part 

720). 

Test Data (Check Below any included in this notice) 

Environmental fate data 0 Yes 	 Other data 181 Yes 

Health effects data 0 Yes 	 Risk assessments 

Environmental effects data 0 Yes 	 Structure/activity relationships 

PhysicaVChemical Properties· 181 Yes 	 Test data not in the possession 

or control ofthe submitter 

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

TYPE OF NOTICE (Check Only One) 

~ PMN (Premanufacture Notice) :;g 

<::":) 

o INTERMEDIATE PMN (submitted in sequence with final product P~ 

C') 

--I o SNUN (Significant New Use Notice) 	 t.,

o TMEA (Test Marketing Exemption Application) - o LVE (Low Volume Exemption) @40CFR 723.50(c)(l) 

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

o LVE Modification WREX Modification 

IS THIS A CONSOLIDATED PMN? Yes 

# ofchemicals .,.--::-__2-::--::-_--,::­

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

EPA FORM 7710·25 (Rev. 5·95) 	 Replaces previous editions of EPA Form 7710-25. 

Cl70!50b 
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 (2070-0012), Washington, D.C. 20503. 

CERTIFICATION 

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

I. 	 The company named in Part I, section A, subsection 1 a ofthis notice fonn 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 infonnation provided in this notice is complete and truthful as ofthe date ofsubmission. 

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

reasonably ascertainable by me as required by §720.50 ofthe Pre manufacture Notification Rule. 

Additional Certification Statements: 

Ifyou are submitting a PMN, Intennediate PMN, Consolidated PMN, or SNUN, check the following user fee certification 

statement that applies: 

I2$'J 	 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$1000 for an lntennediate PMN (defined @40 CFR 

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

o The Company named in Part I Section A is a small business concern under 40 CFR 700.43 and has remitted a fee of$1 00 

in accordance with 40 CFR 700.45(b). 

If you are submitting a low volume exemption (LVE) application in accordance with 40 CFR 723.50(c)(l) 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 tenns of 40 CFR 723.50. 

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

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

o If this application is for an L VE in accordance with 40 CFR 723 .50( c)( I), the manufacturer intends to commence 

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

review period. 

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

described herein. Any knowinl! and willful misinteruretation is subject to criminal penally pursuant to 18 USC 1001. 

Signature and title ofAuthorized Official (Original Signature Required) Dale 

Confidential 

x 

Signature of agenl - (if applicable) Dale 

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

x 
Submitting 

Notice (in U.S.) 

b. Agent (if 

applicable) 

Company 

address (number street) 

State, ZIP Code 

Name of authorized official 

Mailing address (number and street) 

City, State, ZIP Code Telephone 

c. IfyouareSul)miitt~~~~~ea~W1OfaJ<~~hm~km~rnrl~0t~~~::::::::::::::::::::::::::::~::::::::::::~lC:r 

Joint Submitter (if 

applicable) 

Name of 

3. 

Contact (in 

U.S.) 

Mailing address (number and street) 

Company 

(number and street) 

City, State, Code 

notice 

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

the chemical 

substance covered by this notice, enter the exemption number assigned by 

EPA. If you previously submitted a PMN for this substance enter the 

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

5. have submitted a notice Bona 

6. 

for the chemical substance covered by this notice. enter the notice 

assigned by EPA. 

Type of Notice - Mark (X) 

I. Manufacture 

Only

D Binding Option 

Mark 

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

Position 

Confidential 

x 

Mark (X) • D X if none 

Mark (X) • D X if none 

Mark (X) • D X if none 

2. 	 Import 

Only 3. 	 Both 

D 	 Binding Option 


Mark 


Page 3 

D 
Chemical Abstracts (CA) name ofthe substance based on 

nomenclature rules and conventions. 

a. 

b. 

c. 

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

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). 

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 D Method 2 (Other Source) 

report obtained from the CAS Inventory Expert Services must be 

submitted as an attachment to this 

x 

x 

CAS# 

x 

x 

e. For a class correct structure - (I) 

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

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

x 

diallram. a~ comnlete a~ can he known. if one can he rea~nahlv a~certained. 

this box if you attach a continuation sheet. 

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

I. 

a. Class of substance - Mark (X) 

name correct 

c. you 

as an 

d. 

e. 

Mark (X) this box if 

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

that in a sheet. 

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

I D Class I or 2 D Class 2 

Inventory 

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). 

Confidential 

x 

x 

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

report obtained from the CAS Inventory Expert Services must be 

Molecular formula and CAS Registry Number (if a number already exists for the substance) 

x 

x 

and correct structure diagram. For a class 2 substance - ( I) List the immediate 

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

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

di3.llfam. as comnlete a~ can he known. if one can he rea~onahlv a~certained, 

attach a continuation sheet. 

EPA FORM 7710-25 (Rev. 5-95) Page 4-2 
a. Indicate the number-average weight of the lowest molecular weight composition ofthe polymer you intend to manufacture. 

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

below 1,000 absolute molecular weight ofthat composition. 

Describe the methods of measurement or the basis for your estimates: OPC 0 Other 0: (Specify) 

i) lowest number average molecular weight: 

ii) maximum weight % below 500 molecular weight: 

iii) maximum weight % below 1000 molecular weight: 

attach a continuation sheet. 

You must make separate ity claims for monomer or and residual information. 

"Confidential" box next to any item you claim as confidential 

Confidential 

(1) - 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. 

Mark (X) this column ifentry in column (1) is confidential. 

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

(2) -

(3) -

(4) - Mark (X) the identity column if you want a monomer or other reactant used at two weight percent or less to be I isted 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 ofeach monomer or other reactant that may be present as a residual in the polymer as manufactured for 

commercial purposes. 

umn if in column is confidential. 

Monomer or other reactant and CAS Registry Number 

% 

% 

% 

% 

% 

% 

% 

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

o Method 1 (CAS Inventory Expert Service - a copy of the identification report 0 Method 2 (other source) 

obtained from CAS Inventory Expert Service must be submitted as 

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

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

attach a conti nutation sheet. 

% 

% 

% 

% 

% 

% 

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

Number 

x 

(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. Ifthere are unidentified impurities, enter ·'unidentified." 

Estimate the maximum % of each .. %. 

Maximum 

percent dential 

% X 

% X 

% X 

% X 

% 

% 

% 

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

5. 	 1m 

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

you must provide a generic name 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. 

Halogen-substituted oxetane 

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

X 
Part I -- GENERAL INFORMATION -- Continued 

Section C -- PRODUCTION, IMPORT, AND USE INFORMATION: 

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

1. 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 (kglyr) 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 ofthe new chemical substance by function and application .. 

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

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

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

commercial purposes at sites under your control associated with each category 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 (ies) in column (8) is (are) confidential business information (CBI). 

Category of use (1) CBI Binding Produc- CB] %in CBI % of substance expected per use CBI 

Option tion% Form­ (8) 

(by function and application i.e. a dispersive dye Mark (x) ulation Site- Con-'" Indus- Com- Binding (3) for finishing polyester fibers) (2) (4) (5) (6) (7) limited sumer trial mercial Option (9) 

Monomer used in the production of x x x 

reactive oolvmers for surface % % 

coating materials and other - - -% - -% - - - - - - - oolvmer intermediates 

[open I non-<lispersive use] 

% % 

% % 

% % 

% % 

% % 

'" Ifyou have identified a "consumer" use, please provide on a continuation sheet a detailed description of the use(s) of this chemical substance in consumer products. 

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

substance loses its identity in the consumer product. 

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

b. Generic Ifyou 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 

N/A 

I 

I 

. D Mark (X) thIS box Ifyou attach a contmuatlOn sheet. 

Binding 

Option 

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

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

Mark (X) this box if you attach hazard information. 

EPA FORM 77\0-25 (Rev. 5-95) Page 7 
box next to any you 

Complete section A for each type of 1II(I",U"".,u.", pro(:es~,In:g, operation Involving the new chemical substance at industrial sites you 


control. Importers do not have to complete this "n,~r,,·tl""" outside the U.S.; however, you may still have reporting requirements if 


there are further industrial ou must these See in"·trll,,ti£\nC 


Confiof the site at which the on,,,·,:,t,on will occur. 

1. 

dential 

x 

Site address (number and street) 

City, County, State, ZIP code 

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. 


x 

Batches/year x 

x 

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

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

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

(2) 	 Provide the identity, the approximate weight (by kg/day or kg/batch on a 100"!. new chemical substance basis), and entry point ofall starting materials and 

feedstocks (including reactants, solvents, catalysts, etc.), and ofall products, recycle streams, and wastes. Include cleaning chemicals (note frequency ifnot 

used daily or per batch.). 

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

2. Continuous 

this box if you attach a continuation sheet. 

EPA FORM 7710-25 (Rev. 5-95) 	 Page 8 
Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued 

Section A -- INDUSTRIAL SITES CONTROLLED BY mE SUBMITTER -- Continued 

2. Occupational Exposure -- You must make separate confidentiality claims for the description ofworker activity, physical form ofthe new chemical substance, 

number ofworks exposed, and duration of activity. Mark (X) the "Confidential" box next to any item you claim as confidential. 

(l) -- 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 (CBl). 

(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 form(s) of the new chemical substance (e.g., solid: crystal, granule, powder, or dust) and % new chemical substance (if part ofa mixture) 

at the time of exposure. 

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

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

(9) -- Mark (X) this column ifentry in column (8) is confidential business information (CSl). 

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

(\2) -- Mark (X) this column if entries in columns (10) and (II) are confidential business information (CSI). 

Worker activity CBl Protective Equipment! Binding Physical fonns(s) Binding CBI #of CBl Maxirnu duration CBl 

rn 

(i.e., bag dumping, filling drums) Engineering Controls Option and % new Option Worker Hrslday Dayslyr Mark (x) substance Mark (x) s 

(I) (2) (3) (4) (5) (6) (7) Expose (9) (\0) (\I ) (12) 

d 

(8) 

i X 

X 

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

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

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

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

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

(3) -- Mark (X) this column ifentries in columns (1) and (2) are confidential business information (CSI). 

(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 substance 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 ifentries in columns (4) and (5) are confidential business information (CSI). 

(7) --Identify the destination(s) ofreleases 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 CSI Media of Control technology and efficiency (you may wish to optionally attach efficiency data) CBI 

Number released release 

(I) (2a) (2b) e.g, stack sir Binding 

(3) (4) (Sa) Mark (Xl (5b) (6) 

X 

X 

(7) Mark (X) the D POTW provide name(s) below: CBI D Navigable D Other - Specify provide NPDES # CSI 

destination( s) of ,--­ waterway 

releases to water. 

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

EPA FORM 7710-25 (Rev. 5-95) Page 9 
Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued 

INDUSTRIAL SITES CONTROLLED BY OTHERS 

Complete section B for typical processing or use operations involving the new chemical substance at sites you do not control. Importers do not have to complete this 

section for operations outside the U.S.; however, you must report any processing or use activities after import. See the Instructions Manual. Complete a separate 

section B for each type ofprocessing, or use operation involVing the new chemical substance. If the same operation is performed at more than one site describe the 

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

1. Operation Description -- To claim information in this section as confidential, circle or bracket the specific information that you claim as confidential. 

(I) -- Diagram the major unit operation steps and chemical conversions, including interim storage and transport containers (specify - e.g. 5 gallon pails, 55 gallon 

drums, rail cars, tank trucks, etc). On the diagram, identify by letter and briefly describe each worker activity. (2) -- Provide the identity, the approximate weight 

(by kg/day or kglbatch, on an 100% new chemical substance basis), and entry point of all feedstocks (including reactants, solvents and catalysts, etc) and 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 ofthe new chemical substance. (4) Please enter the # of sites (remember to identify the locations of 

these sites on a continuation sheet): 

[ ] 

# of sites 

CONFIDENTIAL BUSINESS INFORMATION: 

[ 

] 

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

2. Worker ExposurelEnvironmental Release 

(I) -­From the diagram above, provide the letter for each worker activity. Complete 2-8 for each worker activity described. 

(2) --Estimate the number of workers exposed for all sites combined. 

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

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

protect workers. 

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

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

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

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

(specify) and control technology, if any, that will be used to limit the release of the new substance to the environment. 

(14) -- Identify byproducts which may result from the operation. 

(3), (5), (8), II), (13) and (IS) ­ Mark (X) this column if any ofthe nroceedin!!. entries are confidential business information (CBI). 

Letter # of CBI Duration CBI Protective Equip. I "loin CBI Release Amount of CBI Media of Release CBI 

of Workers of Engineering Controls! Fonn· Number New & Control 

Act· Exposed Exposure Physical Form and % ulation Substance Technology 

ivity new substance Released 

(I) (2) (3) (4a) (4b) (5) (6) (7) (8) (9) (lOa) (lOb) (Ill (12) (13) 

I 

oducts: (15) 

(X) this box if you attach a continuation sheet, 

EPA FORM 7710-25 (Rev. 5-95) Page 10 
OPTIONAL POLLUTION PREVENTION INFORMATION 

To claim infonnation in this section as confidential circle or bracket the specific infonnation 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 

of the 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 

of risks 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 perfonnance, a decrease in 

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

substances used in similar application; or (6) the extent to 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. 

See Attachment #13 - Pollution Prevention Information. 

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

EPA FORM 7710-25 (Rev. 5-95) Page 11 
Part III -- LIST OF ATTACHMENTS 

Attach continuation sheets for sections ofthe 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 ofthe 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 you claim infonnation as confidential. 

Attachment name 

Material Safetv Data Sheet (MSDS) 

Physical and Chemical Properties Worksheets 

Attachment #1 - CAS Inventory Expert Service Reports 

Attachment #2 

Attachment #3 

Attachment #4 

Attachment #5 

Attachment #6 

Attachment #7 

Attachment #8 

Attachment #9 

Attach ment #10 

Attachment #11 

Attachment #12 

Attachment #13 - Pollution Prevention Information 

Attachment #14 - Toxicological Data on Two Structurally Similar Compounds (Ames Tests) 

Attachment Confi· 

page number(s) dentiaJ 

13 - 24 


25 -26 


27 - 29 


30 - 33 
 x 

34 x 

35 x 

36 x 

37 - 38 x 

39 -41 x 


42 -43 
 x 


44 -46 
 x 

47 x 


48 -49 
 x •

50 - 52 x 


53 - 54 


55 - 101 


l J Mark (X) this box if you attach a continuation sheet. Enter the attachment name and number. 

EPA FORM 7710·25 (Rev. 5·95) Page 12 
ISSUED: MARCH 2000 

PAGE 1 OF6 

[ ] 


MATERIAL SAFETY DATA SHEET 

Section 1. CHEMICAL PRODUCT AND COMPANY IDENTIFICATION 

MANUFACTURER: 

[ ] 

[ ] 

[ ] 

[ ] 

EMERGENCY TELEPHONE: 

CHEMTREC ® (800) 424-9300** 

24 hour info line 

** CHEMTREC ® number to be 

used only in the event of a 

chemical emergency 

ISSUE DATE: March 7,2001 REVISED: 

INFORMATION TELEPHONE NUMBER: 

[ ) 

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

PRODUCT NAME: [ ] 

CHEMICAL FAMILY: Halogen substituted oxetane 

CHEMICAL FORMULA: Proprietary 

DOT PROPER SHIPPING NAME: N/A 

10 NUMBER: N/A 

DOT HAZARD CLASSIFICATION: N/A 

030013 

ISSUED: MARCH 2000 

PAGE20F6 

SECTION 2: COMPOSITIONIINFORMATION ON INGREDIENTS 

COMPONENT PERCENT 

WEIGHT 

EXPOSURE 

LIMIT 

UNITS AGENCY TYPE 

Halogen substituted 

oxetane 

CAS# N/A 

100% None 

Established 

*Indicates toxic chemicals subject to the reporting requirements of section 313 of title III 

and of 40 CFR 372. 

HAZARD EMERGENCY OVERVIEW 

May bea mild skin, eye and respiratory irritant. 

SECTION 3: HAZARDS IDENTIFICATION 

HEALTH HAZARDS/ROUTES OF ENTRY 

EYE CONTACT: YES, MAY BE MILDLY IRRITATING TO EYES. 

SKIN CONTACT: YES, MAY BE MILDLY IRRITATING TO SKIN. 

INHALATION (BREATHING): YES, MAY BE MILDLY IRRITATING TO 

RESPIRATORY TRACT. 

INGESTION: POSSIBLE 

CARCINOGENICITY STATUS: NOT LISTED BY NTP, IARC OR OSHA 

I SECTION 4: FIRST AID MEASURES 


EYE CONTACT: FLUSH WITH WATER FOR 15 MINUTES. GET MEDICAL 


ATTENTION. 


SKIN CONTACT: FLUSH WITH WATER FOR 15 MINUTES. REMOVE 


CONTAMINATED CLOTHING. GET MEDICAL ATTENTION. 


INHALATION: REMOVE TO FRESH AIR. GET MEDICAL ATTENTION. 

INGESTION: INDUCE VICTIM TO VOMIT. GET MEDICAL ATTENTION. 

SECTION 5: FIRE FIGHTING MEASURES 

ISSUED: MARCH 2000 

PAGE30F6 

EXTINGUISHING MEDIA: WATER, CARBON DIOXIDE, DRY CHEMICAL OR FOAM. 

UNUSUAL FIRE AND EXPLOSION HAZARD: May form [ ] when 

decomposing in heat or fire. Material is reactive with Lewis Acids/Strong Acids. 

SPECIAL FIRE FIGHTING PROCEDURES: WEAR POSITIVE PRESSURE SELFCONTAINED BREATHING APPARATUS (SCBA). AVOID COMBUSTION 

PRODUCTS. CONTROL RUN-OFF. 

SECTION 6: ACCIDENTAL RELEASE MEASURES 

SPILL AND LEAK PROCEDURES 

WEAR PERSONAL PROTECTIVE EQUIPMENT TO AVOID EXPOSURE. CONTAIN 

SPILLS; KEEP MATERIAL FROM BEING DISCHARGED TO LAND, WATERWAYS 

OR AIR. PICK UP WITH INERT ABSORVENT; PLACE INTO WASTE CONTAINERS. 

I SECTION 7: HANDLING AND STORAGE 

CONTAINS LIQUIDS. USE GLOVES (NEOPRENE OR NITRILE). STORE IN A 

COOL AND DRY LOCATION. 

I SECTION 8: EXPOSURE CONTROUPERSONAL PROTEC"nON 

VENTILATION: GENERAL ROOM VENTILATION IS ADEQUATE. 

RESPIRATORY PROTECTION: NONE REQUIRED 

PROTECTIVE GLOVES: NEOPRENE OR NITRILE 

EYE PROTECTION: SAFETY GLASSES 

OTHER PROTECTION: N/A 

000015 

] ISSUED: MARCH 2000 

] PAGE40F6 

SECTION 9: PHYSICAL AND CHEMICAL PROPERTIES 


BOILING POINT RANGE: 300 C @ 0.2 mm R!:!: NEUTRAL 

SOLUBILITY IN WATER: < 1% PERCENT VOLATILE: < 1% 

APPEARANCE: Colorless viscous oil ODOR: Sweet odor. 

VAPOR DENSITY: N/A VAPOR PRESSURE: N/A 

EVAPORATION RATE (BUOAC=100): N/A 

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

I SECTION 10: STABILITY AND REACTIVITY 


STABILITY: STABLE 

INCOMPATIBILITY (MATERIALS TO AVOID): WILL REACT WITH LEWIS ACIDS 

AND STRONG ACIDS. 

CONDITIONS TO AVOID: TEMPERATURE ABOVE 1500 C 

HAZARDOUS DECOMPOSITION PRODUCTS: [ 

HAZARDOUS POLYMERIZATION: MAY OCCUR 

I SECTION 11: TOXICOLOGICAL INFORMATION 

NEGATIVE RESULTS FOR MUTAGENICITY ON AMES TEST FOR SIMILAR 

SUBSTANCES. 

I SECTION 12: ECOLOGICAL INFORMATION 


NOT AVAILABLE AT THIS TIME. 


I SECTION 13: DISPOSAL INFORMATION 


DISPOSE IN ACCORDANCE WITH FEDERAL, STATE AND LOCAL REGULATIONS. 

GG0016 
] ISSUED: MARCH 2000 

PAGE50F6 

SECTION 14: TRANSPORT INFORMATION 


NOT A HAZARDOUS MATERIAL AS REGULATED BY DOT. 


I SECTION 15: REGULATORY INFORMATION 


OSHA HAZARD COMMUNICATION RULE 29 CFR 1910.1200: 

Compounds listed are defined as hazardous ingredients according to 

OSHA (Occupational Safety and Health Administration) Regulations. 

Routine handling of this material is not expected to generate airborne 

concentrations in excess of OSHA Permissible Exposure Limits or ACGIH 

(American Conference of Government Industrial Hygienists) TLV 

(Threshold Limit Value). However, each use of this product should be 

evaluated to determine if exposure hazard exists. 

CERCLAISUPERFUND, 40 CFR 117 PART 302: N/A 

TOXIC SUBSTANCES CONTROL ACT (TSCA): This is a proprietary product of 

[ ] and is currently being managed under a Low Volume 

Exemption to TSCA, (40 CFR 723.50(c)(1». 40 CFR 723 limits the use of this material 

to the subsequent production of reactive polymers for coating material and other 

polymer intermediates. 

EUROPEAN INVENTORY OF EXISTING COMMERCIAL CHEMICAL SUBSTANCES 

(EINECS) AND EUROPEAN LIST OF NOTIFIED CHEMICAL SUBSTANCES 

(ELlNCS): This is a proprietary product of [ ] and is currently 

being managed in the European Community under Article 13 paragraph 2. of 

Substances Directive (67/548/EEC) as a substance intended solely for purposes of 

scientific research and development carried out under controlled conditions. This 

product is to be used within the European Community only for research and 

development as permitted by EC and other appropriate jurisdictional regulations. 

CANADIAN ENVIRONMENTAL PROTECTION ACT(CEPA): This is a proprietary 

product of [ ] and is currently being managed in Canada under 

the provisions of the Research and Development Special Use Category as a substance 

intended solely for purposes of scientific research and development carried out under 

controlled conditions. It is not on the DSL or NDSL. This product is to be used only for 

research and development as permitted by the CEPA and other appropriate 

jurisdictional regulations. 

G0001? 
ISSUED: MARCH 2000 

PAGE60F6 

SEC1"ION 16: OTHER INFORMATION 


HAZARD RATINGS: 


NFPA HAZARD CLASS 2 HMIS HAZARD CLASS 3 HAZARD RANKING 

Health Hazard: 2 Health Hazard: 1 0- Least 

Flammability: 0 Flammability: 0 1 - Slight 

Reactivity: 2 Reactivity: 2 2 - Moderate 

Other: - PPE: 3 - High 

4 - Extreme 

2 _ National Fire Protection Association (NFPA) 

Rating identifies the severity of hazards of material during a fire (i.e. "on fire"). 

3_ Hazardous Materials Identification System (HMIS) 

National Paint and Coatings Association ratings apply to product "as Packaged". 

Information on this form is furnished solely for the purpose of compliance with the Occupational Safety 

and Health Act of 1970, and applicable regulations, and should not be used for any other purpose. No 

warranty express or implied, of merchantability, fitness for purpose, or of any other nature regarding this 

data or the product is made herein. 

Issue date: March 7, 2001 Revision date: April 4, 2001 

Written by: [ 

Reviewed by: Awaiting independent toxicological review. 

Approved by: [ 

OUTSIDE THE UNITED STATES, THIS MATERIAL IS PROVIDED FOR RESEARCH 

AND DEVELOPMENT USE ONLY BY THE ORIGINAL RECIPIENT. THIS MATERIAL 

MAY NOT BE TRANSFERRED TO ANY OTHER INDIVIDUAL, CONCERN OR 

ORGANIZATION WITHOUT THE EXPRESS WRITTEN APPROVAL OF [ ]. THE 

U.S. TOXIC SUBSTANCE CONTROL ACT, 40 CFR 723, THE EUROPEAN 

COMMUNITY SUBSTANCES DIRECTIVE (675/548/EEC) AND CANADA'S CEPA 

REGULATIONS LIMITS THE USE OF THIS MATERIAL. 

G00013 

Current Issue: December 18, 2002 

PAGE 1 OF6 

[ ] 


MATERIAL SAFETY DATA SHEET 


Section 1. CHEMICAL PRODUCT AND COMPANY IDENTIFICATION 

MANUFACTURER: 

[ ] 

[ ] 

[ ] 

[ ] 

EMERGENCY TELEPHONE: 

CHEMTREC ® (800) 424-9300** 

24 hour info line 

** CHEMTREC ® number to be 

used only in the event of a 

chemical emergency 

ISSUE DATE: December 18,2002 REVISED: Original 

INFORMATION TELEPHONE NUMBER: 

[ ] 

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

PRODUCT NAME: [ ] 

CHEMICAL FAMILY: Halogen substituted oxetane 

CH EMICAL FORMULA: Proprietary 

DOT PROPER SHIPPING NAME: N/A 

10 NUMBER: N/A 

DOT HAZARD CLASSIFICATION: N/A 

G00019 

1 Current Issue: December 18, 2002

] PAGE20F6 

SECTION 2: COMPOSrnON/INFORMATION ON INGREDIENTS 

COMPONENT PERCENT EXPOSl~RE I UNITS AGENCY TYPE 

WEIGHT LIMIT 

Halogen substituted 100 % None 

oxetane Established 

CAS# N/A 

*Indicates toxic chemicals subject to the reporting requirements of section 313 of title III 

and of 40 CFR 372. 

HAZARD EMERGENCY OVERVIEW 

May be a mild skin, eye and respiratory irritant. 

SECTION 3: HAZARDS IDENTIFICATION 

HEALTH HAZARDS/ROUTES OF ENTRY 

EYE CONTACT: YES, MAY BE MILDLY IRRITATING TO EYES. 

SKIN CONTACT: YES, MAY BE MILDLY IRRITATING TO SKIN. 

INHALATION (BREATHING): YES, MAY BE MILDLY IRRITATING TO 

RESPIRATORY TRACT. 

INGESTION: POSSIBLE 

CARCINOGENICITY STATUS: NOT LISTED BY NTP, IARC OR OSHA 

I SECTION 4: FIRST AID MEASURES 


EYE CONTACT: FLUSH WITH WATER FOR 15 MINUTES. GET MEDICAL 


ATTENTION. 


SKIN CONTACT: FLUSH WITH WATER FOR 15 MINUTES. REMOVE 


CONTAMINATED CLOTHING. GET MEDICAL ATTENTION. 


INHALATION: REMOVE TO FRESH AIR. GET MEDICAL ATTENTION. 

INGESTION: INDUCE VICTIM TO VOMIT. GET MEDICAL ATTENTION. 

SECTION 5: FIRE FIGHTING MEASURES 


Gaoo~~o 
Current Issue: December 18, 2002 

PAGE30F6 

EXTINGUISHING MEDIA: WATER, CARBON DIOXIDE, DRY CHEMICAL OR FOAM. 

UNUSUAL FIRE AND EXPLOSION HAZARD: May form [ ] when 

decomposing in heat or fire. Material is reactive with Lewis Acids/Strong Acids. 

SPECIAL FIRE FIGHTING PROCEDURES: WEAR POSITIVE PRESSURE SELFCONTAINED BREATHING APPARATUS (SCBA). AVOID COMBUSTION 

PRODUCTS. CONTROL RUN-OFF. 

SECTION 6: ACCIDENTAL RELEASE MEASURES 

SPILL AND LEAK PROCEDURES 

WEAR PERSONAL PROTECTIVE EQUIPMENT TO AVOID EXPOSURE. CONTAIN 

SPILLS; KEEP MATERIAL FROM BEING DISCHARGED TO LAND, WATERWAYS 

OR AIR. PICK UP WITH INERT ABSORVENT; PLACE INTO WASTE CONTAINERS. 

I SECTION 7: HANDLING AND STORAGE 

CONTAINS LIQUIDS. USE GLOVES (NEOPRENE OR NITRILE). STORE IN A 

COOL AND DRY LOCATION. 

I SECTION 8: EXPOSURE CONTROUPERSONAL PROTECTION 


VENTILATION: GENERAL ROOM VENTILATION IS ADEQUATE. 

RESPIRATORY PROTECTION: NONE REQUIRED 

PROTECTIVE GLOVES: NEOPRENE OR NITRILE 

EYE PROTECTION: SAFETY GLASSES 

OTHER PROTECTION: N/A 

G~0021 
[ Current Issue: December 18, 2002 

[ PAGE40F6 

SECTION 9: PHYSICAL AND CHEMICAL PROPERTIES 

BOILING POINT RANGE: 300 C @ 0.2 mm .Il!:!: NEUTRAL 

SOLUBILITY IN WATER: < 1% PERCENT VOLATILE: < 1% 

APPEARANCE: Colorless viscous oil ODOR: Sweet odor. 

VAPOR DENSITY: N/A VAPOR PRESSURE: N/A 

EVAPORATION RATE (BUOAC=100): N/A 

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

SECTION 10: STABILITY AND REACTIVITY 


STABILITY: STABLE 

INCOMPATIBILITY (MATERIALS TO AVOID): WILL REACT WITH LEWIS ACIDS 

AND STRONG ACIDS. 

CONDITIONS TO AVOID: TEMPERATURE ABOVE 1500 C 

HAZARDOUS DECOMPOSITION PRODUCTS: [ 

HAZARDOUS POLYMERIZATION: MAY OCCUR 

I SECTION 11: TOXICOLOGICAL INFORMATION 


NEGATIVE RESULTS FOR MUTAGENICITY ON AMES TEST FOR SIMILAR 

SUBSTANCES. 

SECTION 12: ECOLOGICAL INFORMA"rlON 

NOT AVAILABLE AT THIS TIME. 


SECTION 13: DISPOSAL INFORMATION 


DISPOSE IN ACCORDANCE WITH FEDERAL, STATE AND LOCAL REGULATIONS. 


I 

I 
] Current Issue: December 18, 2002 

] PAGE50F6 

SECTION 14: TRANSPORT INFORMATION 


NOT A HAZARDOUS MATERIAL AS REGULATED BY DOT. 


TION 15: REGULATORY INFORMATION 


OSHA HAZARD COMMUNICATION RULE 29 CFR 1910.1200: 

Compounds listed are defined as hazardous ingredients according to OSHA 

(Occupational Safety and Health Administration) Regulations. Routine handling 

of this material is not expected to generate airborne concentrations in excess of 

OSHA Permissible Exposure Limits or ACGIH (American Conference of 

Government Industrial Hygienists) TLV (Threshold Limit Value). However, each 

use of this product should be evaluated to determine if exposure hazard exists. 

CERCLAISUPERFUND, 40 CFR 117 PART 302: N/A 

TOXIC SUBSTANCES CONTROL ACT (TSCA): This product is currently being 

managed under the R&D Exemption to TSCA, (40 CFR 720.36). It is not on the TSCA 

list or the Confidential TSCA list. This product is provided in small quantities only for 

Research and Development and must be used under the direct supervision of a 

Technically Qualified Individual as defined by 40 CFR 720.3 (ee). The recipient of this 

material is expected to conduct its research and development activities in accordance 

with Prudent Laboratory Practices and to inform employees who may be exposed to 

this material of the hazards associated therewith. For use by original recipient only. 

EUROPEAN INVENTORY OF EXISTING COMMERCIAL CHEMICAL SUBSTANCES 

(EINECS) AND EUROPEAN LIST OF NOTIFIED CHEMICAL SUBSTANCES 

(ELlNCS): This product is currently being managed in the European Community under 

Article 13 paragraph 2. of Substances Directive (67/548/EEC) as a substance intended 

solely for purposes of scientific research and development carried out under controlled 

conditions. It is not on the EINECS or ELINCS lists. This product is to be used within 

the European Community only for research and development as permitted by EC and 

other appropriate jurisdictional regulations. 

CANADIAN ENVIRONMENTAL PROTECTION ACT(CEPA): This product is currently 

being managed in Canada under the provisions of the Research and Development 

Special Use Category as a substance intended solely for purposes of scientific research 

and development carried out under controlled conditions. It is not on the DSL or NDSL. 

This product is to be used only for research and development as permitted by the 

CEPA and other appropriate jurisdictional regulations. 

Ga0023 
Current Issue: December 18, 2002 

] PAGE60F6 

Other Jurisdictions: This product has not been notified. registered or listed on any 

new chemical substance list or similar document outside the United States. Use of this 

product in countries other than those listed above shall be in accordance with the 

appropriate jurisdictional regulations concerning new chemical substances. 

SECTION 16: OTHER INFORMATION 


HAZARD RATINGS: 


NFPA HAZARD CLASS 2 HMIS HAZARD CLASS 3 HAZARD RANKING 

Health Hazard: 2 Health Hazard: 1 0- Least 

Flammability: 1 Flammability: 1 1 - Slight 

Reactivity: 2 Reactivity: 2 2 - Moderate 

Other: - PPE: 3 - High 

4 - Extreme 

2 • National Fire Protection Association (NFPA) 

Rating identifies the severity of hazards of material during a fire (i.e. "on fire"). 

3. Hazardous Materials Identification System (HMIS) 

National Paint and Coatings Association ratings apply to product "as Packaged". 

Information on this form is furnished solely for the purpose of compliance with the Occupational Safety 

and Health Act of 1970, and applicable regulations, and should not be used for any other purpose. No 

warranty express or implied, of merchantability, fitness for purpose, or of any other nature regarding this 

data or the product is made herein. 

Issue date: October 18. 2002 Revision date: December 18, 2002 


Written by: [ ] 


Reviewed by: Awaiting independent toxicological review. 


Approved by: [ ] 


G00024 

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. Identity 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 ofthe neat substance should be provided. These measured properties should be for the neat ( I 00% 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 simplity review and ensure that confidential 

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

is not a substitute for submission of test data. 

Property Mark Page Value 

[ 1 (Xl if number 

provided (b) (c) (a) 

Physical state of neat substance X 25 (s) x (I) (g) 

Vapor pressure 

@Temperature °C Torr 

Density/relative density X 25 1.2 g/cm3 

Solubility 

@ Temperature °C 

Solvent gil 

Solubility in water @ Temperature 25 °C X 25 <1% gil:, 

Melting temperature °C 

Boiling / sublimation temperature(a) 0.2 torr pressure X 25 30 °C 

Spectra 

Dissociation constant 

Particle size distribution 

Octanol / water partition coefficient 

Henry's Law constant 

Volitalization from water 

Volitalization from soil 

pH@ concentration 100% X 25 -7 

Flammability 

Explodability 

Adsorption / coefficient 

Other - SpecifY 

Estimate 

(MorE) 

N/A 

M 

M 

M 

I 

! 

M I 

I I 

&00025 

ATTACHMENT 

PAC!I! 13 
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 Mark Page Value lMeasured 0 Confidential 

[ J (X) if 

provided 

(a) 

Physical state of neat substance X 

Vapor pressure 

@ Temperature °C 

Density/relative density X 

Solubility 

@ Temperature °C 

Solvent 

Solubility in water (QJ Temperature 25 °C X 

Melting temperature 

Boiling I sublimation temperature (a) 0.2 torr pressure X 

Spectra 

Dissociation constant 

Particle size distribution 

Octanol I water partition coefficient 

Henry's Law constant 

Volitalization from water I 

Volitalization from soil 

pH(QJ concentration ]00% X 

Flammability 

Explodability 

Adsorption I coefficient 

Other - Specify 

number 

(b) (c) 

26 (s) x (I) 

26 1.4 

26 <1% 

I 

26 30 

26 ~7 

(g) 

Torr 

g/cm3 

gIL 

~ 

°C 

°C 

Estimate Mark (X) 

(M or E) (d) 

N/A 

M 

M 

M 

M 

G00026 

ATTACHMENT 

P'I)"(;I& 13 
Consolidated PMN 

TS-IHlSlM 

Attachment #1 

Part I, Section B.l 

PMNp.4 

asInventory Expert Service Reports 

G00027 

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: answers@cas.org 

INVENTORY EXPERT SERVICE REPORT 

IES-ORDER NUMBER: [ ] 

REGISTRY NUMBER:[ l 

CA INDEX NAME 

[ ] 

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

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

your IES order. Please make the same correction(s) to your PMN before 

submitting it to the EPA. 

G00028 

2540 Olentangy River Road P.O. Box 3343 Columbus. Ohio 43210..()334 
ern Inventory Expert Service 


A division of the American Chemical Society Phone: 800-631-1884, 614-447-3600 (ext. 2308) 

Fax:614w447-3747 

E-mail: answers@cas.org 

INVENTORY EXPERT SERVICE REPORT 

IES-ORDER NUMBER: ] 

REGISTRY NUMBER:~ ] 

CA INDEX NAME 

[ ] 

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

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

your lES order. Please make the same correction(s) to your PNN before 

submitting it to the EPA. 

<;00029 

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

TS-IHlSIM 

Attachment #2 

Part I, Sections A and B 

PMNp.4 

caOO2:0 

G00031 

G00032 


Consolidated PMN 

TS-IHISIM 

Attachment #3 

Part I, Section C.l 

PMNp.7 
Consolidated PMN 

TS·IHlS1M 

Attachment #4 

Part II, Section A.t.c 

PMNp.8 

I 

I 

I 

I 

, 

I 

CGG035 

Consolidated PMN 

TS-IHISIM 

Attachment #5 

Part II, Section A.1 

PMNp.8 

G00036 

Consolidated PMN 

TS"lHlSlM 

Attachment #6 

Part II, Section A.l.d 

PMNp.8 

GOOO~'7 
TS-1H1S1M 

G00038 

Consolidated PMN 

TS-IHISIM 

Attachment #7 

Part II, Sections A.2 and A.3 

PMNp.9 

G00039 

TS-1H1S1M 

Attachment #7 

Worker 

Activity 

(I) 

CBI 

(2) 

Protective 

Equipment! 

Engineering 

Controls 

(3) 

Binding 

Option 

Mark 

(4) 

Physical 

form(s) and 

% new 

chemical 

substance 

(5) 

Binding 

Option 

Mark 

(6) 

CBI 

m 

Number 

of 

Workers 

Exposed 

(8) 

CBI 

(9) 

Maximum duration CBI 

(12) 

Hrs/day 

(10) 

Days/year 

(11) 

X X X X 

X X X X 

X X X X 

I 

X X X X 

X X X X 

X X X X 
TS-IHISIM 

Attachment #7 

Release 

Number 

(1) 

Amount of New Substance 

Released 

CUI 

(3) 

Media of Release 

(4) 

Control Technology and Efficiency CDI 

(6) (2a) (2b) (Sa) 

Bindi 

ng 

Mark (5b) 

X X 

X X 

X X 

X X 

X X 

X X 

X X 

X X 

(7) Mark (X) the o POTW provide name(s) below: 

destination( s) of 

releases to water. 

CBI 

r--­

o Navigable waterway 

o Other 

Provide NPDES # CBI 

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

TS-IHISIM 

Attachment #8 

Part II, Section A.l.d 

PMNp.8 

GGOO<12 

TS-IHlSlM 

L 


C00043 

Consolidated PMN 

TS-IHISIM 

Attachment #9 

Part II, Sections A.2 and A.3 

PMNp.9 

COOO,14 

TS-IHISIM 

Attachment #9 

. 

[ ] 

Worker 

Activity 

(1) 

CBI 

(2) 

Protective 

Equipmentl 

Engineering 

Controls 

(3) 

Binding 

Option 

Mark 

(4) 

Physical 

formes) and 

0/.. new 

chemical 

substance 

(5) 

Binding 

Option 

Mark 

(6) 

CBI 

(7) 

Number 

of 

Workers 

Exposed 

(8) 

CBI 

(9) 

Maximum duration CBI 

(12) 

Hrs/day 

(10) 

Days/year 

(11) 

X X X X 

X X X X 

X X X X 

X X X X 

X X X X 

X X X X 

G()0045 


I 
TS-IHlSlM 

Attachment #9 

[ 


Release 

Number 

(J) 

Amount of New Substance 

Released 

CDI 

(3) 

Media of Release 

(4) 

Control Technology and Efficiency CHI 

(6) (2a) (2b) (58) 

Bind; 

~a~ (5b) 

X X 

X X 

I 

X X 

X X 

X X 

X X 

X X 

X X 

(7) Mark (X) the o POTW provide narne(s) below: 

destination(s) of 

releases to water. 

CBI 

r--­

o Navigable waterway 

o Other 

Provide NPDES # CBl 

• 

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

TS-IHISIM 

Attachment #10 

Part II, Section A.1 

PMNp.8 

000047 

Consolidated PMN 

TS-IHISIM 

Attachment #11 

Part II 

Section A.1.d 

PMNp.8 

G00048 

TS-IHISIM 
2 

Consolidated PMN 

TS-IHISIM 

Attachment #12 

Part II, Sections A.2 and A.3 

PMNp.9 

GOC050 

TS-IHISIM 

Attachment #12 

[ 1 

Worker 

Activity 

(1) 

CBI 

(2) 

Protective 

Equipment! 

Engineering 

Controls 

(3) 

Binding 

Option 

Mark 

(4) 

Physical 

form(s) and 

0/.. new 

chemical 

substance 

(5) 

Binding 

Option 

Mark 

(6) 

CBI 

(7) 

Number 

of 

Workers 

Exposed 

(8) 

CBI 

(9) 

Maximum duration CBI 

(12) 

HrsJday 

(10) 

Days/year 

(11) 

X X X X 

X X X X 

X X X X 

X X X X 

X X X X 

X X X X 

I 

000051 

TS-IHISIM 


Attachment #12 

[ ] 


Release 

Number 

(1) 

Amount of New Substance 

Released 

CDI 

(3) 

Media of Release 

(4) 

Control Technology and Efficiency eBI 

(6) (2a) (2b) (Sa) 

Bindi n, 

Mark (5b) 

X X 

X X 

X X 

X X 

X X 

(7) Mark: (X) the o POTW provide name(s) below: 

destination(s) of 

releases to water. 

CBl 

-

o Navigable waterway 

o Other 

Provide NPDES # eB! 

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

000052 

Consolidated PMN 

TS-IHISIM 

Attachment #13 

PMNp.ll 

Pollution Prevention Infonnation 

G00053 

4 

Consolidated PMN 

TS-IHISIM 

000054 

Consolidated PMN 

TS-IHlSlM 

Attachment #14 

Other Information 

Toxicological Data on Two Structurally Similar Compounds (Ames Tests) 

G~0055 
CONFIDENTIAL MLH 0011984844

SUBJECT CHEMICAL #1 

BACTERIAL MUTATION ASSAY 

Sponsor Research Laboratory 

Huntingdon Life Sciences Ltd., 

P.O. Box 2, 

Huntingdon, 

Cambridgeshire, I l 

PE186ES,

L ENGLAl'rD. 

Report issued 9 December 1998 

Page 1 of23 

G00056 
I'v1LH 0011984844 

CONTENTS 

Page 


COrvrPLlANCE WITH GOOD LABORATORY PRACTICE STANUARDS ............... . 3 


QUALnY ASSURANCE STATEMENT ............. . 4 


SUMMARy .................................................... . 5 


INTRODUCTION ..................................................................................................................... . 6 


TEST SUBSTANCE................................................................................................................... 8 


EXPERllvIENTAL PROCEDURE ............................................................................................. 9 


ASSESSMENT OF RESULTS.................................................................................................. 13 


MAlNTINANCE OF RECORDS................................................................ .............................. 14 


RESULTS.................................................................................................................................... 15 


CONCLUSION. ....... ........................... ...... ..... .... .... ........ ........... ...... ......... ....... .... .................... .... 15 


REFERENCES............................................................................................................................ 16 


TABLES 

1. Subject Chemica] #1 Mutation test 1 ............ ...... .......... ........................................... . 17 


2. Positive control compOlmds - Mutation test 1 ........................ . 19 


3. Subject Chemica] #1 - Mutation test 2 ......................... 20 


4. Positive control compounds - Mutation test 2 ... .. 22 


APPENDIX 


Historical control data . 23 


2 


000057 
MLH 001/984844 

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS 

The study described in this report was conducted in compliance with the following Good Laboratory 

Practice standards and I consider the data generated to be valid. 

The UK Good Laboratory Practice Regulations 1997 (Statutory Instrument No 654). 

OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MCICHEM(98)17. 

EC Council Directive 871l8IEEC of l8 December 1986 (Official Journal No L 15129) 

.1..f).g,~..t. j.1.8.. Date 

Department of Genetic Toxicology, 

Huntingdon Life Sciences Ltd .. 

G00058 

MLH 0011984844 

QUALITY ASSUR;\NCE STATEMENT 

The foIloVving have been inspected or auclited in relation to this study 

Study Phases Inspected Date of Inspection Date of Reporting 

Protocol review 3 November 1998 26 November 1998 

Process Based Inspections 

Preparation & labelling 

Agar } 5 June 1998 } 5 June 1998 preparation 

Test material preparation } 30 June 1998 } 10 Ju1y 1998 Dosing 

Plate counting 3 July 1998 10 July 1998 

Record keeping 9 july 1998 10 July 1998 

S9 prepamtion 4 - 10 November 1998 4 - 10 November 1998 

Report 23 November 1998 26 November 1998 

Protocol: An audit of the protocol for this study was conducted and reported to the Study 

Director and Company Management as inclicated above, 

Process based inspections: At or about the time this study was in progress inspections and 

audits of routine and repetitive procedures employed on this type of study were carried out. 

These were conducted and reported to appropriate Company Management as indicated above, 

Report Audit: This report has been audited by the Quality Assurance Department. This 

audit was conducted and reported to the Study Director and Company Management as 

indicated above. 

The methods, procedures and observations were found to be accurately described and the 

reported results to reflect the raw data, 

"( ~".-"', .1__.::>. IG,qb ......... ~~~........ " .. 


Vanessa E. Grant, F.LA.T., Dip. R.Q.A., Date 


Quality Assurance Unit Head, 


Department of Quality Assurance, 


Huntingdon Life Sciences Ltd. 


4 

cn0059 
MLH 0011984844 

SUMMARY 

In this in vitro assessment of the mutagenic potential of Subject Chemical # I, histidine dependent 

auxotrophic mutants of Salmonella t}phimurium (strains TA1535, TAl TA98 and TAJOO) and a 

tryptophan dependent mutant of Escherichia coli (strain CM89l) were exposed to the test substance 

diluted in dimethyl sulphoxide, which was also used as a negative control. 

Two independent mutation tests were performed in the presence and absence of liver preparations from 

Aroclor 1254-induced rats (S9 mix), The first was a standard plate incorporation assay, the second 

involved a pre-incubation stage, 

Dose levels of up to 5000 /-Lg/plate were tested in the mutation tests, This is the standard limit dose 

reconunended in the regulatory guidelines this assay follows, Other dose levels used were a series of ca 

half-loglO dilutions of the highest concentration, No signs of toxicity were observed towards the tester 

strains ill either mutation test, 

No evidence of mutagenic activity was seen at any dose level of Subject Chemical # I in either mutation 

test, 

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity 

ofthe liver preparations, 

It is concluded that, when tested in dimethyl sulphoxide, Subject Chemical #1 shows no evidence of 

mutagenic activity in this test system, 

5 C00060 

MLH 0011984844 


INTRODUCTION 

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

bacterial system The study was conducted in compliance with the following guidelines 

OECD Guideline for the Testing of Chemicals. (1997) Genetic Toxicology: Bacterial 

Reverse Mutation Test, Guidelines 471. 

EEC Annex to Directive 92/69fEEC. (1992) Part B Methods for Determination of 

Toxicity, B.13. Other effects Mutagenicity Escherichia coli - Reverse Mutation Assay. 

OJ No. L 383 A, 157. 

EEC Annex to Directive 92/69fEEC. (1992) Part B . Methods for Determination of 

Toxicity, B.14. Other effects - Mutagenicity: Salmonella typhimurium - Reverse 

Mutation Assay. OJ No. L 383 A, 160. 

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

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

62, No. 158. 

The method described was also designed to comply with ICH (1996 & 1997), and followed the 

recommendations of the United Kingdom Environmental Mutagen Society (Gatehouse et al 1990). 

The in vitro technique described by Ames and his co-workers, (Ames, McCann and Yamasaki 1975, 

Maron and Ames 1983) enables the mutagenic effect of a test substance to be determined by exposing 

specially selected strains of Salmonella typhimurium to the test substance. Normally S. typhimurium is 

capable of synthesising the essential amino acid, histidine, but the mutant strains used in this test are 

incapable of this function. When these strains are exposed to a mutagen, reverse mutation to the 

original histidine independent form takes place in a proportion of the population. These are referred to 

as revertants, and are readily detected by their ability to grow and form colonies on a histidine deficient 

medium. 

A technique based on similar principles has also been described by Green (1984). This system employs 

mutant strains of Escherichia coli which are incapable of synthesising the amino acid tryptophan 

required for growth. 

The strains used carry additional mutations which render them more sensitive to mutagens. The S 

typhimurium strains have a defective cell coat which allows greater permeability oftest substances into 

the celL All the strains are deficient in normal DNA repair processes. In addition three ofthem possess 

a plasmid (pKt\l[1 01) which introduces an error-prone repair process, resulting in increased sensitivity 

to some mutagens. 

Many substances do not exert a mutagenic effect until the}' have been metabolised by enzyme systems 

not available in the bacterial cell. Therefore the bacteria and test substance are incubated in both the 

absence and presence of a supplemented liver fraction (S9 mix) prepared from rats previously treated 

with a substance (Aroelor 1254) known to induce a high level of enzymic activity. 

6 COOOS! 
IVILH 001/984844 

The protocol was approved by Huntingdon Life Sciences Management on 14 September 1998, the 

Sponsor on 5 October 1998 and by the Study Director on 27 October 1998. 

The experimental phase of the study was conducted between 28 October and 9 November 1998 

7 CC3062 
Identity 

Alternative name: 

Chemical name: 

CAS number: 


Appearance: 


Storage conditions: 


Batch number: 


Expiry date: 


Purity: 


Date received: 


MLH 001/984844 

TEST SUBSTANCE 

Subject Chemical #1 

[ J 


] 

Clear colourless liquid 

Room temperature in the dark, desiccated 

CP077-62B 

Stated to be stable for 3 months on the 5 October 

1998 


>99.5% 


22 October 1998 

: 8 : C00063 

MLH 0011984844 

EXPERIMENTAL PROCEDURE 

BACTERIAL STRAINS 

The following strains were used:­

S typhimunum T AI535 hisG46 rfa uvrB 

s. typhlmurium TAI537 hisC3076 rfa uvrB 

S. typhlmurium TA98 hlsD3052 'fa uvrB pKMIOI 

S. typhimurium TAIOO hisG46 ria uvrB pKMIOI 

E. coli CM891 WP2 trp uvrA pKM 101 

The strains of S. typhimurium were obtained from Professor B.X Ames, University of California, 

Berkeley, California, USA. 

The strain of E. coli was obtained from the National Collections of Industrial and Marine Bacteria. 

Aberdeen, Scotland. 

Batches of the strains were obtained from master stocks held in liquid nitrogen. The test batches were 

aliquots of nutrient broth cultures and were stored at -80°C. Dimethyl sulphoxide (DMSO) was added 

to the cultures at 8% vivas a cryopreservative. Each batch of frozen strain was tested, where 

applicable, for cell membrane penneability (rfa mutation), sensitivity to UV light and the pKM101 

plasmid which confers resistance to ampicillin. The responses of the strains to a series of diagnostic 

mutagens were also assessed. 

For use in tests an aliquot of frozen culture was added to 25 ml of nutrient broth (Merck No.2) and 

incubated, with shaking, at 37°C for 10 hours. These cultures provided at least 2 x 109 cells per ml 

which were measured photometrically. 

POSITIv""E CONTROLS 

In the absence of S9 mix 

Identity: N-Ethyl-N'-nitro-N-nitrosoguanidine 

Supplier: Sigma Chemical 

Batch number: 67F-3700 

Appearance: Pale yellow crystalline powder 

Solvent: DMSO 

Concentration: 5 !J.g/plate for strain T A IS 35 

3 flg/plate for strain TAl 00 

2 llg/plate for strain CM891 

9 G30064 
MLH 001/984844 

Identity 


Supplier: 


Batch number 


Appearance 


Solvent: 


Concentration: 


Identity: 


Supplier: 


Batch number: 


Appearance: 


Solvent: 


Concentration: 


In the presence of S9 mix 

Identity: 


Supplier 


Batch number: 


Appearance: 


Solvent: 


Concentration: 


Identity: 


Supplier: 


Batch nmnber: 


Appearance: 


Solvent: 


Concentration: 


9-Arrunoacridine 

Sigma Chenucal 

35H1408 

Yellow powder 

DMSO 

30 tJ.g/plate for strain TAl537 

2-Nitrofluorene 

Aldrich Cherllical Company 

75108 

Beige powder 

DMSO 

1 t-tg/plate for strain T A98 

2-Arrunoanthracene 

Aldrich Chemical Company 

0013406 

Green powder 

DMSO 

2 tJ.g/plate for strain TAIS35 

10 fJ.g/plate for strain CM891 

Benzo[ a ]pyrene 

Aldrich Chemical Company 

05915DY 

Yellow powder 

DMSO 

5 tJ.gJplate for strains TA1537, TA98 and TAIOO 

PREPAR>\TION OF S9 FRACTION 

Species: 

Sex: 

Strain: 

Source: 

Age: 

Weight: 

Diet: 

Rat 

Male 

Sprague-Dawley derived 

Harlan Olac Ltd 

7-8 weeks 

<300 g 

SDS R+M no. I (modified) nuts 

S9 fraction was prepared from a group of co 20 animals. Mixed function oxidase systems in the rat 

livers were stimulated by Arodor 1254, administered as a single intra-peritoneal injection in Arachis oil 

at a dosage of 500 mglkg bodyweight. On the fifth day after injection, following an overnight 

starvation, the rats were killed and their livers aseptically removed. 

The following steps were carried out at 0-4°C under aseptic conditions. The livers were placed in 

0.15 M KCI (3 ml KCl : 1 g liver) before being transferred to an ultra-Turrax homogeniser. Following 

preparation, the homogenate was centrifuged at 9000 g for 10 minutes. The supernatant fraction 

10 GOOOS5 
MLH 001/984844 

(59 fraction) was dispensed into aliquots and stored at -80°C until required. The efficacy of each batch 

of S9 fraction was tested in a bacterial mutation assay with the mutagenic precursors 7,12­

dimethylbenzanthracene and 2-aminoanthracene before use. The sterility was also checked. 

PREPARATION OF S9 MIX 

S9 mix contained: 59 fraction (10% v/v), MgCI" (8 mM), KCI (33 mM), sodium orthophosphate buffer 

pH 7.4 (l00 mM), glucose-6-phosphate (5 mM). NADP (4 mM). All the cofactors were filtersterilised before use. 

SELECT!ON OF SOL VENT 

Prior to commencing testing, the solubility of the test substance was assessed at 50 mglml in dimethyl 

sulphoxide, in which it dissolved. Water was not assessed as information provided by the Sponsor 

indicated that the test substance was insoluble in this solvent. Therefore dimethyl sulphoxide was used 

as the sol vent for this study. 

MUTATION TEST PROCEDURE 

First test 

The test substance was added to cultures of the five tester strains at seven concentrations separated by 

ca half-loglo intervals. The highest concentration of Subject Chemical #1 tested was 50 mglml in the 

chosen solvent, which provided a final concentration of 5000 /lg/plate. This is the standard limit dose 

recommended in the regulatory guidelines this assay follows. The negative control was the chosen 

solvent, dimethyl sulphoxide. The appropriate positive controls were also included. 

An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S9 mix or 0.5 ml 0.1 M phosphate buffer 

(pH 7.4) were placed in glass bottles. An aliquot of 100 III of the test solution was added, followed 

immediately by 2 m1 of histidine/tryptophan deficient agar. The mixture was thoroughly shaken and 

overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Each petri dish was 

individually labelled with a unique code corresponding to a sheet, identifying the dish's contents. Three 

petri dishes were used for each dose level. Plates were also prepared without the addition of bacteria in 

order to assess the sterility of the test substance, S9 mix and phosphate buffer. All plates were 

incubated at 37°C for ca 72 hours. After this period the appearance of the background bacterial lawn 

was examined and revertant colonies counted using a Seescan automated colony counter. 

Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony 

counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects 

the top concentration normally used in the second test would be the same as that used in the first. If 

toxic effects were observed a lower concentration may be chosen. It should be ensured that if a lower 

concentration was chosen, signs of bactenal inhibition are present at the top concentration. Ideally a 

minimum ofthree non-toxic concentrations should be obtained. 

II GOOOSG 
MLH 0011984844 

Second test 

As a clear negative response was obtained in the first test. a variation to the test procedure was used for 

the second. The variation used was the pre-incubation assay in which the bottles were incubated at 

37°C for 30 minutes with shaking before the addition of the agar overlay 5000 ~g/plate was agam 

chosen as the top concentration but onl} five dose levels were used as it was known from the first test 

that the test substance was non-toxic 

STABILITY AND FORMULATION ANALYSIS 

The stability of the test substance and of the test substance in the solvent were not determined as part of 

this study ..Analysis of achieved concentration was not perfonned as part of this study. 

12 oa0067 
:MLH 001/984844 

ASSESSMENT OF RESLTLTS 

For a test to be considered valid the mean of the solvent control revertant colony numbers for each 

strain should be in the range stated in the appropriate Standard Operating Procedure. These ranges are 

based on the laboratory's historical control values ..Also, the positive control compounds must cause at 

least a doubling of mean revertant colony numbers over the solvent controL 

The mean number of revertant colonies for all treatment groups is compared with those obtained for the 

solvent control groups. The mutagenic activity of a test substance is assessed by applying the 

following criteria: 

(a) 	 If treatment with a test substance produces an increase in revertant colony nwnbers of at least 

twice the concurrent solvent controls, vvith some e-v'idence of a positive dose-relationship, in two 

separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it is 

considered to show evidence of mutagenic activity in this test system. 

(b) 	 If treatment with a test substance does not produce reproducible increases of at least 1.5 times 

the concurrent solvent controls, in either mutation test, it is considered to show no evidence of 

mutagenic activity in this test system. 

In addition the test data were subjected to analysis to determine the statistical significance of any 

observed increases in revertant colony numbers. The statistical procedures used were analysis of 

variance followed by Durmett's test (testing at p:s; 0.01 level), as described by Mahon et al 

(1989). The biological significance of any observed statistically significant increases were also 

considered. 

13 &00068 
:tvlLH 001/984844 

MAINTENANCE OF RECORDS 

All experimental data arising from the study (including documentary raw data, records and other 

materials: collectively defined as the "materials") \'Vill remain the property of the Sponsor 

Huntingdon Life Sciences shan retain the materials in its archive for a period of five years from the date 

of issue of the final report. After such time, the Sponsor will be contacted and their adVIce sought on 

the return, disposal or further retention of the materials. If requested, Huntingdon Life Sciences will 

continue to retain the materials, subject to a reasonable fee being agreed with the Sponsor. 

14 G00069 
MLH 001/984844 

RESULTS 

The mean number of revertant colorues, together with the individual plate counts for Subject Chemical 

# I obtained in the first mutation test with the tester strains are shovvn in Table 1. Positive control 

mutability checks are shown in Table 2. No toxicity was observed towards the tester strains. 

The mean number of revertant colonies. together with the individual plate counts for Subject Chemical 

# I obtained in the second mutation test with the tester strains are shown in Table 3. Positive control 

mutabilIty checks are shown in Table 4 

No substantial increases in revertant colony numbers of any of the tester strains were observed 

following treatment with Subject Chemical #1 at any dose leveL in the presence or absence of S9 mix, 

in either mutation test. 

VVhen the test data was statistically analysed using Dunnett's test a few single significant values were 

shown (marked in results tables) However, none of these values were greater than 1.5 times the 

concurrent negative control and all fell well within the laboratories historical negative control range. 

As there was also no evidence of any dose relationship in the observed significant increases they were 

not seen as having any biological significance. 

The mean revertant colony counts for the solvent controls were within the historical range. The 

concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of 

the liver preparations by causing increases over double the concurrent solvent controL 

CONCLUSION 

It is concluded that, when tested in dimethyl sulphoxide. Subject Chemical #1 shows no evidence of 

mutagenic activity in this test system. 

15 G00070 
lv1LH 001/984844 

REFERENCES 

AMES, B.N., McCANN, J., and YAMASAKI. E. (1975) Methods for detecting carcinogens and 

mutagens WIth the Salmonella/mammalian mIcrosome mutagenicity test. Mutation Research, 31,347. 

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

(1990) Bacterial mutation assays in: KIRKLAND, DJ. (Ed.). UKEMS Sub-committee on 

Guidelines jor Mutagenicity Testing. Report. Part I revised. Basic Mutagenicity Tests: UKEMS 

Recommended Procedures, p.13. Cambridge University Press, Cambridge. 

GREEN, M.H.L. (1984) Mutagen testing using lrp+ reversion in Eschen'chia coli in KILBEY, 

B.L, LEGATOR, M., NICHOLS, W. and RAMEL, C. (Eds.). Handbook ojMutagenicity Test 

Procedures. Second edition, p.161. Elsevier Science Publishers BV, Amsterdam. 

ICH (1996) Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests. 

ICH (1997) Genotoxicity: A Standard Battery of Genotoxicity Testing of Pharmaceuticals. 

MAHON, G.AT., GREEN, M.H.L., MIDDLETON, B., MITCHELL, I.de G., ROBINSON, W.D. 

and TWEATS, OJ. (1989) Analysis of data from microbial colony assay in: KIRKLAND, D.J. (Ed.) 

UKEMS Subcommittee on Guidelines for Mutagenicity Testing Report, Part Ill. Statistical 

Evaluation ofMutageniCity Test Data, p.26. Cambridge University Press, Cambridge. 

MARON, D.M. and AMES, B.N. (1983) Revised methods for the Salmonella mutagenicity test. 

Mutation Research, 113, 173. 

16 G00071 
JVlLH 0011984844 


TABLE 1 


Mutation Test 1 


Subject Chemical #1 - revertant colony counts obtained 


per plate using bacterial strains TA1535, TA1537 and TA98 


Strain Dose level Liver Mean SD Individual revertant 

(j.lg/plate) S-9 
 revertant colony counts 

colony 

counts 

TA 1535 
 5000 
 - 15 
 1 .7 
 17, 14, 14 


1500 
 - 14 
 4 .6 
 11, 11, 19 


500 
 - 17 
 1.5 16, 17, 19 


150 
 - 14 
 2.1 12, 13, 16 


50 
 - 15 
 0.6 14, 15, 15 


15 
 - 14 
 2.5 14, 12, 17 


5 
 - 14 
 2.3 17, 13, 13 


Solvent 
 - 13 
 1.5 15, 12, 13 


5000 
 + 14 
 1.2 13, 15, 13 


1500 
 + 13 
 3.1 16, 12, 10 


500 
 + 18 
 2.9 21, 16, 16 


150 
 + 15 
 0.6 15, 15, 14 


50 
 + 14 
 0.0 14, 14, 14 


15 
 + 18 
 1.0 17, 18, 19 


5 
 + 18 
 2.1 16, 17, 20 


Solvent 
 + 14 
 2.1 16, 13, 12 


TA 1537 
 5000 
 - 8 
 0.6 8, 9, 8 


1500 
 - 10 
 2.0 10, 12, 8 


500 
 - 8 
 1.5 6, 9, 8 


150 
 - 9 
 3.5 7, 7, 13 


50 
 - 13 
 1.2 14, 12, 12 


15 
 - 6 
 2.0 4, 6, 8 


5 
 - 9 
 1.5 7, 10, 9 


Solvent - 11 
 1.0 10, 12, 11 


5000 
 + 8 
 1.0 7, 8, 9 


1500 
 + 9 
 2.5 7, 9, 12 


500 
 + 11 
 1.0 11, 10, 12 


150 
 + 12 
 2.5 12, 10, 15 


50 
 + 11 
 1.5 9, 11, 12 


15 
 + 9 
 3.2 8, 7, 13 


5 
 + 11 
 1.2 10, 10, 12 


Solvent + 14 
 3.6 17, 15, 10 


TA 98 
 5000 
 - 23 
 3.8 19, 25, 26 


1500 
 - 22 
 1.5 22, 21, 24 


500 
 - 26 
 3.1 23, 25, 29 


150 
 - 23 
 2.1 25, 21, 22 


50 
 - 20 
 1.5 19, 22, 20 


15 
 - 22 
 2.0 22, 24, 20 


5 
 - 27 
 3.5 31, 25, 25
 Solvent - 25 
 5.8 22, 22, 32 


5000 
 + 23 
 2.3 22, 26, 22 


1500 
 + 27 
 4 .2 
 32, 26, 24 


500 
 + 27 
 2.5 27, 30, 25 


150 
 + 22 
 1.0 22, 23, 21 


50 
 + 31 
 0.6 31, 31, 30 


15 
 + 27 
 4.5 22, 27, 31 


5 
 + 24 
 1. "7 25, 25, 22


Solvent + 28 
 4 .5 
 33, 28, 24 


Absence 

+ Presence 

SD Standard deviation 

17 000072 
lv1LH 001/984844 

TABLE 1 

(continued) 

Mutation Test 1 

Subject Chemical #1 revertant colony counts obtained 

per plate using bacterial strains TA100 and CM891 

Strain Dose level Liver Mean SD Individual revertant

(llg/plate) 5-9 revertant colony counts 

s 

TA 100 5000 - ** 131 13~1 145, 130, 119

1500 ** 147 14.5 132, 147, 161

500 - 107 3.5 109, 103, 109

150 107 11 1 95, 	 117, 109

50 ll2 2 1 114 111, 110

15 ** 134 4.2 , 137, 129

5 - ll7 9.1 107, 121, 124 

Solvent - 100 7.5 92, 	 107, 101

CM 891 

5000 

1500 

500 

150 

50 

15 

5 

Solvent 

5000 

1500 

500 

150 

50 

15 

5 

Solvent 

5000 

1500 

500 

150 

50 

15 

5 

Solvent 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

-

-

-

-

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 	 i 

114 

100 

III 

107 

113 

106 

115 

120 

234 

333 

322 

327 

330 

294 

316 

290 

261 

362 

361 

357 

348 

371 

365 

324 

9.7 103, 116, 122 

6.1 93, 105, 101 

2.1 109, 112, 113 

5.1 113, 103, 106 

6.1 120, 109, 110 

5.0 105, ll1, 101 

2.3 116, 116, 112 

9.0 121, 129, 111 

41.5 276, 193, 234 

27.5 341, 302, 355 

21. 9 338, 331, 297 

21. 4 318, 351, 311 

15.3 318, 324, 347 

50.3 281, 252, 350 

25.5 344, 310, 294 

13.6 285, 279, 305 

48.1 316, 237, 229 

25.7 369, 334, 384 

7.0 354, 360, 368 

16.3 344, 375, 351 

15.0 363, 348, 333 

43.6 413, 326, 374 

19.5 351, 356, 387 

19.9 310, 316, 347 

Absence 

+ 	 Presence 

SD 	 Standard deviation 

Significant at p:::;O.O 1 using Dunnett's test. 

18 G00073 
MLH 0011984844 

TABLE 2 

Mutation Test 1 

Revertant colony counts obtained per plate with positive controls 

Strain Compound Dose level 

().lg/plate) 

Liver 

5-9 

Mean 

revertant: 

colony 

5D Individual reve rt alCt 

colony counts 

counts 

TA 

TA 

TA 

TA 

CM 

1535 

1537 

98 

100 

891 

ENNG 

9 AC 

NF 

ENNG 

ENNG 

:; 

30 

1 

3 

2 

-

-

-

141 

92 

1 

433 

1877 

6 

6. 

°

20.7 

25.2 

20.0 

135, 141, 147 

87£ 100, 90 

143 184 1 159 

45 437 406 

1 , 6, 1898 

TA 

TA 

TA 

TA 

CM 

1535 

1537 

98 

100 

891 

AA 

BlalP 

B[a]P 

B[al£' 

Af'.. 

2 

5 

5 

5 

10 

.,. 

.,. 

+ 

+ 

86 

39 

139 

601 

1527 

14.8 

8.0 

34.7 

32.1 

21. 9 

70, 99, 90 

31, 39, 47 

99, 157, 161 

584, 581, 638 

1511 , 1552, 1518 

Absence 

+ Presence 

SD Standard deviation 

ENNG N-Ethyl-N'-nitro-N-nitrosoguanidine 

9 AC 9-Aminoacridine 

NF 2-Nitrofluorene 

AA 2-Aminoanthracene 

B[aJP Benzo[aIpyrene 

19 
:tvILH 0011984844 


TABLE 3 


Mutation Test 2 


Subject Chemical #1 - revertant colony counts obtained 


per plate using bacterial strains TA1535, TA1537 and TA98 


Str-ain Dose level Liver Mean SD Individua-1 revertant (]lg/plate) S-9 
 revertant colony counts colony 

counts 

TA 1535 
 5000 
 15 
 4.2 12, 20, 14 


1500 
 - 13 
 1.5 12, 15, 13 


500 
 - 18 
 3 ~ 1 
 21, 15, 19 


150 
 - 14 
 2. 12, 12, 17 


50 
 - 15 
 2.3 16, 16, 12 


Solvent 
 - 18 
 1~2 19, 19, 17 


5000 
 + 15 
 1.2 14, 16, 14 


1500 
 + 16 
 2.0 16, 14, 18 


500 
 + 18 
 5.6 24, 17, 13 


150 
 + 15 
 0.0 15, 15, 15 


50 
 + 16 
 0.6 16, 17, 16 


Solvent + 20 
 2.3 19, 19, 23 


TA 1537 
 5000 
 - 8 
 2.9 10, 10, 5 


1500 
 - 9 
 3.8 13, 7, 6 


500 
 7 
 0.6 7, 7, 6 


150 
 8 
 7, 6, 12 


50 


3.2 

8 
 3.5 10, 4, 10 


Solvent - 10 
 4.7 6, 8, 15


5000 
 + 10 
 .1 
 8, 11, 12 


1500 
 + 9 
 2 5 
 11, 6, 9 


500 
 + 10 
 3.6 11, 6, 13 


150 
 + 13 
 2.1 11, 15, 14 


50 
 + 9 
 2.1 8 7, 11 


Solvent 
 + 12 
 5.5 , 9, 8 


TA 98 
 5000 
 23 
 2.1 22, 25, 21 


1500 
 - 24 
 5 .. 6 
 19, 30, 23 


500 
 - 24 
 2.6 27, 23, 22 


150 
 - 24 
 6. 19, 22, 31 


50 
 - 23 
 5.1 17, 27, 24 


Solver:t 
 - 23 
 3.1 26, 20, 22 


., 5000 
 + 25 
 _.L-~ 23, 24, 29 


1500 
 + 26 
 3.2 25, 24, 30 


500 + 24 2.6 23, 22, 27 


150 + 28 3.2 30, 29, 24 


50 + 26 4.7 24, 31 


Solvent + 
 28 i 4.4 26, , 33
 i 


Absence 

+ Presence 

SD Standard deviation 

20 G00075 
l\1LH 001/984844 

TABLE 3 


( continued) 


Mutation Test 2 


Subject Chemical #1 - revertant colony counts obtained 


per plate using bacterial strains TAl00 and CM891 

Strain Dose level 

(Ilg/plate) I 

Liver 

S-9 

Mean 

revertant 

colony 

counts 

SD Individual revertant 

colony counts 

TA 100 5000 

1500 

500 

150 

50 

Solvent 

-

-

-

-

** 123 

105 

109 

108 

106 

98 

9.5 

7.1 

:J.6 

2.1 

4.0 

7.2 

117, :34, 18 

106, 97, III 

109, 109, 108 

106, 109, 110 

105, 110, 102 

90, 102, 103 

5000 

1500 

500 

150 

50 

Solvent 

+ 

+ 

~ 

.. 

.. 

+ 

118 

::'16 

125 

120 

138 

108 

14.8 

2.0 

4.6 

9.7 

12.7 

16.6 

102, 

114, 

128, 

131, 

124, 

106, 

122, 131 

116, 118 

120, 128 

112, 118 

148, 143 

93, 126 

CM 891 5000 

1500 

500 

150 

50 

Solvent 

-

-

-

-

-

-

159 

186 

207 

189 

169 

184 

5.0 

16.1 

6.0 

14.5 

23.7 

5.8 

160, 

168, 

207, 

204, 

154, 

177, 

164, 

191, 

201, 

175, 

196, 

187, 

154 

199 

213 

188 

156 

187 

5000 

1500 

500 

150 

50 

Solvent 

+ 

+ 

+ 

+ 

+ 

+ 

150 

198 

209 

174 

166 

172 

14.0 

7.6 

3.6 

12.1 

17 .0 

11.0 

134, 

195, 

212, 

17:J, 

157, 

168, 

156, 

207, 

205, 

165, 

186, 

184, 

160 

193 

210 

188 

156 

163 

Absence 

+ Presence 

SD Standard deviation 

Significant at psO.OI using Dunnett's test. 

21 000076
Iv1LH 001/984844 

TABLE 4 


Mutation Test 2 


Revertant colony counts obtained per plate with positive controls 


Strai:1 Compou:1d Dose level 

(]Jg/plate) 

Liver 

S-9 

Mean 

revertant 

colony 

counts 

SD Individua.i revertant 

colony counts 

TA 1535 ENNG 5 230 9.2 238, 232, 220 

TA 1537 9 AC 30 ~ 442 9.1 438, 4Cl5, 452 

TA 98 NF 1 - 89 6.1 84, 96, 88 

TA 100 ENNG 3 437 18.7 4 431, 422 

CM 891 ENNG 2 185 . 92.6 18 , 1910, :744 

TA 1535 AA 2 + 447 61.1 404, 517, 420 

TA 1537 B[a]P 5 + i 59 17 .7 50, 47, 79 

TA 98 B[a]P 5 + 287 28. 303, 254, 305 

T}l. 100 B[a]P 5 + 454 30.4 429, 446, 488 

CM 891 AA Ie - I 2094 I 1.2 2095, 2093, 2093 

Absence 

+ Presence 

SD Standard deviation 

ENNG N-Ethy I-N'-nitro-N -nitrosoguanidine 

9AC 9-Aminoacridine 

NF 2-Nitrofluorene 

AA 2-Aminoanthracene 

B[aJP Benzo[a]pyrene 

22 GG0077 

rvrLH 001/984844 

APPENDIX 

Historical control data 

Presented below are the historical control data from the two years prior to commencing this study 

Dimethyl sulphoxide solvent controls 

Strain TA1535 TA1537 TA98 TAIGO CM891 

S9 mix + + + + + 

Minimum 9 10 5 6 17 20 75 76 134 101 

Maximum 21 22 14 16 33 34 158 154 376 373 

Mean 14.8 16.2 9.9 11.2 24.5 26.5 116.1 122.5 225.8 208.0 

Values 154 162 151 162 152 163 157 167 78 77 

Positive controls 

Strain TAl535 TAl537 TA98 TAlOO CM89 1 

S9 mix + + + + + 

Minimum 80 31 75 28 68 71 178 159 436 351 

Maximum 2489 2171 2094 326 681 747 2564 2137 2221 2192 

Mean 470.2 156.5 497.l 74.4 304.1 192.3 496.0 452.7 1466.4 1256.8 

Values 224 235 39 233 221 239 228 246 III 114 

23 000078 
CONFIDENTIAL MLH 002/984845 


SGBJECT CHEMICAL #2 

BACTERIAL MUTATION ASSAY 

Sponsor Research Laboratory 

Huntingdon Life Sciences Ltd., 

P.O. Box 2, 

Huntingdon, 

Cambridgeshire, 

PE186ES, 

ENGLAt'ID. 

Report issued 9 December 1998 

Page 1 of23 G00079 
MLH 002/984845 

CONTENTS 


Page 


COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS......... , ....... . 3 


TABLES 


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


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


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


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


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


ASSESSMENT OF RESULTS.............. , .................. , ............... , .............. , ....... ,.......................... 13 


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


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


CONCLUSION ., .............. , ... ' ...... '.. ,.,', .... ,..... ,.. '... ,.. , ................... , ........... , .. ,..... , ............ , ..... ,..... , 15 


REFERENCES ................ " ...................... ". ,,' ...................... "... ,.... " ....... , .. , ......... ', ..... ,',. , .... ,..... . 16 


1. Subject Chemical #2 - Mutation test L.,.. 17 


2. Positive control compolU1ds - Mutation test 1 ........ 19 


3, Subject Chemical #2 - Mutation test 2 .. ,.. "" 20 


4, Positive control compounds - Mutation test 2 22 


APPENDIX 

Historical control data ... , ........... ,............. 


: 2 G00080 
1vILH 002/984845 

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS 

The study described in this report was conducted in compliance with the following Good Laboratory 

Practice standards and I consider the data generated to be valid. 

The UK Good Laboratory Practice Regulations 1997 (Statutory Instrument No 654). 

OEeD Principles of Good Laboratory Practice (as revised in 1997), ENVIMCICHEM(98)17. 

EC Council Directive 87/181EEC of 18 December 1986 (Official Journal No L 15/29) 

.. ~.F-"?~:dpL/.!'J.~ J Kite Date 


Study Director, 


Department of Genetic Toxicology, 


Huntingdon Life Sciences Ltd .. 


3 : G00081 
:MLH 002i984845 

QUALITY ASSURANCE STATEMENT 

The follo\',ing have been inspected or audited in relatIOn to this study 

Study Phases Inspected Date of Inspection Date of Reporting 

Protocol review 3 :'\Iovember 1998 26 November 1998 

Process Based Inspections 


Preparation & labelling 
 } 5 June 1998 } 5 June 1998 Agar preparation 


Test material preparation 
 } 30 June 1998 } 10 July 1998 Dosing 

Plate counting 3 July 1998 10 July 1998 

Record keeping 9 July 1998 10 July 1998 

S9 preparation 4 - 10 November 1998 4 - 10 November 1998 

Report 25 November 1998 26 November 1998 

Protocol: An audit of the protocol for this study was conducted and reported to the Study 

Director and Company Management as indicated above. 

Process based inspections: At or about the time this study was in progress inspections and 

audits of routine and repetitive procedures employed on this type of study were carried out. 

These were conducted and reported to appropriate Company Management as indicated above. 

Report Audit: This report has been audited by the Quality Assurance Department. This 

audit was conducted and reported to the Study Director and Company Management as 

indicated above. 

The methods, procedures and observations were found to be accurately described and the 

reported results to reflect the raw data . 

......~.~.......... . 
 --, ~~ \9'="t ~ ........... ~ •••• ~ ....... w •••••••••••••• 


Vanessa E. Grant, F.LA.T., Dip. R.Q.A., Date 


Quality Assurance Unit Head, 


Department of Quality Assurance, 


Huntingdon Life Sciences Ltd. 


: 4 C00082 
:MLH 002/984845 

SUMMARY 

In this in vitro assessment of the mutagenic potential of Subject Chemical #2, histidine dependent 

auxotrophic mutants of Salmonella typhimurium (strams TA1535. TA1537, TA98 and TAlOO) and a 

tryptophan dependent mutant of Escherichia cob (stram CM891) were exposed to the test substance 

diluted in dimethyl sulphoxide, which was also used as a negative control 

Two independent mutation tests were performed in the presence and absence of liver preparations from 

ArocIor 1254-induced rats (S9 mix). The first was a standard plate incorporation assay, the second 

involved a pre-incubation stage. 

Dose levels of up to 5000 IJ-g/plate were tested in the mutation tests. This is the standard limit dose 

recommended in the regulatory guidelines this assay follows Other dose levels used were a series of co 

half-loglO dilutions of the highest concentration. No signs of toxicity were observed towards the tester 

strains in either mutation test. 

No evidence ofmutagenic activity was seen at any dose level of Subject Chemical #2 in either mutation 

test. 

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity 

of the liver preparations. 

It is concluded that., when tested in dimethyl sulphoxide, Subject Chemical #2 shows no evidence of 

mutagenic activity in this test system. 

:5: G00083 
MLH 002/984845 

INTRODUCTION 


This report describes a study designed to assess the mutagenic potential of Subject Chemical #2 in a 

bacterial system. The study was conducted in compliance \A.lth the following guidelines: 

OECD Guideline for the Testing of Chemicals. (1997) Genetic Toxicology Bacterial 

Reverse Mutation Test, Guidelines 471. 

EEC Annex to Directive 92/691EEC. (1992) Part B : Methods for Determination of 

Toxicity, R13. Other effects - Mutagenicity: Escherichia coli - Reverse Mutation Assay 

O.J No. L 383 A, 157. 

EEC Annex to Directive 92/69IEEC. (1992) Part B : Methods for Determination of 

Toxicity, B.14. Other effects - Mutagenicity: Salmonella typhimurium - Reverse 

Mutation Assay. OJ No. L 383 A, 160. 

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

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

62, No. 158. 

The method described was also designed to comply with lCH (1996 & 1997), and followed the 

recommendations of the United Kingdom Environmental Mutagen Society (Gatehouse et al 1990). 

The in vitro technique described by Ames and his co-workers, (Ames, McCann and Yamasaki 1975, 

Maron and Ames 1983) enables the mutagenic effect of a test substance to be determined by exposing 

specially selected strains of Salmonella typhimurium to the test substance. Normally S typhimurium is 

capable of synthesising the essential amino acid, histidine, but the mutant strains used in this test are 

incapable of this function. 'When these strains are exposed to a mutagen, reverse mutation to the 

original histidine independent form takes place in a proportion of the population. These are referred to 

as revertants, and are readily detected by their ability to grow and form colonies on a histidine deficient 

medium. 

A technique based on similar principles has also been described by Green (1984). This system employs 

mutant strains of Escherichia coli which are incapable of synthesising the amino acid tryptophan 

required for groVvth. 

The strains used carry additional mutations which render them more sensitive to mutagens. The S 

lyphimurium strains have a defective cell coat which allows greater permeability oftest substances into 

the celL All the strains are deficient in normal DNA repair processes. In addition three of them possess 

a plasmid (pKMIOI) which introduces an error-prone repair process, resulting in increased sensitivity 

to some mutagens. 

Many substances do not exert a mutagenic effect until they have been metabolised by enzyme systems 

not available in the bacterial celL Therefore the bacteria and test substance are incubated in both the 

absence and presence of a supplemented liver fraction (S9 mix) prepared from rats previously treated 

with a substance (Aroelor 1254) known to induce a high level of enzymic activity. 

6 : 
MLH 002/984845 

The protocol was approved by Huntingdon Life Sciences Management on 14 September 1998, the 


Sponsor on 5 October 1998 and by the Study Duector on 27 October 1998. 


The experimental phase of the study was conducted between 28 October and 9 November 1998. 


7 G00085 

Identity 

Alternative name: 

Chemical name: 

CAS number: 

Appearance: 

Storage conditions: 

Batch number: 

Expiry date: 

Purity: 

Date received: 

I'v1LH 002/984845 

TEST SUBSTANCE 

Subject Chemical #2 

[~ J 

[ ] 

Clear colourless liquid 

Room temperature in the dark, desiccated 

CP077-62A 

Stated to be stable for 3 months on 5 October 1998 

>99.5% 

22 October 1998 

G00086 8 
1v1LH 002/984845 

EXPERIMENTAL PROCEDURE 

BACTERIAL STRAINS 

The following strains were usedS. typhimurium TAl535 hlSG46 ria uvrB 

S. typhimurium TAl537 hisC3076 ria uvrB 

S. typhimurium TA98 hisD3052 ria uvrB pKMlOI 

S. typhimurium TAlOO hisG46 ria uvrB pKMIOl 

E coli CM891 WP2 trp uvrA pKMlOI 

The strains of S. typhimurium were obtained from Professor RN Ames, University of California, 

Berkeley, California, USA. 

The strain of E coli was obtained from the National Collections of Industrial and Marine Bacteria, 

Aberdeen, Scotland. 

Batches of the strains were obtained from master stocks held in liquid nitrogen. The test batches were 

aliquots of nutrient broth cultures and were stored at -80°C. Dimethyl sulphoxide (DMSO) was added 

to the cultures at 8% vivas a cryopreservative. Each batch of frozen strain was tested, where 

applicable, for cell membrane permeability (ria mutation), sensitivity to UV light and the pKMIOl 

plasmid which confers resistance to ampicillin. The responses of the strains to a series of diagnostic 

mutagens were also assessed. 

For use in tests an aliquot of frozen culture was added to 25 ml of nutrient broth (Merck No.2) and 

incubated, with shaking, at 37°C for 10 hours. These cultures provided at least 2 x 109 cells per ml 

which were measured photometrically. 

POSITIVE COl\'TROLS 

In the absence of S9 mix 

Identity: N-Ethyl-N'-nitro-N-nitrosoguanidine 

Supplier Sigma Chemical 

Batch number: 67F-3700 

Appearance: Pale yellow crystalline powder 

Solvent: DMSO 

Concentration: S flg/plate for strain TAIS35 

3 flg/plate for strain TAIOO 

2 flg/plate for strain CM891 

: 9 G00087 
1vfLH 002/984845 

Identity 


Supplier 


Batch number: 


Appearance 


Solvent: 


Concentration: 


Identity: 


Supplier: 


Batch number: 


Appearance: 


Solvent: 


Concentration: 


In the presence of S9 mix 

Identity: 


Supplier: 


Batch number: 


Appearance: 


Solvent: 


Concentration: 


Identity: 


Supplier: 


Batch number: 


Appearance: 


Solvent: 


Concentration: 


9-Amlnoacridine 

Sigma Chemical 

35H1408 

Yellow powder 

DMSO 

30 f.Lg/plate for stram T A 15 3 7 

2-Nitrofluorene 

Aldrich Chemical Company 

75108 

Beige powder 

DMSO 

I f.Lg/plate for strain T A98 

2-Aminoanthracene 

Aldrich Chemical Company 

0013406 

Green powder 

DMSO 

2 lJ.g/plate for strain TA1535 

10 f.Lg/plate for strain CM89 I 

Benzo [a ]pyrene 

Aldrich Chemical Company 

05915DY 

Yellow powder 

DMSO 

5 IJ.g/plate for strains TA1537, TA98 and TAl 00 

PREPARATlON OF S9 FRACTION 

Species: 

Sex: 

Strain 

Source: 

Age: 

Weight: 

Diet: 

Rat 

Male 

Sprague-Dawley derived 

Harlan Olac Ltd 

7-8 weeks 

<300 g 

SDS R-+-M no. 1 (modified) nuts 

S9 fraction was prepared from a group of ca 20 animals. Mixed function oxidase systems in the rat 

livers were stimulated by Aroclor 1254, administered as a single intra-peritoneal injection in Arachis oil 

at a dosage of 500 rnglkg bodyweight. On the fifth day after injection, following an overnight 

starvation, the rats were killed and their livers aseptically removed. 

The follo'W1.ng steps were carried out at 0-4°C under aseptic conditions. The livers were placed in 

0.15 M KCl (3 ml KCl : I g liver) before being transferred to an Ultra-Turrax homogeniser. Following 

preparation, the homogenate was centrifuged at 9000 g for 10 minutes. The supernatant fraction 

10 {}00088 
MLH 002/984845 

(59 fraction) was dispensed into aliquots and stored at -80°C until required. The efficacy of each batch 

of 59 fraction was tested in a bacterial mutation assay \-vith the mutagenic precursors 7.12­

dimethyl benzanthracene and 2-aminoanthracene before use. The sterility was also checked 

PREPARATION OF S9 MIX 

59 mix contained: S9 fraction (10% v/v), MgCI: (8 mM), KCl (33 mM). sodium orthophosphate buffer 

pH 7.4 (100 roM), glucose-6-phosphate (5 mM), NADP (4 mM). .tVI the cofactors were filtersterilised before use. 

SELECTION OF SOL VENT 

Prior to commencing testing, the solubility of the test substance was assessed at 50 mglml in dimethyl 

sulphoxide, in which it dissolved. Water was not assessed as information provided by the Sponsor 

indicated that the test substance was insoluble in this solvent. Therefore dimethyl sulphoxide was used 

as the solvent for this study. 

MUTATION TEST PROCEDURE 

First test 

The test substance was added to cultures of the five tester strains at seven concentrations separated by 

ca half-Ioglo intervals. The highest concentration of Subject Chemical #2 tested was 50 mglml in the 

chosen solvent, which provided a final concentration of 5000 IJ.glplate. This is the standard limit dose 

recommended in the regulatory guidelines this assay follows. The negative control was the chosen 

solvent, dimethyl sulphoxide. The appropriate positive controls were also included. 

An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S 9 mix or 0.5 ml 0.1 M phosphate buffer 

(pH 7.4) were placed in glass bottles. An aliquot of 100 IJ.I of the test solution was added, followed 

immediately by 2 ml of histidineltryptophan deficient agar. The mixture was thoroughly shaken and 

overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Each petri dish was 

individually labelled with a umque code corresponding to a sheet. identifying the dish's contents. Three 

petri dishes were used for each dose level. Plates were also prepared without the addition of bacteria in 

order to assess the sterility of the test substance, S9 mix and phosphate buffer. luI plates were 

incubated at 37°C for ca 72 hours. After this period the appearance of the background bacterial lavm 

was examined and revertant colonies counted using a Seescan automated colony counter. 

Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony 

counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects 

the top concentration normally used in the second test would be the same as that used in the first. If 

toxic effects were observed a lower concentration may be chosen. It should be ensured that if a lower 

concentration was chosen, signs of bacterial inhibition are present at the top concentration. Ideally a 

minimum of three non-toxic concentrations should be obtained. 

11: 000089 
l'v1LH 002/984845 

Second test 

As a clear negative response was obtained in the first test. a variation to the test procedure was used for 

the second. The variation used was the pre-incubation assay in which the bottles were incubated at 

37°C for 30 minutes WIth shaking before the addition of the agar overlay. 5000 ~g/plate was 

chosen as the top concentration but only five dose levels were used as It was known from the first test 

that the test substance was non-to;-;:lc. 

STABILITY AND FORMULATION ANALYSIS 

The stability of the test substance and of the test substance in the solvent were not determined as part of 

this study. Analysis of achieved concentration was not performed as part ofthis study. 

GO{)090 : 12 
:MLH 002/984845 

ASSESSMENT OF RESUL TS 

For a test to be considered vahd the mean of the solvent control revertant colony nwnbers for each 

strain should be in the range stated ill the appropriate Standard Operating Procedure, These ranges are 

based on the laboratory's historical control values, Also, the positive control compounds must cause at 

least a doubling of mean revertant colony nwnbers over the solvent control. 

The mean nwnber of revertant colonies for all treatment groups is compared with those obtained for the 

solvent control groups, The mutagenic activity of a test substance is assessed by applying the 

following criteria: 

(a) 	 If treatment with a test substance produces an increase in revertant colony numbers of at least 

twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two 

separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it is 

considered to show evidence of mutagenic activity in this test system, 

(b) 	 If treatment with a test substance does not produce reproducible increases of at least 1.5 times 

the concurrent solvent controls, in either mutation test, it is considered to show no evidence of 

mutagenic activity in this test system. 

In addition the test data were subjected to analysis to determine the statistical significance of any 

observed increases in revertant colony numbers. The statistical procedures used were analysis of 

variance followed by Durmett's test (testing at p::::: 0.01 level), as described by Mahon et al 

(1989). The biological significance of any observed statistically significant increases were also 

considered. 

000091 : 13 
MLH 002/984845 

MAINTENANCE OF RECORDS 

All experimental data arising from the study (including documentary raw data, records and other 

materials: collectively defined as the "matenals") will remain the property of the Sponsor. 

Huntingdon Life Sciences shall retain the materials m its archive for a period of five years from the date 

of issue of the final report. After such time, the Sponsor will be contacted and their advice sought on 

the return, disposal or further retention of the materials. If requested, Huntingdon Life Sciences will 

continue to retain the materials, subject to a reasonable fee being agreed with the Sponsor. 

14 : G00092 
IvlLH 002/984845 

RESULTS 

The mean number of revertant colorues. together with the individual plate counts for Subject Chemical 

#2 obtained in the first mutation test with the tester strains are shown in Table 1. Positive control 

mutability checks are shown in Table 2. No toxicity was observed towards the tester strains. 

The mean number of revertant colonies, together with the individual plate counts for Subject Chemical 

#2 obtained in the second mutation test with the tester strains are shown in Table 3. Positive control 

mutability checks are shown in Table 4. 

No substantial increases in revertant colony numbers of any of the tester strains were observed 

following treannent with Subject Chemical #2 at any dose level, in the presence or absence of S9 mix, 

in either mutation test. 

When the test data was statistically analysed using Dunnett's test a few single significant values were 

shown (marked in results tables). However, none of these values were greater than 1.5 times the 

concurrent negative control and all fell well v,lthin the laboratories historical negative control range. 

As there was also no evidence of any dose relationship in the observed significant increases they were 

not seen as having any biological significance. 

The mean revertant colony counts for the solvent controls were within the historical range. The 

concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of 

the liver preparations by causing increases over double the concurrent solvent control. 

CONCLUSION 

It is concluded that, when tested in dimethyl sulphoxide, Subject Chemical #2 shows no evidence of 

mutagenic activity in this test system. 

C00093 IS 
MLH 002/984845 

REFERENCES 

AMES, B.N .. McCANN, L and YAMA.SAKL E. (1975) Methods for detecting carcinogens and 

mutagens with the Saimonellalmammalian rrucrosome mutagenicity test, Mutation Research, 31, 347, 

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

(1990) Bacterial mutation assays in: KIRKLAND, OJ. (Ed.). UKEMS Sub-committee on 

Guidelines for Mutagenicity Testing. Repon. Pan 1 revised. Basic Mutagenicity Tests: UKEMS 

Recommended Procedures, p.13. Cambridge University Press, Cambridge. 

GREEN, M.H.L. (1984) Mutagen testing using trp+ reversion in Escherichia coli in KILBEY, 

BJ., LEGATOR, M., NICHOLS, W. and RAMEL, C. (Eds.). Handbook ofMutagenicity Test 

Procedures. Second edition, p.161. Elsevier Science Publishers BV, Amsterdam. 

ICH (1996) Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests. 

ICH (1997) Genotoxicity: A Standard Battery of Genotoxicity Testing of Pharmaceuticals. 

MAHON, GAT., GREEN, M,H.L, MIDDLETON, B" MITCHELL, I.de G., ROBINSON, W.O, 

and TWEATS, OJ, (1989) Analysis of data from microbial colony assay in: KIRKLAND, OJ, (Ed,) 

UKEMS Subcommittee on Guidelines for Mutagenicity Testing. Report. Part m, Statistical 

Evaluation ofMutagenicity Test Data, p.26. Cambridge University Press, Cambridge. 

MARON, O.M. and AMES, B.N. (1983) Revised methods for the Saimonella mutagenicity test. 

Mutation Research, 113, 173. 

C00094 16 
MLH 002/984845 

TABLE 1 


Mutation Test 1 


Subject Chemical #2 - revertant colony counts obtained 


per plate using bacterial strains TA1535, TA1537 and TA98 

I St rain Dose level Liver Mear! SD Individual revertant 

(jlgiplate) S-9 
 revertant colony counts 

TA 1535 
 5000 
 - 13 
 4.4 18, 11, 10


1500 
 15 
 1.5 15, 14, 17


500 
 - 14 
 4.4 19, 12, 11 


150 
 - 12 
 1.2 13, 11, 11


50 
 - 17 
 0.0 17, 17, 17 


15 
 - 10 
 1.2 11, 11, 9 


5 
 - 15 
 1.5 15, 17, 14


Solvent - 13 
 1.5 15, 12, 13 


5000 
 + 16
 2.0 14, 18, 16 


1500 
 + 16 
 3.6 20, 15, 13 


500 
 + 15 
 1.2 14, 16, 14


150 
 + 12 
 0.6 12, 12, 13 


50 
 + 16 
 0.6 16, 17, 16 


15 
 + 12 
 3.6 11, 9, 16 


5 
 + 17
 1.0 17, 16, 18 


Solvent + 14 
 2.1 16, 13, 12 


TA 1537 
 5000 
 7 
 1.2 6, 8, S

1500 
 7 
 0.0 7, 7 7 


500 
 13 
 0.6 12, , 13 


150 
 - 11 
 0.6 11, 11 12 


50 
 - 10 
 3.1 7, 9, 

15 
 - 12 
 2.3 13, 13, 9 


5 
 - 10 
 2.9 7, 12, 12 


Solvent - 11 
 1.0 10, 12, 11 


5000 
 + 10 
 2.0 8, 12, 10 


1500 
 + 10 
 4.2 13, 5, 11 


500 
 + 9 
 2.1 7, 10, 11 


150 
 + 11 
 1.2 10, 10, 12 


50 
 + 14 
 1.0 15, 13, 14 


15 
 + 9 
 2.1 7, 11, 10 


5 
 + 10 
 0 0 10, 10, 10 


Solvent + 14 
 3.6 17, 15, 10 


TJl. 98 
 5000 
 - 23 
 1.5 23, 25 


1500 
 25 
 0.6 2 , 25, 24 


500 
 24 
 5.5 18, 28, 27 


150 
 27 
 6.4 23, 23, 34 


50 
 - 23 4.9 29, 20, 21 


15 
 - 20 2 0 18, 22, 20 


5 
 - 20 2.5 17 22, 20 


Solvent - 25 5.8 22, 32 


5000 
 + 21 
 3.6 24, 17 


1500 
 + 27 
 2.1 2 26, 25 


500 
 + 25 
 3.8 29, 23, 22 


150 
 + 26 
 6.0 32, 20, 25 


50 
 + 22 
 .5 22, 24, 21 


15 
 + 24 
 0.6 24, 24, 23
 5 
 + 25 
 :2.5 25 27, 22 


Solvent + 28 
 4 .5 28, 24 


Absence 

+ Presence 

SD Standard deviation 

17 G00095 

i 
tvlLH 002/984845 


TABLE 1 


(continued) 


Mutation Test I 


Subject Chemical #2 - revertant colony counts obtained 


per plate using bacterial strains TAl00 and CM891 


St rain Dose level Liver Mean SD Individual revertant ().1g/plate) $-9 revertant colony coun1:s 

colony 

counts 

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


1500 
 - 114 
 2.3 111, 115, 115


500 
 .. 138 
 13 .3 
 129, 131, 153 


150 
 115 
 14.8 99, 128 119 


50 
 :25 11.3 131, 1 112 


15 
 - :00 10.8 97, 91 


5 
 ** 145 
 15.€ 161, , 143 


Solvent - 100 
 7.5 92, 107, 101


5000 
 + 109 
 7.8 104, 105, 118 


1500 
 + 127 
 26.8 120, 105, 157 


500 
 + 119 
 35.0 79, 141 


150 
 + :11 6.1 114, 1 , 104 


50 
 + 132 
 22.9 12O, 158, 117 


15 + 
 104 
 13 .1 
 114, 108 


5 
 + 119 
 42.6 83, 166 


Solvent + 120 
 9.0 121, 111 


CM 891 
 5000 
 300 
 52.4 360, 262, 279 


1500 
 293 
 9.8 299, 299, 282 


500 
 358 
 14.7 371, 361, 342 


150 
 287 
 22.7 306, 262, 294 


50 
 272 
 35.8 252, 313, 250 


15 
 - 241 
 13.6 233, 257, 234 


5 
 264 
 lC2 267, 249, 277 


Solvent 290 
 13.6 285, 279, 305 


5000 
 + 323 
 38.4 316, 364 


1500 
 + 330 
 33.5 317, 3 305 


500 
 + 393 
 12.5 399, 402 


150 
 + 338 
 2.6 337, 341 


50 
 + 301 
 22.1 299, 280, 324 


15 
 + 290 
 27.2 265, 319, 286 


5 + 329 
 30.6 300, 361 


Solvent + 324 
 19.9 310, 347 


Absence 

+ 	 Presence 

SD 	 Standard deviation 

Significant at p;:;O.Ol using Dunnett's test. 

18 CG0096 
MLH 002/984845 

TABLE 2 


Mutation Test 1 


Revertant colony counts obtained per plate with positive controls 


Strain Compound Dose level 

(pg/plate) 

Liver 

S-9 

Mean 

revertanT: 

colony 

SD Individual revertant 

colony C:OU:1,"'.::s 

i 

counts 

1'A 

TA 

1'A 

TA 

CM 

1535 

1537 

98 

100 

891 

ENNG 

9 AC 

NF 

ENNG 

ENNG 

5 

30 

1 

3 

2 

-

-

-

141 

92 

162 

433 

1877 

6.0 

6.8 

20.7 

25.2 

20.0 

135, 141, 147 

87, 100, 90 

143, 184, 159 

456, 437, 406 

1858, 1876, 1898 

TA 

l'JI. 

l'JI. 

TA 

CM 

1535 

1537 

98 

100 

891 

All. 

B[a]P 

B[ajP 

B[ajP 

AA 

2 

5 

5 

5 

10 

+ 

+ 

+ 

+ 

+ 

86 

39 

139 

601 

1527 

14.8 

8.0 

34.7 

32.1 

21. 9 

70, 99, 90 

31, 39, 47 

99, 157, 161 

584, 581, 638 

1511 , 1552, 1518 

Absence 

+ Presence 

SD Standard deviation 

ENNG N-Ethyl-N'-nitro-N-nitrosoguanidine 

9 AC 9-Aminoacridine 

NF 2-Nitrofluorene 

AA 2-Aminoanthracene 

BlalP Benzol a]pyrene 

19 G00097 
MLH 002/984845 

TABLE 3 


Mutation Test 2 


Subject Chemical #2 - revertant colony counts obtained 


per plate using bacterial strains TA1535, TAl537 and TA98 

Strain Dose level 

(Ilg i plate) 

Liver 

S-9 

Mean 

revertant 

SD Individual revertant 

colony counts 

TA 1535 

TA 1537 

TA 98 

5000 

1500 

500 

150 

50 

Solvent 

5000 

1500 

500 

150 

50 

Solvent 

5000 

1500 

500 

150 

50 

Solvent 

5000 

1500 

500 

150 

50 

Solvent 

5000 

1500 

500 

150 

50 

Solvent 

sOCIa 

. 500 

500 

150 

50 

Solvent 

-

-

-

-

-

+ 

+ 

+ 

+ 

+ 

+ 

-

-

-

-

-

+ 

+ 

+ 

+ 

+ 

+ 

-

-

-

-

+ 

+ 

+ 

+ 

+ 

+ 

14 

12 

16 

14 

17 

18 

18 

17 

14 

18 

14 

20 

7 

8 

9 

9 

7 

10 

10 

10 

11 

11 

13 

12 

19 

28 

27 

26 

25 

23 

30 

25 

23 

27 

26 

28 

2.9 

1.7 

1.7 

6.4 

2.6 

1.2 

1.2 

0.6 

4.6 

2.1 

2.6 

2.3 

2.5 

1.5 

3.2 

1.5 

2.1 

4.7 

3.5 

3.5 

2.3 

3.0 

1.0 

5.5 

4.7 

6.7 

4.7 

3.2 

1.0 

3.1 

2.5 

5.1 

1.5 

1.5 

3.6 

4.4 

16, 11, 16 

10, 13, 13 

15, 15, 18 

21, 10, 10 

14, 19, 18 

19, 19, 17 

19, 17, 19 

16, 17, 17 

13, 10, 19 

17, 20, 16 

17, 13, 12 

19, 19, 23 

7, 5, 10 

6, 8, 9 

10, 5, 11 

9, 11, B 

5, 9, 6 

6, 8, 15 

lC1, 14, 7 

14, 8, S 

12, 12, S 

11, 8, :4 

13, 12, 14 

:8, 9, 8 

23, 14, 21 

32, 31, 20 

22, 31. 29 

28, 27, 22 

26, 25, 24 

26, 20, 22 

30, 27, 32 

31, 21, 24 

24, 21, 23 

27, 29, 26 

22, 29, 27 

26, 25, 33 

Absence 

+ Presence 

SD Standard deviation 

G00098 20 
1v1LH 002/984845 

TABLE 3 


( continued) 


Mutation Test 2 


Subject Chemical #2 revertant colony counts obtained 


per plate using bacterial strains TAtoO and CM891 

Strain Dose level 

().lg/plate) 

Liver 

$-9 

Mean 

revertant 

SD Individual revertant 

colony counts 

Tl', :00 5000 

1500 

500 

150 

50 

Solvent 

5000 

1500 

500 

150 

50 

Solvent 

-

-

-

-

-

+ 

+ 

+ 

+ 

+ 

+ 

** 

120 

128 

112 

108 

111 

98 

110 

146 

121 

128 

116 

108 

12.5 

12.3 

8.4 

7.6 

7.8 

7.2 

7.2 

9.5 

8.1 

8.4 

17.7 

16 .6 

106, 124, 130 

119, 14 2, 123 

:07, 108, 122 

99, 113, 111 

106, 107, 120 

90, 102, 103 

102, 114, 115 

141, 157, 140 

130, 116, 116 

124, 123, 138 

128, 125, 96 

106, 93, 126 

CM 891 5000 

1500 

500 

150 

50 

Solvent 

5000 

1500 

500 

150 

50 

Solvent 

-

-

-

+ 

+ 

+ 

+ 

+ 

+ 

175 

203 

195 

209 

202 

184 

189 

194 

201 

189 

185 

172 

13.1 

30.5 

8.5 

15.3 

16.4 

5.8 

2.6 

44.1 

29.7 

15.7 

9.5 

lLO 

165, 

173, 

186, 

222, 

198, 

177, 

186, 

151, 

176, 

201, 

175, 

168, 

190, 

201, 

196, 

2:2, 

188, 

187, 

19:, 

191, 

234, 

171, 

194, 

184, 

171 

234 

203 

192 

220 

187 

190 

239 

194 

194 

185 

163 

Absence 

Presence 

SD Standard deviation 

Significant at psO.Ol using Dunnett's test. 

ca0099 

2l 
MLH 002/984845 

TABLE 4 


Mutation Test 2 


Revertant colony counts obtained per plate with positive controls 


St rain Compound Dose level 

(1l9 Iplate) 

Liver: 

$-9 

Mean 

reverta:;t 

cOlony 

SD Individual ::-evertant 

colony counts I 

counts 

'fA 

'fA 

":A 

":A 

eM 

1535 

1537 

98 

100 

891 

ENNG 

9 AC 

NF 

ENNG 

ENNG 

5 

30 

1 

3 

2 

-

-

-

230 

442 

89 

437 

1851 

9.2 

9.1 

6.1 

18.7 

92.6 

238, 232, 220 

438, 435, 452 

84 96, 88 

4 , 431 1 422 

1898, 1910, 1744 

TA 

TA 

'fA 

'fA 

eM 

1535 

1537 

98 

100 

891 

AA 

B[a]P 

B[a]P 

B[a]!' 

AA 

2 

5 

5 

5 

10 

+ 

+ 

+ 

+ 

+ 

447 

59 

287 

454 

2094 

61.1 

17.7 

28.9 

30.4 

1.2 

404, 517, 420 

50, Q7, 79 

303, 254, 305 

429, 446, 488 

2Q 95, 2093, 2093 

Absence 

+ Presence 

SD Standard deviation 

ENNG N-Ethyl-N'-nitro-N-nitrosoguanicline 

9AC 9-Aminoacridine 

NF 2-Nitrofluorene 

AA 2-Aminoanthracene 

B[a]P Benzo[a]pyrene 

: 22 (J00100 
MLH 002/984845 


APPENDIX 

Historical control data 

Presented below are the historical control data from the two years prior to commencing this study. 

Dimethyl sulphoxide solvent controls 

Strain TAl535 TAIS37 TA9S TAIOO CM891 

89 mix + + + + + 

Minimum 9 10 5 6 17 20 75 76 134 101 

Maximum 21 22 14 16 33 34 158 154 376 373 

Mean 14.8 16.2 9.9 11.2 24.5 26.5 116.1 122.5 225.8 208,0 

Values 154 162 151 162 152 163 157 167 78 77 

Positive controls 

Strain TA1535 TA1537 TA98 TA100 CM891 

S9 mix + + + + + 

Minimum 80 31 75 28 68 71 178 159 436 351 

Maximum 2489 2171 2094 326 681 747 2564 2137 2221 2192 

Mean 470.2 156.5 497.1 74.4 304.0 192.3 496.0 452.7 1466.4 1256.8 

Values 224 235 39 233 221 239 228 246 111 114 

23 000101