p. 1

Form Approved. O.'YLB. No. 2070-0012 Approval Expire s 100.31-%.

T.J. S. ENVIRONMENTAL PROTECTION AGENCY AGENCY E ONLY

E PA.

PRE14iANUFACTURE °f recei

p

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r

~~~ ~~~

NOTICE . ,' , - •_

FOR NEW CHEMICAL SUBSTANCES

DOCUMENT CONTROL OFFICER

OFFICE OF POLLUTION Company Sanitind PREVENTION

AND TOXIC SUBSTANCES, 7407

U.S. E.P.A. 401 M STREET, SW

WASHINGTON, D.C. 20460

Enter the total number of pages ~ 1- / ent t:oln

in the Pre manufacture Notice J por-19?

GENERAL INSTRUCTIONS Ts- UDULIAT]

• you must peomide all informatiaa requested in this. form to the ezteak that it is known to or reasonably ascertainable by you. Ivlake teasonable estimatat if you do

not have actual data.

• Before you complete this fonot, you should read the "insttndions Manual for Premmafactare Nottficatloa" (the 7sstsadions Manual is available from the Toxic

Sabstanm control Ad (MCA) Information Sesvinby calling /2(12-66&140{, or faxing 202- 554-560U

• If a user fee has beenremitted for this notice (40 CFE 700.45), indicate in the boxes above the TS-aser fee identification namber yo4 have generated .

Remember, your user fee IDnumber mad also appearonyour corresponding fee ranUtance, which is sent tat EPA, HQ Accounting Opeeatlons Branch -('11F266),

P.O, 960399I►4,P(WbarAh, PA 15251-6399, Attn. TSCA User Fee.

Part I - GENERAL INFORMATION TEST DATA AND OTHER DATA

You mast provide the currently coned Chemical Abstract.(CA) Name of the You are reqviied to submit all test data in yourp6swsion or cnntecl .

new chemical substance, even If you dales the identity as confidential. You may and to provide a dredptioa of all other data known to or reasonably

authorize mother pawn to submit chemical ideality infonaaHon for you, but + out+dnable by you, If tkese data asminted to the health and

your submission will not be complete and the review will not begin until EPA env 4onmaWefforts of theasarfatlatwpioassina, distribution

receives this Informaflon. A i efter In supportofyour submission should In. mmmeeq, rse, or dirposd of the new chemical substance. Standard

rsfemnoe yourTS user fee identification nombeG You m ust'sabstif an od6ina 1 literature aibtionsmay be tnbmilted fordata in the open scientific

and two copies ofthis notice Including all we data . If you cWsaed any literature. Its

information as oonfidantlal, a single sanitized copy inust Won be submitted,

clearly identifywhether test data I.onthe substance atanan analog. Also,

Part II- HUMAN EXPOSURE. AND ENVIRONMENTAL RELEASE the chemical eompodNoa of the tested sabdalWould be characterized.

Following are examples of tut data and otherdsta Data should be

If there are several mannfactmy proaesdng, atuse operations to be described In submitted according to the requirements of 572ASD of the Pmoa,ufaRaie

Part U, sections A and B ofthis noHoe,reproduce tbe sa~iour as aeaded. Notification Rule (40 CPR Paat T!D).

Part III - LIST OF ATTACHMENTS Test Data (Chrlc Below any Included in this notice?

Attach additional sheets if there is not enough space to answer a question fully.

Label each continuation skeet with the-mnesponding section keadfng. In Part • Fsivlrunmentai fate data a Yd • Otherdata 13 Yes

IM Hat these attachmentt, any -tat data or other data and any optional

information included In the notice. . • He-K16 Winds data Yes Risk maessmeab

OPTIONAL INFORMATION • Eavfionnteatal etfecb data ~ Yes relationships

You may Include any information that you wantEPA to consider in evaluating • I'hydaJlCYeainl Piopatler' ~ Yes Teddata not in the possession

the new sabstanes On page 11 of thb foram, spaas ban ben provided for you to or mnteoi of the snbmitt.e

describe pollution prevention and recycling infonsstlonyou may have regarding

the new snbstancs 'A physical and citrid-peopntio woelobeel is locsbd on the last page

ofthis foatr

So-called "binding" bmcs are indaded throughout this inns for you to indie dt TYPOF NOTICE (Check. Onl to certain shbmeab yoaaWin thfsnoicsr such y your willingness to be bound

ause, production volume, prroteatiye eqotpment. . . This option is intended to

uce delays that routinely accompany the datelopzNaR -of consent ordain or

Significant New Use Rules. Eapin the case of exeaptionappihatbn (suck as ® Ph NPneanafaclue Natlad Z~

TMEA, LVE, LORE70 where certain lafon~a provided in such notification Is 13 INTERMEDIATE PMN. (abmitEsd in .W. with f6vQ,ysodq~ binding on the wbmiiterrvhen the Agency appiv►as the exemption ~kafloy PM14

checking a binding box in this notice dgimia g by Itself prohibit The snbmielsr

from Later deviating from the inborout~ (e~cnpt chemical IdeaNty) reported in ~ SKIN (Sipifkut New U611 Notfael

Pla (7) 0 TN>BA (restMadceting Bxmptian AppllaliaeJ

CONF CONFIDENTIALITY CLAIMS

❑ LVB (Lowr VolumeBurrptiosJ . 40 t.1 R7?S..lo (cX1110

you may dais any infoauNosi in thisnod= as confidential. To awed a datm

on the 6aeat, mask 0 0 the confidential box neat bIh 0 LOAM (Lme lbissrs[owBpoonrBzebpto) 0 Zr0eK3 . information hysdda

a confidential. To armed a dais is an altaeimeat, drd@ or bndnt the

~ LVE MadlHotbt ~ LCli87(Modl$afi~ Information you dales as confidential.

IDnfidentialy ouu mnd aL pravide a

Ifno d2ha 1"forma

ktswa ~mioe ef IN notlae (Ied dlao

~ Far additional insbudiou on daimin6lnfoeeeaties s

aonfideetia{, rend the InriracHons Manual. IS THIS A CONSOLIDATED PMN? ~ Y49

Mark pq if any information in.this notice is dalmed as mnfldentiat. of ehesaiCRls

tPtsmtias Coaminicatba # required, anier # on page 3)

EPA FORM 7J10•25 .(Rev. 5-95) Replaces prevlous ediHow of EPA Form 7710•25 .

f
2

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

Mana erit and Budget, Paork Reduction Act (2070-0012), Washington, D .C. 20503. -- -

CERTIFICATION

I certify that to the best of my knowledge and belief1 , The company named in Part I. section A, subsection la of this notice form intends to manufacture or import for a

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

Part I, Section B.

2 All information prbvided in this notice is complete and truthful as of the d'ate of submission

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

or reasonably ascertainable by me as required by §720 .50 of the Premanufacture Notification Rule.

Additional Certification Statements:

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

statement that applies:

® The Company named in Part I, Section A has remitted the fee-of $2500 specified in 40 CFR 700 .45 (b), or

The Company named in Part I, Section A has remitted the fee of '$1000 for an Intermediate PMN (defined ® 40 CFR 700 .43) in

accordance with 40 CFR 700.45(b), or

❑ The Company named in Part I, Section A is a small business concern under 40 CFR 700.43 and has remitted

a fee of $100 in accordance with 40 CFR 700 .45 (b)•

If you are submitting a low volume exemption (LVE) application in accordance with 40 CFR 723.50 (c) (1) or a Low

release and low exposure exemption (LoRBaQ application in accordance with 40 CFR 723 .50 (c) (Z), check the following

certification statements:

❑ The manufacturer submitting this notice intends to manufacture or import 'the new chemical substance for

commercial purposes, other than in small quantities solely for research and development, under the terms

of 40 CFR 723.50.

❑ 'fhe manufacturer is familiar with the terms of this section and will comply with those terms; and

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

❑ If this application is for an LVE in accordance with 40 CFR 72350 ( c)(1), the manufacturer intends to commence

manufacture of the exemptedsubstatree for commercial purposes within 1, year of the date of the expiration of the

30 day review period. .

The aqcuracyr of the statements you make in this notice should reflect your best Prediction of the anticipated facts regarding the demiat

substanee described herein . Any knowing andwillful misinterpretation ffi.subject to criminal penalty pursuant to 18 USC I MI. Confidential

Signature and title uthorized (Original Signature Required) Date

4 v ~

C .-

gnature of agent -(if applicable)- Date .

FORM EPA 7710-25 (Rev. 5-45) Page 2
p. 3

Part I - GENERAL INFORMATIO N

Section A - SUBMTITER IDENTIFICATION ConfiMark (X) the "Confidential" box next to any subsection you claim as confidential

. dential

Name of authorized official Position

la. Person Mr

. . 'H. Miyachi President SubmWirgNotice Company

(in U.S

.) Marubeni Specialty Chemicals Inc .

Mailing address (number and street)

10 Bank Street, Suite 740

City, State, ZIP Code

White Plains, New York 10606

b. Agent (iF Naaie of au)fiorized offlcial _- Poeition

applicable)

Company

Mailing address (number and street)

City, State, ZIP Code Area Code Number

Telephone

C. If you are submitting this notice as part of a joint submission, mark OQ this box.

joint Name of authorized officLaL Position

Submitte

r (if applicable)

Company

Mailing address (number and street)

City, State, ZII' Code ' Area Code Number

Telephone

2 . Technical Name Position

Contact

(inU-ql) Dr . George S . Dominguez/D . Kuter President/Regulatory-Affairs Mana er

Company

Regulatory Assistance Corporation

Mailing address (number and street)

17 Clearview Circle

City;State, 23P Code. ArwCode Number

Telephone I

Hopewell Junction, Necv_York 12533 i 845 i 896-4340

3 . If you havehad a prenodae .cammunication (PC) concerning this notice

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

if none

4. If you previously submitted an exemption application for the chemical

substance covered by thisnotioe, enter the exemption number ieadgned Mar .- If you previously submitted a PMN for this substance enter the ~ek by EPA ~

PT~M! number assigned by EPA (La. withdrawn or iacampieEe). . ~

5 . If you have submitted a

the chemical substance covered by this

notice of Bona fide intent to manufacture

notlae, mber the

of Mark 00 impoet for if rwm

notice number assigned by EPA

1 ~ impoct i

Type of Notice - Mark 04 1

Manufacture

❑ On1y { 2.

~--~ Only 3. Both

❑ Mark

g(

4

Option ) ❑

~~g~

.~ption 1 ❑

FORM EPA 7110-25 (Rev. 5-95) Page 3
Part I - GENERAL INFORMATION - Continue d

Section B-- CHEMICAL IDENTITY INFORMATION: You must provide a currently correct Chemical Abstracts (CA) name of the substance

based on the ninth Collective Index (9a) of CA nomenclature rules and conventions .

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

Complete either item 1(Class 1 or 2 substances) or 2 (Polymers) as appropriate. Complete a ll other items.

If another person will submit chemical identity information for you (for either item 1 or 2), mark (X) the box at the right . ConfiIdentif the name, company, and address of that p_erson in a continuation sheet. -~'❑ dential

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

a . class of substance - Mark ()4 1E3 Class 1 or 2 Class 2

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

For Class 1 substances a CA Index Name must be provided . For Class 2 substances either a CA Index Name or CA Preferred Name must

be provided, whichever is appropriate based on CA 9CI nomenclature rules and conventions .).

a Please identify which method you used to develop or obtain the specified chemical indentity information reported in this notice:

(check one).

Method 1(CAS Inventory Expert Service - a copy of the Identification [_]Method 2(Ckher Source)

report obtained from theCAS Inventory Expert Service must be

submitted as an attachment to two notice)

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

CAS s

X

e. For a class I substance, provide a complete and correct chemical structure diagram. For a class 2 substance - (1) List the immediate

precursor substances with their respective CAS Registry Numbers. (2) Describe the nature of the reaction or prooeu. (3) indkate the

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

structure diagram, as complete as can be known, if orie can be reasonably ascertained.

❑ Mark ()q this box if you attach a continuation sh eet

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

Part I-- GENERAL INFORMATION -- Continued

Section B - CHENIICAL IDENTITY INFORMATION -- Continue d

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

a

. Indicate the `<number-average weight of tlie lowest molecular weight composition of the polymer you inte]nd to manufacture

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

500 and below 1,000 absolute molecular weight of that composition. a

Describe the methods of measurement or the basis for your estimates: GPC ❑ Otherr❑ : (Specify)

t) lowest number average molecvlar weight

ii) maximum weight % below 50p molecu)ar weight

"iii) maximum weight % below 1000 molecular weight

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

b . You must make separate confidentiality claims for monomer or other reactant identity, composition information, and residual information . Mark 00 the "Confidential" box next to any item you claim as 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. li

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

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

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

polymer description on the TSCA Chemical Substance Inventory.

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

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

manufactured for commercial purposes.

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

Monomer or other reactant and CAS Registry Number Confi Typical Identity Conf~ Maximum Confi-

(1) dential composition Mark ()q dential residual dent~tal

(Z (3) (4) (5) (6) • . (7)

9f, %

9b

96

~ ~

.

96 70

%

. 96 96

❑ Mark (X) this box if you attach a aontinuat km sheet.

c. Please identify which methodyou used to de velop or obtain the spedfied dtemical identity informatibn reported in thisnotioe. (check one).

Method 1(C.AS Inventory Expert Service - a copy of the identification report Method 2 (other s+ourae)

obtained from

C

AS Inventory ExpertService mug be submitted as

an attachment to this notice)

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

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

L

❑ Mark pQ this box if you attach a continuation sheet

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

Part I - GENERAL INFORMATION - Continued

Section B - CHEMICAL IDENTITY INFORMATION - Continued

3 Impurities

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

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

(b) -Estimate the maadmnm weight % of each impurity. If there an unidentified imparities, estimate their total weight !G .

Impurity and CAS Registry Number Maximum Confl-

(a) Percent dential

%

9'0

%

9'0

. 96

%

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

4. Synonyms - Enter any chemical synonyms for the new chemical substance Identified in subsection 1 or Z con&

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

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

Newcol 7075k' -

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

6 . Generic chemical name - If you claim chemical identity as confidential, you must provide a generic chemical name for your substance

that reveals the specific chemical Identity of the new chemical substance to the ataidmum extent possibleRefer to the'15CA Chemical Substance Inventory, 1995 Edition, Appendix B for guidance on deveioping

generic names. .

Polyoxyethylene polyalkylarylphenylether sulfate ammonium salt

❑ Mark Qq this box if you attach a continuation sheet.

7. Byproducts - Describe any byproducts resulting hued the manufacttue, proeessfrtg, use, or disposal of the new chemical substance. Provide the CAS

Registry Number if availabic

Byproduct CAS Registry Number Confidential

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

FORM EPA 7710-2S (Rev. 5-95) Pa@*6
p. 7

. Part I -- GENERAL INFORMATION - Continued

Section C-- PRODUCTION, I1MPORT, 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

1009'o 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 volyme.

Maximum first 12-month production (kg/yr) Maximum 12-month production (kg/yr) con& BOptioa

b`~

(100%'o new chemical substance basis) (1009'a new chemical substance basis) dential

M&A W

12,000 - • 30,000

.

I J - L

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

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

(2) - Mark (7q this column if entry in column (1) is con fidential business information (CBI).

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

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

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

(6) - Estimate the percent of the new substance as formulated in mixtures, suspensiona„ 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 (Xy this column if entry(ies) in column (8) is (are) confidential business information (CBI).

Category of use (1) CBI Binding Produc- CBI % in I % ofsubstance expected per use I

(by function and application i.e. a dispersive dye for Option tion % Form- ~ W (8)

finishing polyester fibers) ulation Site- huhs- Corn- Bindin g

(2) (3) (4) (5) (6) (7) limited sume r trial merdal Option (9)

X g6 X yy X X

X % X % X

X % X % X X

% %.,

% q6

% %

y6 g6

'"If you have identified a"consumer" use, please .prpvide 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.

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

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

use category. Read the Inatiactions Manual for examples of generic use descriptions. •

description

Surface active agent for emulsion polymerization

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

3. Hazard Information - Include in the notice a copy of reasonable facsimile of any hazard warning statement, Iabet, 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 Mark W equipment or practic es for the safe handling, transport, use, or disposal of the new substance. List in pat III hazard Information you include.

clkia;~ this box if you attach infortnaft . -

FORM EPA 7710-25 (Rev. 5-95) Page 7
p. 8

Part II -- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

Section A - INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER Mark (X) the "Confidentlal" box next to any item you

claim as confidentia l

Complete section A for each type of manufacture, processing, or use operation involving the new chemical substance at industrial sites yo u control.

Import ers do not have to complete this section for operations outside the U.S. ; however, you may stillhave reporting requirements if there are

further iad u ettia~ processing or use operations after import You must describe these operations . See inatractiortit manuaL

1 . Operation description CMfi 40

a. Identity - Enter the identity of the site at which the operation will occur. d ential

Name

Site address (number and street)

City; Count};'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.

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

b. Type - * Mark 00 ❑ Manufacturing ❑ Processing use

c. Amount and Duration - Complete 1 or 2 as appropriate

Ma ximum kg/batchasa s asw cbmaie Hours/batc,h Batches/year •

1 . Batch

Maximum kg/day aoo w d-h-, Hours/day Days/yew

2 . Continuous

d. Process description ❑ Mark 00 to indicate your willingness to have your process description biadina,

(1) Diagram the major unit operation steps and chemical oonvessiozu . 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 approximateweight (by kg/day or kg/batch an a 100% new chemical substance basis), and entry point of all starting

materials andfeedstocks (including reactants, solvents, and catalysts, etc .), and of all products, recycle streaau, and wastes. Include cleaning

chemicals (note frequency if not used daily or per batch.).

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

Since the, .PMN substance is not going to be manufactured in-the

United States we are .not providing this information .

~ . .~

❑ Mark 00 this box if you attach a continuation shee t

FORM EPA 771725 (Rev. 5-95) Page 8
p. 9

Part II -- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE - Continue d

Section A - INDUSTRIAL SITES CONTROLLED BY THE SUBMTTTER - Continued

2. O pational Expoeure - You must make separate confidentiallty daims for the desaiptian of worker activit~g~}ysical forac~f :the new chemical

F

suance, nt~►ber of workers exposed, and duration of activity. Mark (X) the "Con6dential" box next to any itZ;ur you daim as confidentiaL

(1) Des~itie'the activities (i .e bag durriping, tote filling, unloacllng drums, sampling, deaning, etc.) in which workers may be exposed to the

substance.

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

(3) - Describe any protective equipment and engineering controls used to protect workers.

(4) and (6) - Indicate you willingness to have the information provided in column (3) or (5) binding .

(5) - indicate the physical form(s) and % new chemical substance (if in mixture) of the new chemical substance at'the dnie of exposure (i.e .; solid:

crystal, granule, powder, or dust) and % new chemical substance (if in mixture) .

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

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

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

(10) and (11) - Estimate the maximum duration of the activity for any worker in hours per day and days per year. -

12 - Mark this column if enTries in columns (10) and (11) are confidential business information (CB 0 .

Worker activity CBI Protective Equtpa,ent/ Binding Physical form(s atndiag CBI # of CBI Maxdm duration CBI

(i.e. . bag dumping, filling drums) Engineering Controls Option and % new Option Workers Mark W substance Mark W Exposed Iirs/day Days/yr

(1) (2) (3) (4) (5) (8) (9) (10) (11) (12)

❑ 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 of the new chemical

substance released and other release and disposal informatlon. Mark 00 the "Confldentlal" box next to each item youclaim as confidential.

(1) - Enter thenumber of each release point identified in the kwoce- ft description, part 11, section A, subsection ld(3).

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

1(3) - Mark (X) this column if entries in columns (1) and f2) are confidential business lnfaatation (CBI).

(4) - Identify themedia (stack air, fugitive air (optional-eee Instruction Manual), surface water, on-site o r. off-site land or indneration, POTW, or

other (specify)) to which .the new substance will be released from that release padnt.

(5) - a. Describe control technology, if any, ,andcontrol 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 tertiaryon-site treatment. b. Estimate the amount released to the environment after control technology (in kg/day).

(6) - Mark 00 this column if entries in oolumtis (4) and (5) are aonfldentiaTbuatnees information (CBI).

(7) - Identify the destinatlon(s) of releases to water. Please supply NPDES (Natianal Pollutant Discharge Elimination System) numbers for direct

dischargers or NPDES numbers of the POTW (Publicly Owned Treatment Worka). Mark 00 If the PO'IW name or NPDES tk is confidential

business information (CBI).

Release Amount of new substance CZ Meftot Control technology and efficiency (you may wish to optionally attach effidency data) CBI

Number, roles wd

(1) (7a) Qbl (g) (4) (Sa)

.nek .k t~e

~~ (Sb) (b)

• . ~

(7) Mark (?Q the ❑ PO'TW provide name(a) below: 011 ❑N&vlpbk ❑ Other - Specify provide NRDCB S CBI

destination(s) of waterway

releases to water.

❑Mark (7q this box if you attach a continuation sheet.

FORM EPA 7710•25 (Rev. 5-95) Pap 9
P. 10

Part II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued

Section B - INDUSTRIAL SITES CONTROLLED BY OTHER S

Complete section B for typical processing or use operations involving the new chemical substance at sites you do not control- Optional for typical

manufacturing or processing operations involving the new chemical substance at sites outside the US. that you do not control-," Instructions

ManuaL Complete a separate section B for each type of processing, or use operation involving the new chemical`substance . If the same operation is

performed atmore than one site describe the typical operation common to these sites . Identify additionalsibes on a continuation sheet. Optional for

sites outside the United States.- See Instructions Manual.

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

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

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

the approximate weight (by kg/day or kg/batch, 100% new chemical .subetance basis), and entry point of all feedstocks (including reactants,

solvents and catalysts, etc) and of all products, recycle streams, and wastes. include cleaning chemicals (note frequency if not used daily or per

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

Enter of site

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

2 . Worker Exposure/Environmental Release

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

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

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

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

used to protect workers.

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

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

(10) - Estimate the amount of the new substance released (a) directly to the environment or (b) into control technology to the environment

(in kg/ day or kg/batch).

(12) - Describe media of release (stack air, fugitive air (optional-see Instructions Manual), surface water, on-site or off-site land or incineration, P01W,

or other (specify)) and control technology, If any, that will beused to limit the release of the new substance to the environmen t

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

(3), (5), (8), (11), (13) and (15) - Mark ()q this codu.npn if any of the p:oceeding entries are confidential business information (CBA :

Letter it of CBI Duration CBI Protective Equip. / % in CBffRelease Amount of CBI Control . ®I

of Workers Of . EngineeringContrda/ Form- Number New Technology

Act- Exposed Exposure Physical Form and % ulation Substance

ivity new substance Released

(1) (2) 3) (4a) 4b (5) -(6) (7) (8) (9) (10a) 1 106 1 (12) (13

. ~

) ,

. .

-

.

,

(14) - Byproducts: (15)

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

FORM EPA 7710-25 (Rev. 5-95) Page 10 _
P. 11

OPTIONAL POLLUTION PREVENTION INFORMATIO N

To claim information in this section as confidential circle or bracket the specific information that you claim as confidential.

In this section you may provide information not reported elsewhere in thi sf9xm regarding your

efforts to reduce or'minimize potential risks associated with activities surroundin g

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

this 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 of the PMN substance to potential alternatives may be provided .

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

of this substance. See Instructions Manual and Pollution V'revention Guidance manual for

guidance and examples.

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

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

means; (4) a reduction'in potential toxicity or human exposure and/or environmental release; (5) an increase in product

performance, a decrease in the cost of production and/or Improved operation efficiency of the now chemical substance in

comparison to existing chemical substances used in similar applications; 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 .

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

FORM EPA 7710-25 OW.s *M Pap 11
p. 12

Part III -- LIST OF ATTACHMENT S

Attach continuation sheets for sections of the form and test data and other data (including physical/chemical properties and

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

which it relatea;=ifappropriate. Number consecutively the pages of the attachments. In the column below,-enter the inclusive page

numbers of each attachment. Mark ()0 the "Confidential" box next to any attachment name you claim as confidential. Read the Instructions Misaml for

guidance on how to claim any information in an attachment as confidential. You must include with the sanitized copy of the noti ce

form a sanitized version of any attachment in which you claim information as confidential.

Attachment name Attachment Conflpapnumber(s) dential

1 . Eye Irritation To The Rabbit 14 - 26

2 . Skin Irritation To The Rabbit 27 - 39

3 . Acute Oral Toxicity To The Rat 40 - 53

4 . Acute Toxicity To Medaka 54 - 72

5 . Bacterial Mutation Assay 73 - 94

6 . MSDS 95 - 98

D Mark (X) this box if you attach a continuation sh,eet. .Enter the attadtnait rmw mid number.

FORM EPA T110-25 (Rev. 5-95) Page 12
p. 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 W--:RAN substance-in _).You

are novfequired to submit this worksheet; however, EPA strongly recommends that you do. so, as it wi ll 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 (X} if Page Value M°"`ztd Cong_

dentiai (a) provided, number (o) ~ Mark 00

Physical state of neat substanoa (s) (1) (q~

Vapor pressure - OC @ Temperature Torr

Density/relative density 1 . 055 g/cm3

Solubility

@ Temperature

°C 100G

Solvent

Tn 1 nPn P- T1iP 1'~► /L

Solubility in water *Temperature

0C °C.

Melting temperature 0C

Boilin /sublimatioa temperature ~ t"'r prm=

Spectra

Dissociation constant

Par ticle size distribution

Octanol/water partition coefficient

H 's Law constant

Vofltalization from water

Volltalization from soil

pH ® concentration 7.5

Flammabilify

Explodability .

Adsorption/coefHdent

Other-Specify Viscosity 150mPas

-,1 3w ~
p. 14

NEWCOL 707SF CONC

EYE IRRITATION TO THE RABBIT

Sponsor Testing facility

Huntingdon J,ife Sciences Ltd

P .O. Box 2

Huntingdon

Cambridgeshire

PE18 5ES

ENGLAND

Report issued 22 May 1997

Page l of 13

~~
p. 15

CONTENTS =- _

Page

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

QUALITY ASSURANCE STATEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

RESPONSIBLE PERSONNEL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

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

EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

TABLE

1 . Ocular reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

: 2 :

~J
p. 16

COMPLIANCE WITH GOOD LABORATORY PRACTICE STAND S

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

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

Good Laboratory Practice, The United Kingdom Compliance Programme, Department

of Health 1989, and subsequently the United Kingdom Good Laborato ry Practice

Regulations 1997 .

EC Council Directive, 87/18 EEC of 18 December 1986, (No . L 15/29).

Good Laboratory Practice in the testing of Chemicals OECD, ISBN 92-64-12367-9, Paris

1982, subsequently republished OECD Environment Monograph No . 45, 1992.

United States Environmental Protection Agency, (TSCA), Title 40 Code of Federal

Regulations Part 792, Federal Register, 29 November 1983 and subsequent amendment

Federal Register 17 August 1989 . ,

Japan Ministry of International Trade and Industry, Directive 31 March 1984 (Kanpogyo

No . 39 Environmental Agency, Kikyoku No. 85 MITI) .

~ - -

c~ ces-R 22 ~ ~ ~cv:y

Brenda I. Parcell, M.I.A.T., Date

Study Director ,

Huntingdon Life Sciences Ltd.

: 3 :

Ao
p. 17

QUALITY ASSURANCE STATEMENT

This report , has been audited by Huntingdon Life Sciences Quality Assurance Department

(Huntingdon). The Inetfiods, practices and procedures reported herein are an accurate description of

those employed at Huntingdon during the course of the study . Observations and results presented in

this final,xeporLform a true and accurate representation of the raw data generated during the conduct

of the study at:Huntiogdon.

Certain studies such as that described in this report, are conducted at Huntingdon in a setting which

involves frequent repetition of similar or identical procedures . At or about the time the study

described in this report was in progress, `process-based' inspections were made by the Quality

Assurance Department of critical procedures relevant to this study type . The findings of these

inspections were reported promptly to the Study Director and to Management, Huntingdon Life

Sciences .

Date(s) of inspection 10-13 September 1996

Date(s) of repo rting inspection findings

to the Study Director and Management 18 September 1996

Date of repo rting audit findings-to th e

Study Director and M anagement 9 October 1996

Peter Watson, Date

Quality Assur ance Unit Head,

Department of Quality Assurance,

Huntingdon Life Sciences Ltd .

: 4 :

~7
p. 18

RESPONSIBLE PERSONNEL

Brenda I . Parcell, M.I.A.T.,

Study Director,

Department of Acute Toxicology . `"~'s-...,, ., . : ...., . .: .. . ..: :: ::. .: . :

{

:5 :
P. 19

SUMMARY

A study was performed to assess the eye irritation potential of NEWCOL 707SF Cone to the rabbit.

The method followed w as that described in OECD Guideline for Testing of Chemicals No . 405,

"Acute Eye Irritation/Corrosion", Adopted: 24 Februa ry 1987.

Three rabbits= were--each ad ministered a single ocular dose of 0 .1 ml of the test substance and

observed for up to seven days after instillation.

A single instillation of NEWCOL 707SF Cone into the eye of the rabbit elicited well-defined

conjunctival irritation only. All reactions had resolved four or seven days after instillation .

:6 :

~7
p. 20

= INTRODUCTION

The study was designed to assess eye irritation potential of NEWCOL 707SF Conc following-a singlec

instillation into the eye of the rabbit. The test substance may come into contact with the eye during

handling or use .

The study was coi%ducted in compliance with OECD Guideline for Testing of Chemicals No . 405,

"Acute Eye'irritation/Corrosion",`Adopted : 24 February 1987.

The albino rabbit was chosen as it has been shown to be a suitable model for eye irritation studies

and is the animal recommended in the test guideline .

The amount of test substance instilled was chosen in compliance with the guideline .

The protocol was approved by Huntingdon Life Sciences Management on 5 August 1996, by the

Sponsor on 15 August 1996 and by the Study Director on 6 September 1996 .

The experimental phase of the study was undertaken between 10'and 19 September 1996 .

~J

. / .

vV
~ ~ ~ Y•~~~~ ; :, ;;~ ~ ~ p. 29

TEST SUBSTANCE

Identity : _ NEWCOL 707SF Cone

Lot number: 741

Expiry: 20 July 1998

Purity : Not advised

Appear ance: Viscous liquid ,

Storage conditions: Room temperature in dark

Date received: 26 July 1996
p. 22

. . , <:•, ,: - - _ _ '~~ ~~~~~. ; „- ..„ ._, . . ._ . .: . . .: ,,

: : . . . ..

~~t~,~kEo~~v~'L"?~e

~~.as., ~ •

.';F, P _ ~, . FL~•. _ -ti',`Eli zra ~id,A~~:~^'a~

EXPERIMENTAL PROCEDURE

ANIMAL MANAGEMENT

Three healthy adult rabbits of the- New Zealand White strain were obtained from Regal Group (UK)

Ltd, Great Bookham, Surrey, Engl and.

The animals were in the weight range of 3 .0 to 3 .7 kg and approximately 13 to 17 weeks of age,

prior to treatment (Day 1). All rabbits were acclimatised to the experimental environment .

The rabbits were selected without conscious bias for the study. They were housed individually in

metal cages with perforated floors in Building R14 Room 2 .

A st andard laboratory diet SDS Stanrab (P) Rabbit Diet and drinking water were provided ad libitum.

The batch of diet used for the study was not analysed for nutrients, contaminants or micro-organisms .

Results of routine physical and chemical examination of drinking water as conducted by the supplier,

are made available to Huntingdon Life Sciences Ltd as quarterly summaries .

Animal room temperature was maintained at approximately 19°C and relative humidity at 30 - 70% .

These environmental parameters were recorded daily . Any slight deviation from these values that

may have occurred was not considered to have affected the integrity or validity of the study . Air

exchange was maintained at approximately 19 air changes per hour and lighting was controlled by

means of a time switch to give 12 hours of a rtificial light (0700 - 1900 hours) in each 24 hours

period .

Each animal was identified by a numbered aluminium tag placed through the edge of one ear . This

number was unique within the Huntingdon Life Sciences Acute Toxicology Department throughout

the duration of the study . Each cage was identified by a coloured label displaying the study schedule

number, animal number and initials of the Study Director and Home Office licensee .

TEST SUBSTANCE PREPARATION

NEWCOL 707SF Conc was administered as supplied by the Sponsor .

The absorption of the test substance was not determined .

The homogeneity, stability and purity of the test subst ance were the responsibility of the Sponsor .

o~~
a p.23 ~

~ t., a s

.~~ e t~ ~ . „^~ - _' .`~i. .,•~«~'t

TREATMENT PROCEDURE

The eyes of each animal were examined prior to instillation of the test substance to ensure that there

was no pre-existing corneal damage, iridial or conjunctival inflammation .

One animal ,was treated in advance of the others, to ensure that if a severe response was produced,

no further animals would be exposed (pilot animal see Table 1) .

Approximately 0.1 ml of the test substance was placed into the lower everted lid of one eye of each

animal .

The eyelids were then gently held together for one second before releasing . The contralateral eye

remained untreated .

OBSERVATIONS

Clinical signs

All animals were observed daily for signs. of ill health or toxicity.

Ocular responses

Examination of the eyes was made after 1 hour and 1, 2, 3 days (equivalent to 24, 48 and 72 hours)

after instillation. Additional observations were made four and up to seven days after treatment. Observation of the eyes was aided by the use of a h andheld light.

Ocular irritation was assessed using the prescribed numerical system :

Cornea

Opacity: degree of -density (area most dense taken for reading )

No ulceration or opacity 0

Scattered or diffuse areas of opacity (other than slight dulling of normal lustre) ,

details of iris clearly visible I

Easily discernible translucent areas, details of iris slightly obscured 2

Nacreous areas, no details of iris visible, size of pup il barely discernible 3

Opaque cornea, iris not discernible through the opacity 4

Iris

Normal 0

Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia or

injection, any of these or combination of any thereof, iris still reacting to light

(sluggish reaction is positive) 1

No reaction to light, haemorrhage, gross destruction (any or all of these) 2

g 3
p. 24

Conjundivae

Redness (refers to the most severe reading of palpebral and bulbar conjunctivae, as compared

to the control eye)

Blood vessels normal 0

Some blood vessels definitely hyperaemic (injected) I

Diffuse, crimson colour, individual vessels not easily discernible 2

Diffuse beefy-red 3

Chemosis {lids and/or nictating membranes)

No swelling 0

Any swelling above normal (includes nictating membr anes) 1

Obvious swelling with partial eversion of lids 2

Swelling with lids about half-closed 3

Swelling with lids more than half-closed 4

ARCIfiIVES

All raw data and study related documents generated during the course of the study at Huntingdon Life

Sciences, together with a copy of the final report will be lodged in the Huntingdon Life Sciences Ltd

Archives, Huntingdon .

Such records will be retained for a minimum period of five years from the date of issue of the final

report. At the end of the five year retention period the client will be contacted and advice sought on

the future requirements . Under no circumstances will any item be discarded without the client's

knowledge .

DEVIATIONS FROM PROTOCOL

There were no deviations from the protocol.

a
p. 25

AhA

RESULTS

CLINICAL SIGNS

, .

There were no signs of toxicity or ill health in any rabbit during the observation period .

OCULAR RESPONSES

The numerical values given to the ocular reactions elicited by NEWCOL 707SF Conc are shown in

Table 1 .

No corneal damage or iridial inflammation was obse rved .

A diffuse crimson colouration of the conjunctivae with swelling with partial eversion of the eyelids

was seen. These reactions gradually ameliorated and the eyes were normal four or seven days after

instillation.

CONCLUSION

Instillation of NEWCOL 707SF Conc into the rabbit eye elicited well-defined conjunctival irritation

only.

12

a&,
p. 26

TABLE 1

Ocular reactions

Rabbit One Day after instillation

number Region of eye

and-sex hour 1 2 3 4 7

Cornea Density 0 0 0 0 0 0

559 d* Iris 0 0 0 0 0 0

Redness 2 2 1 2 2 0

Conjunctiva

Chemosis 1 1 1 1 1 0

Cornea Density 0 0 0 0 0

560 9 Iris 0 0 0 0 0

Redness 2 2• 2 1 0

Conjunctiva

Chemosis 2 2 1 1 0

Cornea Density 0 0 0 0 0 0

561 d his 0 0 0 0 0 0

Redness 2 2 2 2 2 0

Conjunctiva

Chemosis 2 2 1 1 0 0

* Pilot animal

13

o~~o
p. 27

~._ QUALITY ASSURANCE STATEMEN

T This report has been audited by Huntingdon Life Sciences ,Quality Assurance Departmen t

(Huntingdon) . The methods, practices and procedures reported herein are an accurate description of

those employed at Huntingdon during the course of the study . Observations and results presented in

this final report form a true and accurate representation of the raw data generated during the conduct

of the study at. Huntingdon. _r

Certain studies such as that described in this report, are conducted at Huntingdon in a setting which

involves frequent repetition of similar or identical procedures . At or about the time the study

described in this report was in progress, `process-based' inspections were made by the Quality

Assurance Department of critical procedures relevant to this study type . The findings of these

inspections were reported promptly to the Study Director and to Management, Huntingdon Life

Sciences.

Date(s) of inspection 3-7 June 1996

Date(s) of reporting inspection findings

to the Study Director and Management 7 June 1996

Date of reporting audit findings to th e

Study Director and Management 23 September 1996

2:2 «' :9L .. . : ,

Peter Watson, Date

Quality Assurance Unit Head,

Department of Quality Assurance,

Huntingdon Life Sciences Ltd .

4

97
p. 28

RESPONSIBLE PERSONNEL

Brenda I. Parcell, M .I .A ..T .,

Study Dir'ectof,

Department of Acute Toxicology .

: 5 :
p. 29

SUMMARY

4 This study was performed to assess the skin irritation potential of NEWCOL 707SF Conc to the

rabbit. The method followed was that described in OECD Guideline for Testing of Chemicals No .

404 "Acute Dermal Irritation/Corrosion" . Adopted: 17 July 1992 .

Three rabbits :were each administered a single dermal dose of 0 .5 ml of the test substance and

observed for four days.

A single semi-occlusive application of NEWCOL 707SF Conc to intact rabbit skin for four hours

elicited transient very slight dermal irritation in one animal only, resolving completely by Day 4 .

6 :

~~
p. 30

INTRODUCTION

The study was designed to assess skin irritation potential of NEWCOL 707SF Cone following a single

dermal application to rabbits. The test substance may come into contact with skin during handling

or use.

The study was conducted in compliance with OECD Guideline for Testing of Chemicals No . 404

"Acute Dermal IrritationJCorrosion" . Adopted: 17 July 1992 .

The albino rabbit was chosen as it has been shown to be a suitable model for skin irritation studies

and is the animal recommended in the test guideline .

The amount of test substance administered was chosen in compliance with the guideline .

The protocol was approved by Huntingdon Life Sciences Management on 5 August 1996, by the

Sponsor on 15 August 1996 and by the Study Director on 2 September 1996 .

The experimental phase of the study was undertaken between and 6 September 1996 .

:7 :

30
p. 31

- - t_ -~_ - - TEST SLTBSTANCE

Identity : NEWCOL 707SF Conc

Lot number: 741

Expiry : 20 July 1998

Purity : Not advised

Appearance : Viscous liquid

Storage conditions: Room temperature in dark

Date received : 26 July 1996

1

: 8 :

311
p. 32

EXPERIMENTAL PROCEDURE

ANIMAL MANAGEMENT

Three healthy adult rabbits of the New Zealand White strain were obtained from Regal Group UK,

Great Bookham, Surrey, England .

They were in the weight range of 3 .2 to 3 .5 kg and approximately 13 to 14 weeks of age, prior to

treatment (Day 1). All rabbits were acclimatised to the experimental environment .

The rabbits were selected without conscious bias for the study . They were housed individually in

metal cages with perforated floors in Building R14 Room 2.

A standard laboratory diet SDS Stanrab (P) Rabbit Diet and drinking water were provided ad libitum.

The batch of diet used for the study was not analysed for nutrients, contaminants or micro-organisms .

• .

Results of routine physical and chemical examination of drinking water, as conducted by the supplier ,

are made available to Huntingdon Life Sciences Ltd as quarterly summaries.

Animal room temperature was maintained at approximately 19'C and relative humidity at 30 - 70% .

These environmental parameters were recorded daily . Air exchange was maintained at approximately

19 air changes per hour and lighting was controlled by means of a time switch to give 12 hours of

artificial light (0700 - 1900 hours) in each 24 hours period .

Each animal was identified by a numbered aluminium tag placed through the edge of one ear. This

number was unique within the Huntingdon Life Sciences Acute Toxicology Department throughout

the duration of the study . Each cage was identified by a coloured label displaying the study schedule

number, animal number and initials of the Study Director and Home Office licensee .

TEST SUBSTANCE PREPARATION

NEWCOL 707SF Conc was administered as supplied by the Sponsor .

The absorption of the test substance was not determined .

The homogeneity, stability and purity of the test substance were the responsibility of the Sponsor .

TREATMENT PROCEDUR E

Approximately 24 hours prior to application of the test substance, hair was removed with electric

clippers from the dorso-lumbar region of each rabbit exposing an area of skin approximately

100 mm x 100 mm.

9

v ~
p. 33

`0.5-;nrl of the test substance was applied under a 25 mm x 25 mm gauze pad'to one intact skin site

on each animal.

Each treatment site was. covered with "Elastoplast" elastic adhesive dressing for four hours. The

animals were not restrained during the exposure period and were returned to their cages immediately

after treatment.

At the end of the exposure period, the semi-occlusive dressing and gauze pad were removed and the

treatment site was washed with warm water (30° to 40°C) to remove any residual test substance . The

treated area was blotted dry with absorbent paper.

OBSERVATIONS

Clinical signs

All animals were observed daily for signs of ill health or toxicity .

Dermal responses

Examination of the treated skin was made on Day 1 (ie approximately 60 minutes after removal of

the dressings) and on Days 2, 3 and 4 (equivalent to 24, 48 and 72 hours after exposure) .

Local dermal irritation was assessed using the prescribed numerical system:

Erythema and eschar formation:

No erythema 0

Very slight erythema (barely perceptible) 1

Well-defined erythema 2

Moderate to severe erythema 3

Severe erythema (beet redness) to slight eschar formation (injuries in depth )

preventing erythema reading 4

Oedema formation :

No oedema 0

Very slight oedema (barely perceptible) I

Slight oedema (edges of area well-defined by definite raising) 2

Moderate oedema (raised approximately 1 millimetre) 3

Severe oedema (raised more than I 'millimetre and extendin g

beyond the area of exposure) 4

10 :
p. 34

ARQIIVES

All raw data and study related documents generated during the course of the study at Huntingdon,

together with a copy of the final report will be lodged in the Huntingdon Life Sciences Ltd Archives,

Huntingdon .

Such records will be retained for a minimum period of five years from the date of issue of the final

report. At the end of the five year retention period the client will be contacted and advice sought on

the future requirements . Under--no circumstances will any item be discarded without the client's

knowledge.

DEVIATIONS FROM PROTOCOL

Animals were housed in Building R14 Room 2, not Room 4 or 5 as stated in the protocol . This

deviation was not considered to have affected the integrity or validity of the study .

. 11
p. 3

S 5 ~ RESULT

CLINICAL SIGNS

There were no signs of toxicity or ill health in any rabbit during the observation period .

DERMAL RESPONSE S

The numerical values given to the dermal reactions elicited by NEWCOL 707SF Conc are shown in

Table 1 .

Very slight erythema only was observed in one animal, resolving completely by Day 4 . No dermal

response to treatment was noted in the remaining two rabbits .

CONCLUSION

A single semi-occlusive application of NEWCOL 707SF Conc to intact rabbit skin for four hours

elicited transient very slight dermal irritation in one animal only .

: 1?
p. 36

TABLE 1

Dermal reactions

Rabbit E = Erythema Day

number O = Oedema

and sex i* 2 3 4

541 E 0 0 0 0

0 0 0 0 0

542 Q E 1 1 1 0

O 0 0 0 0

543 ~ E 0 0 0 0

O 0 0 0 0

* Approximately 60 minutes after removal of the dressing

? ..

1 3

30
p. 37

. - .

~

.. .- _~ . . ._ . . . , . ., ,. . . . . . .. . ` . .

- ~.

NEWCOL 707SF CONC

ACUTE ORAL TOXICITY TO THE RAT

Sponsor Research Laboratory

• Huntingdon Life Sciences Ltd

P.O . Box 2

Huntingdon

Cambridgeshire

PE18 6ES

ENGLAND

Report issued 22 May 1997

Page 1 of 14

37
p. 38

CONTENTS

Page

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS . . . . . . . . . . . . . .

.

. . .

QUALITY ASSURANCE STATEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

RESPONSIBLE PERSONNEL 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . .

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

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

EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

CONCLUSION 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

TABLES

1 . Signs of reaction to treatment,,,,,,,,,,,,,,,13 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2. Individual and group mean bodyweights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

3 . Individual bodyweight changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . 14

:2 :

~~
p. 39

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS

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

Laboratory Practite 'standards and with the exception noted below, I consider the data generated to

be valid.

United States -EnvironmentaL. Protection Agency, (TSCA), Title 40 Code of Federal

Regulatrons Paft 792, Federal-Register, 29 November 1983 and subsequent amendment

Federal Register 17 August, 1989 .

Good Laboratory Practice in the testing of Chemicals OECD, ISBN 92-64-12367-9, Paris 1982,

subsequently republished OECD Environment Monograph No. 45, 1992 .

Japan Ministry of International Trade and Industry, Directive 31 March 1984 (Kanpogyo

No. 39 Environmental Agency, Kikyoku No . 85 MITI).

Good Laborato ry Practice, The United Kingdom Compliance Programme, Department of

Health 1989, and subsequently the United Kingdom . Good Laboratory Practice

Regulations 1997.

EC Council Directive, 87/18 EEC of 18 December 1986, (No. L 15/29).

Information regarding test substance characterisatian, namely for purity was not made available to

Huntingdon Life Sciences for compliance with the Good Laboratory Practice standards given

above.

. . .. . . . . .

.

. . . . . .. . . . .. . . . . . . .. . . ._ . . . . . . . . . . . . .. . . . . .. . . . . . .. . . . . . . .. . . . . . . ... .. . . . .

Lewis A. McRae, H.N.C., M.I.A.T., M.I.Sc.T., Date

Study Director,

Huntingdon Life Sciences Ltd .

:3 : 39
p. 40

QUALITY ASSURANCE STATEMENT

This report has been audited by Huntingdon Life Sciences Quality Assurance Department

(Huntingdon). The methods; practices and procedures repo rted herein-are an accurate description of

those employed at Huntingdon during the course of the study . Observations and results presented in

this final report form a true and accurate representation of the raw data generated during the conduct of

the stady at Huntingdon. _

Certain studies such as that described in this report, are conducted at Huntingdon in a setting which

involves frequent repetition of similar or identical procedures . At or about the time the study described

in this report was in progress, 'process-based' inspections were made by the Quality Assurance

Department of critical procedures relevant to this study type . The findings of these inspections were

reported promptly to the Study Director and to Management, Huntingdon Life Sciences .

Date(s) of inspection 29 July- 7 August 1996

Date(s) of repo rting inspection findings

to the Study Director and Management 9 August 1996

Date of reporting audit findings to the

Study Director and Management 31 October 1996

~~.G~~~~:.. . ..~ . . ... ._ ._ . . ~ _ . . ... . . .. . . .._ . .. . . ~..~~...~~._~' . .. . . . :. .

Jacq e Cahill, Date

Audit Team Supervisor,

Department of Quality Assurance,

Huntingdon Life Sciences Ltd.

~

.y
p. 41

RESPONSIBLE PERSONNE L

Lewis A. McRae, H.NC., M.I:A.T., M.I.Sc .T.,

Study Director,

Department of Acute Toxicology.

$

Ny.

5 .
p. 42

SUMMARY

This study was performed to assess the acute oral toxicity of Newcol. 707SF Conc to the rat. The

method followed was that described in the OECD Guideline for Testing"6f Chemicals No . 401, 'Acute

Oral Toxicity". Adopted: 24 February 1987 .

A group often fasted rats (five_males and five females) received a single oral gavage dose of the test

substance, formulated in distilled water, administered at a dose level of 2000 mg/kg bodyweight . All

animals were ki ll ed and examined macroscopically on Day 15, the end of the obse rvation period.

Clinical signs of reaction to treatment comprised piloerection seen in all rats throughout Day 1

accompanied by hunched posture, soft to liquid faeces and ungroomed appearance . Increased

salivation afer dosing was noted for all male rats on Day 1 . No other clinical signs were observed

and all animals had fully recovered by Day 5 (females) or Day 7 (males) .

All animals achieved satisfacto ry bodyweight gains throughout the study .

No abnormalities were observed at the study termination necropsy .

The acute lethal oral dose to rats of Newcol 707SF Conc was demonstrated to be greater than 2000

mg/kg bodyweight.

:6 :

1 ~
p. 43

INTRODUCTION

This study was designed to assess the toxicity of Newcol 707SF Conc fo llowing a single oral dose in

the rat. The rats were dosed by oral gavage as the test subst ance may be ingested "a.cciderttially .

The study was conducted in compliance with the OECD Guideline for Testing of Chemicals No. 401

"Acute Oral Toxicity" . Adopted: 24 February 1987.

The rat was chosen as it has been shown to be a suitable model for this type of study and is the animal

recommended in the test guideline .

The dose level for the study was chosen in compliance with the guideline .

The protocol was approved by Huntingdon Life Sciences Management on 5 August 1996 the Sponsor

on 15 August 1996 and by the Study Director on 16 September 1996 .

The experimental phase of the study was undertaken between 19 September and 3 October 1996 .

~

:7 :

7 J
p. 44

TEST SUBSTANCE

Identity: Newcol 7.07SF Conc

Intended use : Not advised

Appearance: Viscousliquid

Storage conditions : Room temperature in the dark

Lot number : 741

Expiry: 20 July 1998

•

Composition: Not advised

Date received : 26 July 1996

iT .

8

-1i(i(
p. 45

~ - --

EXPERIMENTAL PROCEDURE

ANIMAL MANAGEMENT

Equal numbers of healthy male and female CD rats of Sprague-Dawley origin (Hsd:SpragueDawley(CD)) were obtained from Harlan U.K. Ltd., Bicester, Oxon, England.

They were in-the weight range of 95 to 115 g and approximately four to seven weeks of age prior to

dosing (Day i) . AR-the rats were acclimatised to the experimental environment for a period of seven

days prior to the start of the study .

Rats were allocated without conscious bias to cages within the treatment groups . They were housed in

groups of up to five rats of the same sex in metal cages with wire mesh floors in Building R14 Room 6 .

A standard laboratory rodent diet (SDS LAD 1) and clrinking water were provided ad libitum. Access

to food only was prevented overnight prior to and approximately 4 hours after dosing .

Each batch of diet used for the study was analysed for certain nutrients, possible contaminants and

rnicro-organisms.

Results of routine physical and chemical examination of drinking water, as conducted by the supplier

are made available to Huntingdon Life Sciences Ltd . as quarterly summaries.

Animal room temperature was set to achieve a temperature of 22 f 3°C . Relative humidity was not

controlled but was anticipated to be in the range 30 - 70% RH . Permanent daily recordings of these

parameters were made and these are archived with other Department raw data . Air exchange was

maintained at 10 to 15 air changes per hour and lighting controlled by means of a time switch to

provide 12 hours of artificial light (0700 - 1900 hours) in each 24-hour period ,

Each animal was identified by cage number and ear punching . Each cage was identified by a coloured

label displaying the dose level, study schedule number, animal mark and the initials of the Study

Director and Home Office licensee.

TEST SUBSTANCE PREPARATION

Newcol 707SF Conc was formulated at 20% w/v in distilled water, and administered at a volume of 10

mUkg bodyweight .

The absorption of the test substance wap,-uot determined ,

The homogeneity, stability and purity of the test substance were not undertaken as part of this study

and remained the responsibility of the Sponsor .

:9 :

Ll
p. 46

= _ - 4

MA&&&

_

TREATMENT PROCEDURE

A group of ten rats (five males and five females) was treated at 2000 m g/kg bodyweight.

Control animals

No control animals were included in this study in accordance with the test guideline .

ADMINISTRATION OF TEST SUBSTANCE

The appropriate dose volume of the test substance was administered to each rat by oral gavage using a

syringe and plastic catheter (8 choke) .

The day of dosing was designated Day 1 .

OBSERVATIONS

Mortality

Cages of rats were checked at least twice daily for any mortalities.

Clinical signs

Animals were observed soon after dosing and at frequent intervals for the remainder of Day 1(a period

of approximately five hours). On subsequent days animals were observed once in the morning and

again at the end of the experimental day (with the exception of Day 15 - morning only). This latter

observation was at approximately 16 .30 hours on week days or 11 .30 hours on Saturdays and Sundays.

The nature and severity of the clinical signs and time were recorded at each observation .

All animals were observed for 14 days after dosing .

Bodyweight

The bodyweight of each rat was recorded on Days 1(prior to dosing), 8 and 15 . Individual weekly

bodyweight changes and group mean bodyweights were calculated.

TERMINAL STUDIES

Termination

All animals were killed on Day 15 by cervical dislocation.

Macroscopic pathology

All animals were subjected to a macroscopic examination which consisted of opening the thoracic and

1 0

1i 6
p. 47

abdominal cavities. The cranial cavity was not examined as observations did not indicate neurotoxic

activity. The macroscopic appearance of all examined organs, was recorded .

ARCHIVES

All raw data and study related documents generated during the course of the study at Huntingdon,

together with a copy of the final report will be lodged in the Huntingdon Life Sciences Ltd . Archives,

Huntingdon. - _-

Such records will be retained for a minimum period of five years from the date of issue of the final

report. At the end of the five year retention period the client will be contacted and advice sought on the

future requirements. Under no circumstances will any item be discarded without the client's knowledge .

DEVIATIONS FROM PROTOCO L

There were no deviations from protocol .

~~:f .
p. 48

_ _ _= - ~o~rwr

RESULTS

MORTALITY

There were no deaths following a single oral dose of Newcol 707SF Cone to a group of ten rats (five

males and five females) at a dosage of 2000 mg/kg bodyweight .

CLINICAL SIGNS (Table 1 )

Piloerection (males and females) and increased salivation (males) were observed within five minutes of

dosing. Piloerection was persistent after this time and was accompanied later on Day I by hunched

posture (also on Day 2 and 3), soft to liquid faeces and ungroomed appearance . Recovery, as

judged by external appearance and behaviour, was complete in all animals by Day 5(females) or Day

7 (males).

BODYWEIGHT (Tables 2 and 3 )

All rats achieved satisfactory bodyweight gains throughout the study .

MACROSCOPIC EXAMINATION

No abnormalities were observed at the study termination necropsy .

CONCLUSION

The acute lethal oral dose to rats of Newcol 707SF Cone was demonstrated to be greater than 2000

mg/kg bodyweight. - "

: 12 :

n _, ~t~
p. 49

TABLE 1

Signs of reaction to treatment

No. of rats in group of 5

showing signs

S igns Dose ( 2000 mg/kg)

Male Female

Piloerection 5 5

Hunched posture 5 5

Soft to liquid faeces 5 5

Ungroomed appearance 5 5

$

: 13 :

., yq
p. 50

{

TABLE 2 =

Individual and group mean bodyweights (g)

Sex Dose Animal Bodyweight (g) at Day

(mg/kg) Number

1 8 15

1 111 188 250

2 104 178 236

Male 2000 3 115 192 247

4 105 179 240

5 95 161 214

Mean 106 180 237

6 107 152 183

7 114 162 187

Female 2000 8 107 156 186

9 98 128 148

10 100 137 157

Mean 105 ••l47 172

TABLE 3

Individual bodyweight changes (g )

Sex Dose Animal Bodyweight gains (g)

(mg/kg) Number at Day

8 15

1 77 62

2 74 58

Male 2000 3 77 55

4 74 61

5 66 53

-- 6 45 31

7 48 25

Female 2000 - 8 49 30

9 30 20

10 37 20

Hw .

;~ . . 14

~.

. .

~~
p. 51

- - . ~~

NEWCOL 707SF CONC

• ACUTE TOXICITY TO MEDAKA

(Oryzias lWpes)

Sponsor - Testing facili ty

= Huntingdon Life Sciences Ltd .,

P .O. Box 2,

Huntingdon,

Cambridgeshire,

PE18 6ES,

ENGLAND.

Report issued: 4 July 1997

i

~ - a

u

ru.

Page 1 of 1 9

~

.1 V ~
p. 52

CONTENTS

Page

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

QUALITY ASSURANCE STATEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . 4 RESPONSIBLE PERSONNEL ~ 5

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

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

EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

RESULTS . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

TABLES

; 1. Cumulative mortality data for medaka . . . . . . . . . . . . . . . . . . . . . . . . . 13

2. Observations on sub-lethal effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

3. Mean environmental parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

APPENDICES - -

1 . Individual environmental measurements . . . . . . . . . . . . . . . . . . . . . . . . 16

2. Typical water quality characteristics of the diluent supply . . . . . . . . . . . . . 17

2
p. 53

---

i = COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARD S

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 .

Good Laboratory Practice,-.The United Kingdom Compliance Programme, Department

of Health 1~89, and subsequently the United Kingdom Good Laboratory Practice

Regulations 1997 .

EC Council Directive, 87/18 EEC of 18 December 1986, (No . L 15/29) .

Good Laboratory Practice in the Testing of Chemicals OECD, ISBN 92-64-12367-9,

Paris 1982, subsequently republished OECD Environment Monograph No . 45, 1992 .

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

Craig Kelly, Ph

Study Director,

.D., Date

Huntingdon Life Sciences Ltd .

3

s3
p. 54

• ~

-_-

~

QUALITY ASSURANCE STATEMENT

This, report has been aud#ed • .by Huntingdon Life Sciences Quality Assurance -Department~

(Huntingdon). The methods, practices and procedures reported herein are an accurate description of

those employed at Huntingdon during the course of the study. Observations and results presented in

this final-report-form. a true and accurate representation of the raw data generated during the conduct

of the study at-Huntingdon .

Certain studies such as that described in this report, are conducted at Huntingdon in a setting which

involves frequent repetition of similar or identical procedures . At or about the time the study

described in this report was in progress, `process-based' inspections were made by the Quality

Assurance Department of critical procedures relevant to this study type . The findings of these

inspections were reported promptly to the Study Director and to Management, Huntingdon Life

Sciences.

Date(s) of inspection 8 - 10 October 1996

3 a

Date(s) of reporting inspection finding s

to the Study Director and Management 11 October 1996

Date of reporting audit findings-to the

Study Director and Management 23 December 1996

A

J. . . .d. . . . . . . .i. ~.. .. . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . .

Caroline Sheets, Date

Audit Team Supervisor,

Department of Quality Assurance,

Huntingdon Life Sciences Ltd .

4 .

sy
p. 55

RESPONSIBLE PERSONNEL

Craig Kelly, Ph

Study Director,

.D .,, ~ - ~

Department of Ecotoxicology . .. . . . . . .. .. . . . . . .. . . . . . . . . . . . . .. . . . . .

Karen M. Thirkettle, M.Sc. ,

Scientific Officer,

Department of Ecotoxicology . . . . . . . . . .. . . . . . . .. . . . . . . . .

..

• 5 • >~~
p. 56

- . ---

SUMMARY

A study was performed to assess the acute toxicity of NEWCOL 707SF Cone to medaka

(Oryzias latipes) under static conditions .

The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex

to Directive 92/62[EEC (O .J. No . L383A, 29 .12.92) Part C, Method 1"Acute toxicity for Fish" and

the OECD Guideline for Testing of Chemicals No . 203 "Fish, Acute Toxicity Test" .

Groups of seven juvenile fish were exposed to a range of concentrations of NEWCOL 707SF Cone,

nominally 4.6 - 100 mg/l, dispersed in water . Observations were made on the numbers of dead fish

and the incidence of sub-lethal effects after 3 . 6, 24, 48, 72 and 96 hours exposure . The following

values were determined :

. .

Time (h) LC50 (mg/1) 95% confidence limits (mg/1 )

fi •

3-96 > 100 -

Highest test concentration resulting in 0% mortality : 46 mg/l

"No-observed effect level" : 46 mg/I

Thehighest concentration tested, nominally 100 mg/1, resulted in only 14% mortality . All results are

expressed in terms of nominal concentration .

~.~;_

:~

. 6

.

. 57d
p. 57

INTRODUCTION =--

This study was designed to assess the acute toxicity of NEWCOL 707SF Conc. to medaka

(Oryzias latipes) under static conditions.

The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex

to Directive 92/69/EEC (O.J. No.-L383A, 29 .12.92) Part C, Method 1"Acute toxicity for Fish" and

the OECD Guideline for Testing of Chemicals No . 203 "Fish, Acute Toxicity Test" .

The protocol was approved by Huntingdon Life Sciences Management on 6 August 1996, by the

Sponsor on 26 August 1996 and by the Study Director on 17 October 1996 .

The experimental phase of the definitive study was conducted between 3 and 7 December 1996 .

7 •

67
p. 58

TEST SUBSTANCE

Identity: NEWCOL 707SF Conc

Lot number: 741

Expiry: 20 July 1998

Composition: Volatile content > 98%

Appearance : Viscous liqui d

Storage conditions: In darkness at room temperature

Date received : 14 October 1996

: 8 :

If it
p. 59

EXPERIlVIENTAL PROCEDURE -

TEST SPECIES

Name

Medaka (06zias latipes). -

Source

Neal Hardy Aquatics, Essex, UK (see "Deviation from Protocol") .

Acclimatisation

The stock of fish was obtained from the supplier on 20 November 1996 and acclimatised to test

conditions from that date. Temperature remained within the range 20 ± 1°C and dissolved oxygen

within the range 8 .0 ± 0.5 mgO2/1 in the 14 day period immediately prior to the study . The fish

were fed daily to repletion with Tetramin .® flake food but food' was not given during the 24 hour

period immediately prior to exposure or during the exposure period itself . No medication was given

during the acclimatisation period and mortalities were recorded as 2% in the 7 days prior to the test .

Control fish were measured at the end of the exposure period . The mean standard length was found

to be 2.74 cm (SD = 0 .29 cm) and the mean weight determined as 0 .34 g (SD = 0 .13 g).

DILUENT WATER

The water used for the study was laboratory tap water filtered, dechlorinated and softened by passage

through an Elga® water purification system. The water passes in sequence through a high pressure

= sand filter to remove colloids and particulate matter and a high grade activated carbon filter to remov e

chlorine and any organic contaminants . A proportion of the supply then passes through a water

softener before final reverse osmosis treatment to produce a highly purified water supply . The two

grades of dechlorinated water are then remixed in the ratio of approximately 1 : 1 to give a supply

with the desired water hardness .

Chlorine levels remained below the limit of detection throughout the seven days of the acclimatisation

and exposure periods. The mean hardness level, calculated from daily measurements during the

exposure period, was determined as 134 mgCaCO3(1(SD = 7 .6 mgCaCO3 /1). Typical water quality

characteristics of the diluent supply are given in Appendix 2 .

9 .

~~'
p. 60

TEST-SUBSTANCE PREPARATION

Method of preparation

The test substance was dispersed'directly in diluent water to give an initial stock solution o f

2000 mg/1 . Serial dilutions of this stock solution were prepared with diluent water to give the desired

series of exposure levels, nominally 4 .6, 10, 22, 46 and 100 mg/I .

Stability of test concentrations

Test concentrations were not verified by chemical analysis, at the request of the Sponsor .

EXPOSURE CONDITIONS

Experimental design

Five test concentrations were prepared plus one control'. Seven animals were added to each test

concentration .

Animals were placed at random in glass aquaria containing prepared test media or diluent water only.

Each vessel contained 20 litres of medium to a depth of 19 cm (approximate dimensions of vessel :

25 x 46 x 25 cm). This provided an initial loading of 0 .12 g bodyweight/litre (static volume) .

Test concentrations

Nominal test concentrations : 4.6, 10, 22, 46 and 100 mg/1

Medium renewal

Animals were exposed to the test or control conditions for a period of 96 hours without renewal of

test media.

Environmental conditions

Treatment and control groups were maintained at 20 ± 1°C throughout the exposure period (see

"Deviation from Protocol").

Supplementary aeration was provided via narrow bore glass tubes in this study . A photoperiod of 16

h light :$ h dark was established and daily records of temperature, pH and dissolved oxygen were

maintained for each control and test vessel (see Table 3 and Appendix 1) . The fish were'not fed

during the 96 hour exposure period .

10

'~r. .
p. 61

Criterii: of effect, - - =-- -

The criteria of death employed in this study were (i) absence of respiratory movement and (ii) absence

of response to external stimulation of the caudal peduncle .

In addition to observations on mortality at 3, 6, 24, 48, 72 and 96 hours, subjective assessments were

also made on the type and incidence of sub-lethal effects compared with control fish .

EVALUATION OF DATA

The "no-observed effect level" is the highest concentration tested at which no significant mortality

or sub-lethal adverse reaction to exposure was observed . An incidence rate of more than one affected

fish out of seven is considered to be significant .

DEVIATIONS FROM PROTOCOL

During the in-life phase of the study, the following deviations occurred :

1 . The source of the fish is named in the protocol as Charles River UK Ltd ., Kent, England. The

fish used in the study were obtained from Neal Hardy Aquatics, Essex, England .

2. The protocol states that the temperature in the exposure period will be maintained at ± 2°C

within the range 20 - 24 °C . The temperature remained within the range 20 ± 1°C.

The above deviations from protocol were not considered to have affected the validity of the study .

ARCHIVES

All specimens, raw data and study related documents generated during the course of the study at

Huntingdon Life Sciences, together with a copy of the final report will be lodged in the Huntingdon

Life Sciences, Huntingdon Archive ._ _

Such specimens and records will be -retained for a minimum .period of five years from the date of

issue of the final report. At the end of the five year retention period the Sponsor will be contacted

and advice sought on the future requirements . Under no circumstances will any item be discarded

without the Sponsor's knowledge .

~;-

61
p. 62

RESULTS

Cumulative mortality data . are given in Table 1 . All results are expressed in terms of nominal

concentration.

Analysis of the mortafity data gave the following results :

Time (h) LC,, (mg/1) 95% confidence limits (mg/1)

3-96 > 100 -

Highest test concentration resulting in 0% mortality : 46 mg/1

"No-observed effect level" . 46 mg/1

The highest concentration tested, nominally 100 mg/l, resulted in only 14% mortality .

The environmental parameters (pH and mgO,) remained within acceptable limits throughout the

duration of the study ., Mean values are given in Table 3 and individual values are tabulated in

Appendix 1 . The temperature was slightly outside the range stated in the protocol ,

but this was not considered to have affected the results .

Observations

Marked reactions to exposure (other than death) were swimming at surface and lying at bottom of the

test vessel.

A chronological record of observations is given in Table 2.

CONCLUSION

The 96 h LC.,, value for NEWCOL 707SF Cone with medaka was > 100 mg/1 .

The "no-observed effect level" was 46 mg/1 .

Yt~.

1 2

~ ~~
p. 63

.Ab"&-AA

TABLE 1 = -_- _

Cumulative mortality data for medaka

exposed for 96 hours to NEWCOL 707SF Cone

Nominal Cumulative mortality

concentration (initial population :7)

= mgn 3h 6h 24h 48h 72h 96h

Control 0 0 0 0 0 0

4.6 0 0 0 0 0 0

10 0 0 0 0 0 0

22 0 0 0 0 0 0

46 0 0 0 0 0 0

100 0 0 0 0 1 1

;~ .

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(P3
p. 64

{

TABLE 2

Observations on sub-lethal effects of medaka exposed to NEWCOL 707SF Cone for 96 hours

Nominal Time(hours)

concentration

me/1 3 6 24 48 72 96

Abnormalit y

Control No abnormalities detected

4.6 No abnormalities detected

10 No abnormalities detected

22 No abnormalities detected

46 No abnormalities detected

100 Lying on bottom 3/7 1/6

Swimming at surface 1/7

x/y Number of fish affected/Number of fish surviving

: 14
p. 65

TABLE 3

Environmental parameters

Mean values for pH, temperature and dissolved oxygen

Nominal Mean values

concentration

mgLl pH m-0,/1 T°C

Control 7.9 8.7 20

4.6 7.9 8.7 20

10 7.9 8.7 20

22 7.9 8.7 20

46 8.0 8.8 20

100 8.0 8.8 20

• .

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p. 66

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16

66
p. 67

APPENDIX 2

Typical water quality characteristics of the diluent supply

ASSOCIATED LABORATOR Y

SERVICES LIMITED

CHRISTY HOUSE, CHURCH LANE

SOCKING. BRAINTREE. ESSEX, CtM7 5R X

Telephone: Braintree (01376) 328646 rOr C?.•+[K[ •+O MtSrRr

Fax :-Braintree (013761 5529 2 3

6th September 1996

Huntingdon Research Centre

PO BOX 2

Huntingdon

Cambridgeshire PEI 8 6ES

TEST REPORT N0.11047

Sample Identification Water

Sample Received 19th August 1996

Sample Reference RO Treated/Blended Water ex Fish Stock Room

ALS Sample Number 6295 8

ANALYSIS

Colour - Colourless Odour - Odourless

pa-a-~eters Result

pH units 7.5

Conductivity YS/cm 380

To:aI Hardness as CaC03 mgA 140

Total Alkalinity as CaCO3 mgll 68

Dissolved Solids mg/I 260

Nitrate as N mgfl 1.9

Nitrite as N mg/1 <0.02

Ammonia as N mg/I <0.05

Perm Value mgA <0.2

Silica as Si02 mgA 2.1

Fluoride as F mg/1 0.11

Iron as Fe mgA <0.02

Zinc as Zn mg/l 0.017

Copper as Cu mgA 0.015

Manganese as Mn mgA <0.005

Lead as Pb mgA <0.01

Softm as Na mgA 120

Potassium as K mgA 3.7

Magnesium as Mg mg/I 3.8

Calcium as Ca mgA 52

Sulphate as S04 mgA 57

Residual Chlorine mgA <0.02

Suspended Solids mg/1 <5

Phenols mgA <0.005

Total Cyanide mgA <0.01

Nickel as Ni mgA <0.01

Chromium as Cr mg/I <0.01

Selenium as Se mgA <0.001

Mercury as Hg mgA <0.0002

Sheet 1 of 3

17
p. 68

APPENDIX 2

(continued)

TEST REPORT NO.9'I047cont . . . . f2h SeplembAr 19aA

= Pafaq3etprs Results

Arsenic as As mg/I <0.001

Cadmium as Co mgll <0.001

Aluminium as At mg/I <0.02

Hydrocarbons mg/1 <2

PCB's (Arachlor 1254) ug/l <0.02

Haloforms

Bromodichloromethane ug/I . 1.3

Bromoform }igll <1.0

Chloroform pgA 2 .9

Dibromochtoromethane pgA 0 .3

Total Trfiatomethanes pgA 5

Triazine Herbicide s

Simazine yg/l <0.05

Atrazine pgA <0.05

Propazine pgA <0.05

Qrpanoohosohorus Pesticides

Carbofenothion ugA <0.02

Chlorpyrifos pgA <0 .02

Chtorfenvinfos pgA <0.02

Diazinon Ng/I <0.02

Dichlorvos Ng/I <0.02

Dimethoate Ng/1 <0.02

Fenitrothion pgA <0.02

tvsalaathion • pgA <0.02

Parathion ugff <0.02

Pyrimiphos methyl Ng/1 <0.02

Orcanq;,blorine Pest :cide s

a BHC ygA <0.02

BHC - Ng/1 <0.02

X BHC pgA <0.02

Heptachlor pgA <0.02

Aldrin pgA <0.02

Hea tactilor epoxide 4jgA <0.02

op TOE pgA <0.02

op DDE pgA <0.02

Dietdnn pgA <0.02

op DOT yg/I <0.02

Endrin tsg/f <0.02

pp TDE yg/I <0.02

pp DDT Ng/1 <0.02

Sheet 2of3

! 8

6~'
p. 69

APPENDIX 2

(continued)

TEST REPORT NQ_11Od7cont . . . . 6th Seotember1996

Parameters Results

FAH'

Fluoranthene yg/l <0.01

Benzo (b) Fluoranthene NgA <0.01

$enzo ( k) Fluoranthene ygll <0.01

$erzo (a ) Pyrene I,gA <0.01

Indeno (123c>i) Pyrene pgA c0.02

Benzo (gtv)Perytene ygll <0.01

Total PAi's ligli <0,04

Bacte:iatnoi:ai

Total viable count

1 day ,a_ 37'C per ml cfu 6

3 days Q 21'C perml cfu 1300

Total Colfarm s per 100 ml Absent

raeca? .".of.forr.

.s per 100 rml Absent

Absent

The wa:e- is =;ear a-~ c; ' r.e:rsai rea-z Son . It is so.`, in ;h ara -- er as *.pi5ed by re.atively low

hardness and a; ;ta' :n; :•. .

ine ~^xa*raaon cf =-issolved minerals. me:a's and organi;s are not significant and the

oa ae=o:a:i al qua: ) 5 sa:5j'a~ Jy.

-ies_-s are a: s:^:.a- ie+eis : o orev :o ::s levels. ezxv, that sadiun hasin ; reased.

in :ne es_r,s inci`a :e a wa:er of saa`ac- : f qualry, su;aNe for drinking and

Co mes ,= :,,- r^ses.

^ora:r.onxra:'_,

lSSaCin ~~ ! DRY SERVICES Llmil=O

IS~yr -- --

K SAx= Sheet 3 of 3

Aj

~~
p. 70

NEWCOL 707SF CONC

BACTERIAL MUTATION ASSAY

; ;.

,, .

Sponsor Testing facility

Huntingdon Life Sciences Ltd .,

P.O. Box 2,

Huntingdon,

Cambridgeshire,

PE18 6ES,

ENGLAND .

Report issued 29 May 1997 °

Page 1 of 22

70
p. 71

CONTENTS

Page

COMPLIANCE WITH GOOD. LABORATORY PRACTICE STANDARDS .. . 3

QUALITY ASSURANCE STATEMENT . . . . . . . . . . . . . . . . . . . . . . .

.

. . . . 4

RESPONSIBLE PERSONNEL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

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

EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

TABLES

1 . NEWCOL 707SF Conc - Preliminary toxicity test . . . . . . . . . . . . . . . . . . 16

2. NEWCOL 707SF Conc - Mutation test 1 . . . . . . . . . . . . . . . . . . . . . . . 17

3 . Positive control materials - Mutation test 1 . . . . . . . . . . . . . . . . . . . . . . 19

4. NEWCOL 707SF Conc - Mutation test 2 . . . . . . . . . . . . . . . . . . . . . . . 20

5 . Positive control mater t ,ials - Mutation test 2 . . . . . . . . . . . . . r

. . . . . . . 22

~.~

2 :

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p. 72

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 .

Good Laboratory Practice, The United Kingdom Compliance Programme, Department

of Health 19-89, and subsequently the United Kingdom Good Laboratory Practice

Regulations 1997 .

EC Council Directive, 87/18 EEC of 18 December 1986, (No . L 15/29).

Good Laboratory Practice in the testing of Chemicals OECD, ISBN 92-64-12367-9, Paris

1982, subsequently republished OECD Environment Monograph No . 45, 1992.

United States Environmental Protection Agency, (FIFRA), Title 40 Code of Federal

Regulations Part 160, Federal Register, 29 November 1983 and subsequent amendment

Federal Register 17 August 1989.

Japan Ministry of Agriculture, Forestry and Fisheries, 59 NohSan, Notification No.

3850, Agricultural Production Bureau, 10 August 1984 .

United States Environmental Protection Agency, (TSCA), Title 40 Code of Federal

Regulations Part 792, Federal Register, 29 November 1983 and subsequent amendment

Federal Register 17 August 1989.

Japan Ministry of International Trade and Industry, Directive 31 March 1984 (Kanpogyo

No . 39 Environmental Agency, Kikyoku No . 85 MITI).

United States Food and Drug Administration, Title 21 Code of Federal Regulations Pa rt

58, Federal Register, 22 December 1978, and subsequent amendments.

Japan Ministry of Health and Welfare, Noti fication No . Yakuhatsu 313 Pharmaceutical

Affairs Bureau, 31 March 1982 and subsequent amendment Notification No . Yakuhatsu

870, Pharmaceutical Affairs Bureau, 5 October 1988 .

Japan Ministry of Labour, Notification No . 76, 1 September 1988.

. . .. . .. .. . . . . . . .. . . . . . . . . . . . . . . .. . .. . . . : . .. . . .. . . . . . .. . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . .. . . . . . . .. . . . . . .. . . . . . . . ..~. . . . .. . . . . .

Ricarda A. Gant, B.Sc. (Hons.), Date

Study Director ,

Huntingdon Life Sciences Ltd.

3

. ~_,;: ? ?.t.
p. 73

QUALITY ASSURANCE STATEMENT

This report has:, been , audited by Huntingdon Life Sciences Quality- Assurance Department

(Huntingdon). The methods, practices and procedures reported herein are an accurate description of

those employed at Huntingdon during the course of the study . Observations and results presented in

this final report form a true and accurate representation of the raw data generated during the conduct

of the study at Hunfingdon.

Certain studies such as that described in this report, are conducted at Huntingdon in a setting which

involves frequent repetition of similar or identical procedures. At or about the time the study

described in this report was in progress, `process-based' inspections were made by the Quality

Assurance Department of critical procedures relevant to this study type . The findings of these

inspections were reported promptly to the Study Director and to Management, Huntingdon Life

Sciences .

Date(s) of inspection 4 - 11 November 1996

Date(s) of reporting inspection findings

to the Study Director and Management 12 November 1996

Date of reporting audit findings to th e

Study Director and Management 25 November 1996

~°i. . '_S..'~1.'1. . .. . . .

Rod Scammell, Date

Audit Team Supervisor,

Department of Quality Assurance,

..L Huntingdon Life Sciences Ltd .

: 4 :

~3
p. 74

RESPONSIBLE PERSONNEL

Rxcarda A., .Gant, .B.Sc . Tong.,)r . .,,,,

Study Director;

Department of Cellular Toxicology . .

5

;~- -1Y
p. 75

- - -

SUMMARY = ._ - - - _^

In this in vitro assessment of the - mutagenic potential of NEWCOL 707SF Conc, histidine dependent

auxotrophic mutants of Salmonella typhimuriwn (strains TA 1535, TA 1531, 'TA 98 and TA 100)

were exposed to the test substance, diluted in water 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.

In the preliminary toxicity test with dose levels of up to 5000 μg/plate no toxicity was observed. A

top dose level of 5000 ug/plate was chosen for the subsequent mutation study . Other dose levels used

in the mutation assays were : 1500, 500, 150 and 50 μg/plate .

No evidence of mutagenic activity was seen at any dose level of NEWCOL 707SF Cone in either

mutation test.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the

metabolising activity of the liver preparations .

It is concluded that, when tested in water, NEWCOL 707SF Conc shows no evidence of mutagenic

activity in this bacterial system .

6 .

.7s
p. 7

N 6 { INTRODUCTIO

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

Maron' and Ames 1983) .mutagenic effects are determined by exposing mutant strains ofSalmon4ia

typhimurium to various concentrations of the test substance. Normally S. typhimurium is capable of

synthesising the amino acid histidine it requires for growth but the mutant strains used in this test are

incapable of this function. When large populations of these strains are exposed to a mutagen, reverse

mutation tothe original histidine independent form takes place in a proportion of 'the bacterial

population. These colonies (revertant) are readily detectable due to their ability to grow on a histidine

deficient medium .

The strains of S. typhimurzum routinely used carry additional mutations which render them more

sensitive to mutagens. All strains have a defective cell coat which allows greater permeability of test

substances into the cell. All strains are deficient in normal DNA repair processes. In addition,

strains TA 98 and TA 100 possess a plasmid (pKM1 01) which introduces an error-prone repair

process, resulting in increased sensitivity to mutagens .

Since many substances do not exert their mutagenic effect until they have been metabolised by enzyme

systems not available in the bacterial cell, the test substance and the bacteria are incubated in the

presence of a supplemented liver fraction (S-9 mix) prepared from rats previously treated with a

compound (Aroclor 1254) known to induce a high level of enzymic activity .

This report describes a study designed to comply with the following guidelines :

OECD Guidelines for Testing of Chemicals No . 471 : Genetic Toxicology: Salmonella

typhimurium, Reverse Mutation Assay, 26 May 1983 ,

EEC Methods for Determination of Toxicity, Annex to Directive 92169/EEC, (OS No .

L383A, 29.12.92), Part B, Method B. 14. Other effects - Mutagenicity : Salmonella

typhimurium - Reverse Mutation Assay ,

US Environmental Protection Agency, Method : HG-Gene Muta -,S. typhimurium: The

Salmonella typhimurium reverse mutation assay, 1984 .

The protocol was approved by the Study Director on 15 August 1996, by Huntingdon Life Sciences

Management on 8 August 1996 and by the Sponsor on 15 August 1995 .

The experimental phase of the study was conducted between 16 and 27 September 1996 .

7

76
p. 77

TEST SUBSTANCE

Idenfity : NEWCOL 707-SF Cone

Chemical nam: Information not available to Huntingdon Life

Sciences

Intended use : Information not available to Huntingdon Life

Sciences

Appearance: Clear viscous amber liquid

Storage conditions: Room temperature in the dark

9 Batch number: 741

Expiry date : 20 July 1998

Purity: Assumed to be 100 % fortesting purposes

Date received : 26 July 1996

Supplier :

t, .' .

8

77
p. 78

EXPERIMENTAL PROCEDURE

BACTERIAL STRAIN S

The following strains of S. typhimurium were used in the test :

S. iyph,imunum TA 1535 hisG46 rfa uvrB

S. typhimunum TA 1537 hisC3076 rfa uvrB

S. typhimurium TA 98 hisD3052 rfa uvrB pKM101

S. typhimurium TA 100 hisG46 rfa uvrB pKM101

The strains were obtained from Professor B.N. Ames, University of California, California, USA .

Batches of the strains were stored at -80°C . Each batch of frozen strain was tested for cell

membrane permeability and, where applicable, for the pKM101 plasmid which confers resistance to

ampicillin. The response of the strains to a series of diagnostic mutagens was also assessed .

For use in tests an aliquot of frozen culture was added to 25 mT of nutrient broth (DAB 7, Merck)

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 CONTROL COMPOUNDS

In the absence of S-9 mix

Identity N-Ethyl N'-nitro-1V nitrosoguanidine

Supplier Sigma

Batch 67F-3700

Appearance Pale yellow crystalline powder

Solvent Dimethyl sulphoxide

Concentration 5 μg/plate for strain TA 1535

3 μg/plate for strain TA 100

Identity 9-Aminoacridine

Supplier Sigma

Batch 96F-05641

Appearance Yellow powder

Solvent Dimethyl sulphoxide

Concentration 80 μg/plate for strain TA 1537

9 .

71
p. 79

I#entity 2-Nitrofluorene -- -

Supplier Aldrich

Batch 61896

Appearance Beige powder

° Solvent Dimethyl sulphoxide

Concentration 2 jig/plate for strain TA 1538

1 μg/plate for strain TA 98

In the presence of S--9 mix

Identity 2-Aminoanthracene

Supplier Aldrich

Batch 0013406

Appearance Green powder

Solvent Dimethyl sulphoxide

Concentration 2 μg/plate for strains TA 1535 and TA 1537

0.5 μg/plate for strain TA 98

1 μg/plate for strain TA 100

PREPARATION OF S-9 FRACTION

Species Rat

Sex Male

Strain Sprague-Dawley derived

Source Harlan Olac Ltd

Age 7-8 weeks

Weight <300 g

Diet Biosure Rodent Diet LAD 1

Mixed function oxidase systems in the livers of a group of rats were stimulated by Aroclor 1254,

administered as a single intra-peritoneal injection in Arachis oil at a dosage of 500 mg/kg 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 KCl (3 ml KCl : 1 g liver} before being transferred to an Ultra-Turrax homogeniser .

Following preparation, the homogenates were centrifuged at 9000 g for 10 minutes. The supernatant

fraction (S-9 fraction) was dispensed into aliquots and stored at -80°C until required . The efficacy

of each batch of S-9 fraction was tested with the carcinogens 7,12-dimethylbenzanthracene and

2-aminoanthracene before use . '

10 .

?9,
p. 80

° --

PREPARATION OF S-9 MIX

S-9 mix contained: S-9 fraction ( 10 % v/v), MgC12 (8 mM), KCI (33 mM), sodium phosphate buffer

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

SELECTION OF-SOLVENT -

Prior to commencing testing the solubility of the test substance was assessed . At 50 mg/ml

NEWCOL 707SF Cone was soluble in water, the Sponsor's recommended solvent . Therefore water

was the chosen solvent for use in the subsequent tests.

PRELIMINARY TOXICITY TEST

Four concentrations of test substance were assessed for toxicity using the four tester strains . The

highest concentration was 50 mg/ml of test substance in the chosen solvent, which provided a final

concentration of 5000 μg/plate . Three 10-fold serial dilutions of the highest concentration were also

tested . The chosen solvent, water, was used as the negative control .

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 0.1 ml of the test solution was added,

followed immediately by 2 ml of histidine de ficient agar. The mixture was. thoroughly shaken and

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

used for each dose level . Plates were also prepared without the addition of bacteria in order to assess

the sterility of the test substance, S-9 mix and phosphate buffer . All plates were incubated at 37°C

for 3 days. After this period the appearance of the background bacterial lawn was examined .

Revertant colonies were counted using a Seescan Automatic Colony Counter .

Any toxic effects of the test substance can 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 used in the main tests is the same as that used in the preliminary toxicity test .

If toxic effects are observed a lower concentration may be chosen for the main assays . Ideally the

concentrations chosen for the mutation tests should include a minimum of three non-toxic

concentrations.

MUTATION TEST PROCEDURE

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

ca half-logio intervals . The highest concentration of NEWCOL 707SF Cone used was 5000 ,ug/plate .

The negative control was the chosen solvent, water . The positive control compounds were also

included.

~

~..
p. 81

An aliquot of 0,1 ml of a 10 hour bacterial culture and 0 .5 ml S-9 mix or 0.5 -ml O.=T=M phosphate

buffer. (pH 7.4) were placed in glass bottles . An aliquot of 0.1 ml of the test solution was added,

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

overlaid onto previously prepared petri dishes containing 25 ml minimal agar . Three petri dishes

were used for each dose level . A set of plates were also prepared containing only bacterial culture

and S-9 mix or phosphate buffer (0 μg/plate). Plates were also prepared without the addition of

bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer . All plates

were inqubated at 37°C for 3 days. After this period revertant colonies were counted-using a Seesc an

Automatic Colony Counter .

At a later date the main test was repeated using the procedures described above with the same

concentrations of test substance .

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 pa rt of this study .

ASSESSMENT OF RESULTS

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

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 S-9

mix, it is considered to show evidence of mutagenic activity in this test system . No statistical

analysis is performed .

(b) If treatment with a test substance does not produce reproducible increases of at least 1 .5 times

the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to

show no evidence of mutagenic activity in this test system . No statistical analysis is performed.

(c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response

given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue

of the substance's mutagenic activi ty in this test system.

(i) Repeat tests may be performed using modifications of the experimental method . These

modifications include (but are not restricted to), the use of a narrower dose range than

that already tested ; the use of different levels of liver homogenate S-9 fraction in the S-9

mix. Should an increase in revertant colony numbers be obse rved which satisfies

paragraph (a) the substance is considered to show evidence of mutagenic activity in this

test system. No statistical analysis is performed.

12 .

: ~~
p. 82

If no clear "positive" response can be obtained the test data may be subjected to analysis

to determine the statistical significance of any observed increases in revertant colony

numbers. The statistical procedures used will be those described by Mahon et al. (1989)

and will usually be analysis of variance followed by Dunnett's test .

ARCHIVES

All data are kept in-a loose-leaved-Taboratory notebook which is held in the Department of Cellular

Toxicology and later transferred, together with a copy of the final report, to the Archive Department,

Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, UK for a minimum period of five years .

At the end of the five year retention period the client will be contacted and advice sought on the

future requirements. Under no circumstances will any item be discarded without the client's

knowledge .

Kt;

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13

~ ~~
p. 83

, _ j _`__ -. _•- -

RESULTS

The revertant colony counts for NEWCOL 707SF Cone obtained in .the prel.iminary toxicity test are.

shown in Table 1. NEWCOL 707SF Cone was not toxic towards the tester strains. Therefore

5000 jug/plate was chosen as the top dose level in the mutation tests .

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

707SF Cone obtained in the first mutation test with the tester strains are shown in Table 2. Positive

control mutability checks are shown in Table 3 .

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

707SF Cone obtained in the second mutation test with the tester strains are shown in Table 4 .

Positive control mutability checks are shown in Table 5 .

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

following treatment with NEWCOL 707SF Cone at any dose level, in the presence or absence of S-9

mix, in either mutation test .

The concurrent positive control compounds demonstrated the sensitivity of the assay and the

metabolising activity of the liver preparations.

CONCLUSION

It is concluded that, when tested in water, NEWCOL 707SF Cone shows no evidence of mutagenic

activity in this bacterial system .

_--

$i'r

~.

14

0AP
p. 84

REFERENCES

AMES, B.N ., MCCANN, J: ; and YAMASAKI, E. (1975) .N1elhods for detecting carcinogens and

mutagegs with the -Salmonella/mammalian microsome mutagenicity test . Mutation Research, 31, 347.

EEC (1993) EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC, (OJ No .

L383A, 29. .12.92), Part B, Method B. 14. Other effects - Mutagenicity: Salmonella typhimurium -

Reverse MfitatioifAssay . -

EPA (1984) US Environmental Protection Agency, Method : HG-Gene Muta - S. typhimurium: The

Salmonella typhimurium reverse mutation assay, 1984.

MAHON, G.A.T., GREEN, M.H.L., MIDDLETON, B ., MITCHELL, I.DE G., ROBINSON, W.D .

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

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

Evaluation of Mutagenicity 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 .

OECD (1983) OECD Guidelines for Testing of Chemicals No. 471 : Genetic Toxicology : Salmonella

typhimurium, Reverse Mutation Assay, 26 May 1983 .

,. 4

~.

: 15

. ~
p. 85

TABLE 1

Preliminary toxicity test on NEWCOL 707SF Conc

- revertant colony counts obtained with bacterial strains

TA 1535, TA 1537, TA 98 and TA 100

. _ , , . . . ~. . ``t;

.

Dose Level Liver Bacterial Strains :-

(μg/plate) S-9

= c. TA 1535 TA 1537 TA 98 TA 100

5000 - 12 3 24 111

500 - 19 3 22 157

50 - 19 8 26 154

5 - 14 6 23 152

Solvent - 16 6 40 134

5000 + 15 4 25 131

500 + 19 4 27 164

50 + 16 6 31 137

5 + 22 5 39 159

Solvent + 17 5 38 152

Absence

+ Presence

~--

~.

~

16

' ~~
p. 86

= ', - - __ _- - - - --

TABLE 2

Mutation test 1

NEWCOL 707SF Conc - revertant colony counts obtained per plate using

bacterial strains TA 1535, TA 1537, TA 98 and TA 10 0

Stra-in Dose level _Liver Mean SD Individual revertant

({cg/plate) S-9 revertant colony counts

colony

count s

TA 1535 5000 - 17 3.1 14, 20, 16

1500 - 12 2.3 11, 11, 15

500 - 15 4.0 19, 14, 11

150 - 13 3.5 17, 13, 10

50 - 10 4.0 15, 8, 8

0 - 12 2.6 15, 10, 1 1

Solvent - 16 5.0 21, 15, 11

5000 + 15 3.5 13, 13, 19

1500 + 15 2.1 17,' 13, 14

500 + 13 3.1 16, 14., 10

150 + 13 5.5 19, 10, 9

50 + 12 4.0 8, 16, 1 2

0 + 18 3.8 16, 15, 22

Solvent + 14 3.2 13, 12, 18

TA 1537 5000 8 2.1 10, 9, 6

1500 - 8 2.5 10, 5, 8

500 - 9 0.6 9, 10, 9

150 - 7 0.6 6, 7, 7

50 - 9 5.1 5, 15, 8

0 - 9 2.1 11, 10, 7

Solvent - 8 1 .0 7, 8, 9

5000 + 11 3.1 8, 14, 12

1500 + 9 1.5 9, 11, 8

500 + 9 2.6 12, 7, 8

150 + 10 1.5 10, 9, 12

50 + 9 1.5 8, 9, 1 1

0 + 10 3.8 8, 14, 7

Solvent + 10 1.2 11, 11, 9

- Absence

+ Presence

SD Standard deviation

~;.

'.:

;- : 17 .

v~4. ~
p. 87

TABLE 2

(continued)

Mutation test 1

NEWCOL 707SF Conc - revertant colony counts obtained per plate using

bacterial strains TA 1535, TA 1537, TA 98 and TA 100

Strain Dose level Liver Mean SD Individual revertant

(μg/plate) S-9 revertant colony counts

colony

count s

TA 98 5000 - 24 4.0 28, 23, 20

1500 - 25 6.5 31, 25, 18

500 - 24 7.5 23, 32, 17

150 - 22 5.1 23, 16, 26

50 - 25 6.4 21, 21, 32

0 - 25 3 .1 22, 28, 26

Solvent - 22 3.1 25, 23, 19

5000 + 28 5.1 22, 99, 32

1500 + 27 2.3 24, 28, 28

500 + 26 2.9 24, 24, 29

150 + 26 4.0 28, 28, 21

50 + 22 4.6 25, 25, 1 7

0 + 25 2.5 25, 22, 27

Solvent + 27 1.0 26, 27, 28

TA 100 5000 - 120 12.3 125, 106, 129

1500 - 123 9.2 115, 133, 121

500 - 125 21.0 146, 104, 124

150 - 142 18.5 161, 142, 124

50 - 122 12.7 108, 132, 12 7

0 - 151 8.7 146, 146, 161

Solvent - 136 6.1 129, 137, 141

5000 + 118 31.4 106, 95, 154

1500 + 129 11.0 136, 134, 116

500 + 130 6.1 135, 123, 131

150 + 141 5.5 137, 138, 147

50 + 128 11.5 139, 116, 12 8

0 + 146 11-8 149, 156, 133

Solvent + 150 6.5 157, 144, 150

- Absence

+ Presence -

SD Standard deviation

~. .

18
p. 88

- _,-_

TABLE 3

Mutation test 1

Mutability tests with baeterial strains TA 1535, TA 1537, TA 98 and TA 100

Strain Compound Dose Level Liver Mean SD Individual revertant

- (,%g/plate) S-9 revertant colony count s

colony

counts

TA 1535 ENNG 5.0 - 283 21 .1 269, 272, 307

TA 1537 9 AC 80 .0 - - - X, X, X

TA 98 NF 1.0 - 384 10 .4 381, 396, 376

TA 100 ENNG 3.0 - 534 18.6 549, 539, 513

TA 1535 AA 2.0 + 168 24.7 161, 147, 195

TA 1537 AA 2.0 + 51 9.9 44, 62, 46

TA 98 AA 0.5 + 212 33.7 243, 216, 176

TA 100 AA 1.0 + 462 14.7 445, 467, 473

- Absence

+ Presence

SD Standard deviation

x Too many colonies to count accurately

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

9 AC 9-Anainoacridine

NF 2-Nitrofluorene

AA 2-Aminoanthraceue

~.. ~- .

:~.

,~., •

19 .

~~+'
p. 89

=- = =

TABLE 4

Mutation test 2

NEWCOL 707SF Conc - revertant colony counts obtained per plate Osing

bacterial strains TA 1535, TA 1537, TA 98 and TA 100

Strain Dose level _ Liver Mean SD Individual revertant

(ryg/plate) - S-9 revertant colony counts

colony

count s

TA 1535 5000 - 15 2 .5 18, 15, 13

1500 - 11 2.0 11, 13, 9

500 - 11 2.0 11, 9, 13

150 - 14 3.8 16, 17, 10

50 - 14 0.6 14, 13, 1 4

0 - 13 1.5 11, 14, 13

Solvent - 16 5.2 19, 19, 10

5000 + 12 3.0 12, 9, 15

1500 + 14 5.8 11, 11, 21

500 + 13 7.0 12, 20, 6

150 + 17 3.5 17,'21, 14

50 + 18 1 .2 17, 19, 1 7

0 + 12 1.2 11, 13, 13

Solvent + 13 3.2 12, 17, 11

TA 1537 5000 - 9 3.8 5, 11, 1 2

1500 - 8 3.5 12, 8, 5

500 11 4.6 8, 8, 16

150 - 7 4.2 6, 4, 12

50 - 8 3.1 5, 11, 7

0 - 8 1.7 7, 7, 10

Solvent - 9 1.2 10, 8, 8

5000 + 8 2.1 7, 6, 10

1500 + 12 1.7 13, 13, 1 0

500 + 8 1.2 7, 9, 9

150 + 9 1.5 10, 9, 7

50 + 13 5.3 11, 19, 9

0 + 10 1.5 10, 11, 8

Solvent + 9 4.6 5, 14, 8

- Absence

+ Presence

SD Standard deviation

:; .

ti

20 .

~~
P. 90

TABLE 4

(continued)

Mntation test 2

NEWCOL 707SF Conc - revertant colony counts obtained per plate using

bacterial strains TA 1535, TA 1537, TA 98 and TA 100

Strain Dose level Liver Mean SD Individual revertant

(jig/plate) S-9 revertant colony counts

colony

count s

TA 98 5000 - 24 2 .1 26, 25, 22

1500 - 22 1 .5 20, 23, 22

500 - 26 3.6 23, 30, 25

150 - 23 1 .5 22, 25, 23

50 - 21 6.1 20, 16, 2 8

0 - 26 2.6 29, 25, 24

Solvent - 23 3.5 27, 20, 23

5000 + 25 7.0 32, 1 8, 26

1500 + 28 2.5 25, 28, 30

500 + 27 3.2 28, 23, 29

150 + 25 6.1 28, 29, 18

50 + 28 3.5 30, 30, 24

0 + 27 2.5 25, 30, 27

Solvent + 25 4.0 29, 21, 24

=.i

TA 100 5000 - 118 8.1 114, 112, 127

1500 - 115 24.8 88, 119, 137

500 - 123 32.9 85, 141, 143

150 - 138 9.5 133, 149, 132

50 - 136 19.1 156, 118, 13 4

0 - 149 4.5 153, 149, 144

Solvent - 122 4.2 119, 127, 121

5000 + 118 9.5 124, 107, 123

1500 + 133 8.5 133, 141, 124

500 + 130 3.1 131, 133, 127

150 + 125 4.9 127, 119, 128

50 + 152 14.4 144, 144, 169

0 + 139 18.1 120, 141, 156

Solvent + 137 20 .5 142, 114, 154

- Absence

+ Presence -

SD Standard deviation

21
P. 91

TABLE 5 -

:

. _

.

Mutation test 2

Mutability tests with bacte rial strains TA 1535, TA 1537, TA 98 and, TA, .100

. :StrairL Compound Dose level Liver Mean SD Individual revertant

(A91 plate) S-9 revertant colony counts

colony

counts

TA 1535 ENNG 5 .0 - 261 22.8 275, 235, 274

TA 1537 9 AC 80.0 - - - x, X, X

TA 98 NF 1.0 - 392 34.0 365, 380, 430

TA 100 ENNG 3.0 - 559 68.5 633, 545, 498

TA 1538 NF 2.0 - 448 47.4 426, 502, 415

TA 1535 AA 2.0 + 188 4.0 184, 192, 189

TA 1537 AA 2.0 + 95 7.0 100, 98, 87

TA 98 AA 0.5 + 256 38.5 298, 249, 222

TA 100 AA 1.0 + 622 13.5 618, 611, 637

TA 1538 AA 0.5 + 124 15.2 138, 127, 108

- Absence

+ Presence

SD Standard deviation

X Too many colonies to count accurately

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

9 AC 9-Aminoacridine

NF 2-Nitro fluorene

AA 2-Aminoanthracene

:22 : -

7r
p. 92

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