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 rece

## ip

## ` r



## NOTICE ~~~ ~~~ . ,' , - •_

FOR NEW CHEMICAL SUBSTANCE S DOCUMENT CONTROL OFFICER 

## Company Sanitind OFFICE OF POLLUTION PREVENTIO N AND TOXIC SUBSTANCES, 7407 U .S . E .P .A . 401 M STREET, SW WASHINGTON, D .C . 2046 0 Enter the total number of pages ~ 

# 1- / ent t :ol n in the Pre manufacture No ti ce 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(1 2-66 & 140{, or faxing 202 - 5 54-560U • If a user fee has been remitted 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 ID number mad also appearon your 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 DAT A You mast provide the currently coned Chemical Abstract. (CA) Name of the You are reqviied to submit all test data in your p6swsion 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 as minted to the health and your submission will not be complete and the review will not begin until EPA env 4onmaW efforts of the asarfatlat w pioassina, distribution receives this Informaflon . A i efter In support of your submission should In. mmmeeq, rse, or dirposd of the new chemical substance. Standard rsfemnoe your TS user fee identification nombeG You m ust ' sabstif an od6ina 1 literature aibtions may be tnbmilted for data in the open scientific and two copies of this notice Including all we data . If y ou cWsaed any literature . Its information as oonfidantlal, a single sanitized copy inust Won be submitted, clearly identify whether test data I . on the substance at an an analog . Also, Part II- HUMAN EXPOSURE . AND ENVIRONMENTAL RELEASE the chemical eompodNoa of the tested sabdal Would be characterized. Following are examples of tut data and other dsta Data should be If there are several mannfactmy proaesdng, at use operations to be described In submitted according to the requirements of 572ASD of the P moa,ufaRaie Part U, sections A and B of this 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 • Other data 

# 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 want EPA to consider in evaluating • I'hydaJlCYeainl Piopatler' ~ Yes Ted data not in the possession the new sabstane s On page 11 of thb fora m, spaas ban ben provided for you to or mnteoi of the snbmitt .e describe pollution prevention and recycling infonsstlon you may have regardin g the new snbstancs 'A physical and citrid -peopntio woelobeel is locsbd on the last page of this foatr So-called "binding" bmcs are indaded throughout this inns for you to indie dt TYPOF NOTICE (Check. Only your willingness to be bound to certain shbmeab yoaaWin thfsnoicsr suc h ause, production volume, prroteatiye eqotpment . . . This option is intended t o uce delays that routinely accompany the datelopzNaR -of consent ordain o r 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 f6vQ,ysodq~ 

# 13 INTERMEDIATE PMN. (abmitEsd in .W . with binding on the wbmiiterrvhen the Agency appiv ► as the exemption ~kafloy PM1 4 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 (rest Madceting Bxmptian AppllaliaeJ CONF CONFIDENTIALITY CLAIMS ❑ LVB (Lowr Volume BurrptiosJ . 40 t .1 R 7?S ..lo (cX1110 you may dais any infoauNosi in this nod= as confidential . To awed a datm 0 LOAM (Lme lbissrs[owBpoonrBzebpto) 0 Zr0eK3 on the 6aeat, mask 0 0 the confidential box neat bIh . information hysdd a a confidential . To armed a dais is an altaeimeat, dr d@ or bndnt the ~ LVE MadlHotbt ~ LCli87( Modl$afi~ Information you dales as confidential. IDnfidentialy ouu mnd aL pravide a If no d2ha 1"forma ktswa ~mioe ef IN notlae (Ied dla o ~ Far additional insbudiou on daimin6lnfoeeeati es 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 ehesai CRls tPtsmtias Coaminicatba # required, anie r # on page 3) EPA FORM 7J10•25 .(Rev . 5-95) Replaces prevlous ediHow of EPA Form 7710•25 . 

# f2

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 . -- - CERTIFICATIONI 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 submissio n 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 quanti ti es 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 ; an d 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 demia t substanee described herein . Any knowing and willful misinterpretation ffi. subject to criminal penalty pursuant to 18 USC I MI . Confi dential Signature and title uthorized ( Original Signature Required) Date
4 v ~ C 

### .- Date . gnature of agent -(if applicable) - FORM EPA 7710-25 ( Rev . 5-45) P age 2

### p . 3 Part I - GENERAL INFORMATIO NSection A - SUBMTITER IDENTIFICATION ConfiMark (X) the "Confidential" box next to any subsection you claim as confidential . dential Name of authorized official Positio n la . Person Mr

. . 'H . Miyachi PresidentSubmWirgNotice 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 1060 6 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 Submitter (if applicable) Company

Mailing address (number and street ) City, State, ZII' Code ' Area Code Number Telephone 2 . Technical Name Positio n 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 Circl e City ; State, 23P Code . Arw Code Number Telephone I Hopewell Junction, Necv_York 12533 i 845 i 896-4340 3 . If you have had 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 Mark by EPA .- If you previously submitted a PMN for this substance enter the ~e ~ PT~M! number assigned by EPA (La . withdrawn or iacampieEe) . . ~ 5 . If you have submitted a notice of Bona fi de intent to manufacture of Mark 0 0 impoet for the chemical substance covered by this notlae, mber the 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 the CAS 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 p artial 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 Pa rt I-- GENERAL INFORMATION -- Continue d 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 li manufacture of the polymer . (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 Sub stance 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 method you used to de velop or obtain the spedfied dtemical identity informatibn reported in this notioe . (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 Expert Service mug be s ub m itted 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 struct ure diagram, as complete as can be known, if one can be reasonably aso artained .

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 - Continue d 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 y ou 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 name s . . 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 availabi c Byproduct CAS Regist ry 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 - Continue d 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& B Optioa b` ~ (100%'o new chemical substance basis) (1009'a new chemical substance basis) dential M&A W 12,000 - • 30,00 0



### . 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 informatio n. 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 catego

### ry 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 fo r 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) CB I Binding Produc- CBI % in I % of substance 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 g 6 '"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 con centration of the new chemical substance as expected in consumer products and describe the chemical r eactions by which this substance loses its identity in the consumer product. ❑ Mark (X) this box if you attach a 

### continuation s

### hee

### t b . Generic If 

### you claim any category of use desci P

### don in subsection 2a 

### as oonfidential, 

### enter a g

### eneric 

### 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 RELEAS E 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 sti ll have reporting requirements if th ere are further iad u ettia~ processing or use operations after import You must describe these operations . See inatractiortit manua L 1 . Operation description CMfi 40 a. Iden tity - Enter the identity of the site at which the operation wi ll occur . d ential

Nam e Site address (number and street) City ; Count} ;'State` ZIP Code If the same operation wi ll 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) thi s 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/y ew 2 . Continuou s 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 approxima te weight (by kg/day or kg/batch an a 100% new chemical substance basis), and ent ry point of all starting materials and feedstocks ( including reactants, solvents, and catalysts, etc .), and of all prod ucts, 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-th e 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 - Continue d pational Expoeure - You must make separate confidentiallty daims for the desaiptian of worker activit~g~}ysical forac~f :the new chemical 2 . O 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

ie'the activities (i .e bag durriping, tote filling, unloacllng drums, sampling, deaning, etc .) in which workers may be exposed to the (1) Des~it 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 business information (CBI) . ( 8) - Estimate the maximum number of workers involved in each activity for all sites combined . (9) - Mark (X) this column if entry in column (8) is confidential business information (CBI) . (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 busine ss 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 Worker s Mark W substance Mark W Exposed Iirs/day Days/y r (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 clai m s 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 you claim as confidential . (1) - Enter the n

umber of each release point identified in the kwoce- ft description, part 1 1 , 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 Instructi on 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, ,and control efficiency that will be used to limit the release of the new substance to the environment. For releases disposed of on land, characterize the disposal method and state whether it is approved for disposal of RCRA hazardous waste. On a continuation sheet, for each site describe any additional disposal methods that will be used and whether the waste is subject to secondary or tertiary on-site treatment. b. Estimate the amount released to the environment after control technology (in kg/day) . (6) - Mark 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) CB I Number, roles wd .nek .k t~e (1) (7a) Qbl ( g) (4) (Sa) ~~ (Sb) (b)

• . ~

(7) Mark (?Q the ❑ PO'TW provide name(a) below : 011 ❑ N&vlpbk ❑ Other - Specify p rovide NRD C B S CBI destination(s) of waterwa y releases to water . ❑ Mark (7q this box if you attach a continuation sheet . FORM EPA 7710•25 ( Rev . 5-95) Pap 9P . 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 additional sibes 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 worker s exposed for all sites combined . (4) - Estimate the typical duration of expos ure per worker in (a) hours per day and (b) days per year . (6) - Describe physical form of exposure and % new chemical substance (if in mixture), and any protective equipment and engineering controls, if any used to protect workers . (7) - Estimate the percent of the new substance as formulated when packaged or used as a final'product . (9) - From the process diagram above, enter the number of each release polnt . Complete 9-13 f or 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 be used 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 . Engin eering Contrda/ Form- Number New Technolog y 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 sec ti

## on you may provide information not reported elsewhere in thi s f9 xm 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 efficie n cy 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 sh eet. 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 relate

a;=if appropriate. 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 Conflpap number(s) dential 1 . Eye Irritation To The Rabbit 14 - 2 6 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 12p . 13 PHYSICAL AND CHEMICAL PROPERTIES WORKSHEE T To assist EPA's review of physical and chemical properties data, please complete the fo llowing 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 th e 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 submi ssion 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 - O C @ Temperature Torr Density/relative density 1 . 055 g/cm3 Solubility ° C 100G @ Temperatur e Solvent Tn 1 nPn P- T1iP 1'~ ► /L Solubility in water *Temperature 0 C °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 Flammabilif

y Explodability . Adsorption /coefHden t Other-Specify Viscosity 150mPa s 

## -,1 3 w ~

### p . 14

NEWCOL 707SF CON C 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 1 3



### ~~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 TABL E 1 . Ocular reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

: 2 : ~Jp . 16

COMPLIANCE WITH GOOD LABORATORY PRACTICE STAND S The study described in this report w as conducted in compliartce with the following Good Laboratory -- Practice st andards and I consider the data generated to be valid . Good Laboratory Practice, The United Kingdom Compli ance 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 Environment al 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 an d Industry, Directive 31 March 1984 (K anpogyo No . 39 Environmental Agency, Kikyoku No . 85 MITI) .

~ - -



# c~ ces-R 22 ~ ~ ~ cv :y Brenda I . Parcell, M .I .A .T ., Dat e Study Director , Huntingdon Life Sciences Ltd .



### : 3 : A op . 17

QUALITY ASSURANCE STATEMEN T This report , has been audited by Huntingdon Life Sciences Quality Assurance Department (Huntingdon) . The Inetfiods, practices an d procedures report ed 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 an d accurate representation of the raw data generated during the conduct of the study at :Hunti o

gdon . Cert ain studies such as that described in this report, are conducted at Huntingdon in a se tt ing which involves fr equent 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 Assur ance Department of critic al procedures relev ant to this study type . The findings of these inspections were reported promptly to the Study Director an d to M an agement, Huntingdon Life Sciences .

Date(s) of inspection 10-13 September 1996
Date(s) of repo rting inspection finding s to the Study Director and M an agement 18 September 1996
Date of repo rting audit findings-to th e Study Director and M an agement 9 October 1996

Peter Watson, Dat e Quality Assur an ce Unit Head, Department of Quality Assur ance, Huntingdon Life Sciences Ltd .

: 4 : 

# ~7p . 18

RESPONSIBLE PERSONNE L Brenda I . Parcell, M .I .A .T ., Study Director , De

## p artment of Acute Toxicology . 

## `"~'s-...,, ., . : . . . ., . .: .. . .. : :: : : . .: . :

{

:5 :P . 19

SUMMARY A study w as performed to as sess 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 Febru a ry 1987 . Three rabbits= were--each ad ministered a single ocular dose of 0 .1 ml of the test subst ance 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 : 

### ~7p . 20

= INTRODUCTIO N 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 SUBSTANC E Identity : _ NEWCOL 707SF Cone Lot number : 74 1 Expiry : 20 July 1998 Purity : Not advised Appear an ce : Viscous liquid , Storage conditions : Room temperature in dark Date received : 26 July 1996p . 22

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

: : . . . . . ~~t~,~k

Eo~~v~'L"?~e ~~ .as ., ~ • .' ;F, P _ ~, . FL~• . _ -ti',` zra ~id,A~~ :~^'a ~ Eli

EX PERI MENTAL PROCEDUR E 

### ANIMAL MANAGE MENT 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 tr eatment (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 fl oors in Building R14 Room 2 . A st an dard laboratory diet SDS Stanrab ( P) Rabbit Diet and drinking water were provided ad libitum . The batch of diet used for the study was not an alysed for nutrients, contamin ants 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 identi fi ed 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 th e 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 an ce 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 subst ance to ensure that there was no pre-existing co rneal 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 an imals would be exposed ( pilot animal see Table 1) . Approximately 0 .1 ml of the test subst ance was placed into the lower eve rted 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 obse rv ed 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 inst il lation . Additional observ ations 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 th an slight dulling of normal lustre) , details of iris clearly visible I Easily discernible translucent are as , detail s 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 Iri s Normal 0 Markedly deepened ru gae, congestion, swelling, moderate circumcorneal hyperaemia or injection, any of these or combination of any thereof, iris still reacting to ligh t (sluggish reaction is positive) 1 

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



# g 3p . 24

Conjun divae Redness (refers to the most severe reading of palpebral an d bulbar conjunctivae, as compared to the control eye) Blood vessels normal 0 Some blood vessels de

finitely hyperaemic (injected) I Diffuse, crimson colour, individual vessels not easily discernible 2 Diffuse beef

y- red 3 Chemosis {lids an d/or nictating membranes) No swelling 0 Any swelling above normal ( includes nictating membr an es) 1 Obvious swell ing 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 repo rt 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 circumst ances will any item be discarded without the client's knowledge . DEVIATIONS FROM PROTOCOL There were no deviations fr om the protocol .



# ap . 25

Ah A 

### RESULTS CLINICAL SIGNS , . There were no signs of toxicity or ill health in any rabbit during the obse rv ation period . OCULAR RESPONSE S 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 rv ed . 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 ey e and-sex hour 1 2 3 4 7

Cornea Densi ty 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 Densi ty 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~~op . 27

~._ QUALITY ASSURANCE STATEMENT 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 repo rt 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 finding s 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, Dat e Quality Assurance Unit Head, Department of Quality Assurance, Huntingdon Life Sciences Ltd .


### 4



# 97p . 28

RESPONSIBLE PERSONNE L 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

INTRODUCTIO N 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 : 30p . 31

- - t_ -~_ - - 

### TEST SLTBSTANC E Identity : NEWCOL 707SF Conc Lot number : 74 1 Expiry : 20 July 1998 Purity : Not advised Appearance : Viscous liqui d Storage conditions : Room temperature in dark Date received : 26 July 199 6



### 1

: 8 : 

# 3 11p . 32

EXPERIMENTAL PROCEDUR E
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 PREPARATIO N 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

ARQIIVE S 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 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 .



### . 11p . 3

5 ~ RESULT S 

### CLINICAL SIGN S 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 reaction s Rabbit E = Erythema Day number O = Oedem a 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 dressin g ? . .

1 3 

# 30p . 37

. - . ~

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

- ~ .

NEWCOL 707SF CONC ACUTE ORAL TOXICITY TO THE RA T

Sponsor Research Laborator y 

### • Huntingdon Life Sciences Ltd

P .O . Box 2 Huntingdon Cambridgeshire PE18 6ES ENGLAND Report issued 22 May 1997
Page 1 of 1 4 

# 37p . 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

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TABLE S 1 . Signs of reaction to treatment,,,,,,,,,,,,,,,13 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Individual and group mean bodyweights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 3 . Individual bodyweight changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4

:2 :

~~p . 39

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARD S The study described in this report was conducted in compliance with the. . following Good Laboratory Practit e ' 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 Laborato ry Practice in the testing of Chemicals OECD, ISBN 92-64-12367-9, Paris 1982, subsequently repub lished 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 Comp liance 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) . Information regarding test substance characterisatian, namely for purity was not made available to Huntingdon Life Sciences for compliance with the Good Laborato ry 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 : 

# 39p . 40

QUALITY ASSURANCE STATEMEN T This repo rt has been audited by Huntingdon Life Sciences Quality A ssurance 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 st ady at Huntingdon . _ Certain studies such as that described in this repo rt , are conducted at Huntingdon in a setting which involves frequent repetition of similar or identical procedures . At or about the time the study descri bed in this repo rt was in progress, 'process-based' inspections were made by the Quality Assurance Department of c ritical procedures relevant to this study type . The fi ndings 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 fi ndings to the Study Director and Management 9 August 1996 Date of reporting audit findings to the Study Director and Management 31 October 199 6



## ~~ .G~~~~ :.. . . .~ . . ... ._ ._ . . ~ _ . . ... . . .. . . .._ . .. . . ~ . .~~ .. .~~ ._~' . .. . . . : . . Jacq e Cahill, Date Audit Team Supervisor, Department of Quality Assur ance, Huntingdon Life Sciences Ltd .

~

.yp . 41

RESPONSIBLE PERSONNE L Lewis A . McRae, H .NC ., M .I :A .T ., M .I .Sc .T ., Study Director , Department of Acute Toxicology.

$

N y .

5 .p . 42

SUMMARY This study w as 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 "6 f Chemicals No . 401, 'Acute Oral Toxicity" . Adopted : 24 February 1987 . A group o ften fasted rats ( five_males an d fi ve females) received a single oral gavage dose of the test substan ce, formulated i n disti ll ed 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 peri od . Clinical signs of reaction to treatment comp rised piloerection seen in all rats throughout Day 1 accomp anied 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 obse rv ed an d all an imals had fully recovered by Day 5 (females) or Day 7 (males) . All animals achieved satisfacto ry bodyweight gains throughout the study . No abnormalities were obse rv ed 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

INTRODUCTIO N 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 an ce may be ingested "a .cciderttially . The study was conducted in comp li ance with the OECD Guideline for Testing of Chemicals No . 401 "Acute Oral Toxicity" . Adopted : 24 February 1987 . The rat was chosen as it h as 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 w as chosen in compliance wi th 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 Jp . 44

TEST SUBSTANC E Identity : Newcol 7 .07SF Conc Intended use : Not advised Appearance : Viscous liquid Storage conditions : Room temperature in the dark Lot number : 74 1 Expi ry : 2 0 July 1998 • Composition : Not advised Date received : 26 July 199 6

iT . 8 -1i( 

# i(p . 4 5

- -- ~ 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 PREPARATIO N 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 : Llp . 46 

# = _ - 4

MA&&&



# _ TREATMENT PROCEDUR E A group of ten rats (five males and five females) was treated at 2000 m g/kg bodyweight . Control animal s No control animals were included in this study in accordance with the test guideline . ADMINISTRATION OF TEST SUBSTANC E 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 . OBSERVATION S 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 patholog y All animals were subjected to a macroscopic examination which consisted of opening the thoracic and
1 0 

# 1i 6p . 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 . 4 8 

# _ _ _= - ~o~rw r RESULTS MORTALIT Y 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 treatmen t

No . of rats in group of 5 showing sign s 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 : 

# ., yqp . 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 23 6 Male 2000 3 115 192 247 4 105 179 240 5 95 161 21 4 Mean 106 180 237

6 107 152 183 7 114 162 18 7 Female 2000 8 107 156 186 9 98 128 148 10 100 137 15 7 Mean 105 ••l47 172 TABLE 3 Individual bodyweight changes (g ) Sex Dose Animal Bodyweight gains (g)

(mg/kg) Number at Da y 8 15 1 77 62 2 74 5 8 Male 2000 3 77 55 4 74 61 5 66 5 3 -- 6 45 31

7 48 25 Female 2000 - 8 49 3 0 9 30 20 10 37 20

Hw .;~ . . 14

~ . . .

~~p . 51

- - . ~~



### NEWCOL 707SF CONC • ACUTE TOXICITY TO MEDAKA

(O ryzias lW

p es )

Sponsor - Testing facili ty = Huntingdon Life Sciences Ltd ., P .O . Box 2 , Huntingdon, Camb ri dgeshire, PE18 6ES, ENGLAND . Repo rt issued : 4 July 199 7 

### ~ - a

i 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 TABLE S ; 1 . Cumulative mortality data for medaka . . . . . . . . . . . . . . . . . . . . . . . . . 13

2 . Observ ations on sub-lethal effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 3 . Me an environmental parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 APPENDICES - - 1 . Individual environmental measurements . . . . . . . . . . . . . . . . . . . . . . . . 16 2 . Typical water quality characteristics of the diluent supply . . . . . . . . . . . . . 17

2p . 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 .D ., Date Study Director , Huntingdon Life Sciences Ltd .



### 3



# s3p . 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 th e 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 .



# syp . 55

RESPONSIBLE PERSONNEL 

### Craig Kelly, Ph .D .,, ~ - ~ Study Director , 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 ) 3-96 > 100 - fi •

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 .~
_ 

### :~.. 57dp . 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 • 

# 67p . 58

TEST SUBSTANCE Identity : NEWCOL 707SF Conc Lot number : 74 1 Expiry : 20 July 1998 Composition : Volatile content > 98% Appearance : Viscous liqui d Storage conditions : In darkness at room temperature Date received : 14 October 199 6

: 8 : If itp . 59

EXPERIlVIENTAL PROCEDUR E

- TEST SPECIES Nam e Medaka (06zias latipes) . - Sourc e Neal Hardy Aquatics, Essex, UK (see "Deviation from Protocol") . Acclimatisatio n 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 WATE

R 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 PREPARATIO N Method of preparatio n 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 concentration s Test concentrations were not verified by chemical analysis, at the request of the Sponsor . EXPOSURE CONDITION S 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 concentration s 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 condition s 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) absenc e 

### 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 DAT A 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 . ARCHIVE

S 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 .

~;- 

# 61p . 62

RESULT S Cumulative mortality data . are given in Table 1 . A ll 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 . Observation s 

### 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 (ini tial 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

;~ . ,~ .

- : t

~ : . 

### 1 3



# (P3p . 64 { 

### TABLE 2

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

Nominal Time (hours ) concentration Abnormalit y me/1 3 6 24 48 72 96 Control No abnormali ties detected 4 .6 No abnormalities detected 10 No abnormalities detected 22 No abnormalities detected 46 No abnormalities detecte d 100 Lying on bottom 3/7 1/6

Swimming at surface 1/7 x/y Number of fish affected/Number of fish surviving

: 14p . 65

TABLE 3 Environmental parameter s Mean values for pH, temperature and dissolved oxygen

Nominal Mean value s concentration mgLl pH m-0,/1 T°C Control 7 .9 8 .7 2 0 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 2 0



## • .

{

%, .

{f'

Jt , 'tt~ry .

R~r i~~' ' ~ 

### 15 •p . 66

U v . o\ o , 0\ 0\ r n y {-+ , L o O , 0 0 00 00 O : 00 00 co 00 00 00 CO 

### ~ x ~ o N 00 00 00 00 00 Do U o 0 0 0 0 0 F N N N N N N 4 . o eq t o CO CC 00 00 00 00 y h ~ 0!7 09 0o O O~ O O C C h" I`- 00 ( - 00 00 ~ •

~ U py , 0 00000 . N N N N N N' I . . d '~ y ~ 0 N h I`~ l~ t~• ['~ h ~ ~ 06 06 06 06 to 0 0 •N 6, 00 S 09 cl~ C\ Q1 (7\ a\

y r f~ [~ I• t~ t ~
+ ; rn o\ rn o, v . a\
ZZ h h r~ r oo t ~ ° O 

### tb co 

### 00 0 0 0

### 0 cc co ~ Q ir N O L ~+ oo O~ O+ a~ O O [ 

### - cc cc 

### 0 N N N N N

. .7 00 p O 0 0 00 00 T co ON •"- CA 0

0 OQ 00 00 00 00, C O V ] x h oooot~oooo -

- . I`~ r f~ t~• C~ h ° ' o ~ o e, '" ~ N ~ O C T '„~,t ;• • •- t-0 •- X

~ U o



### 16 66p . 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 Wate r Sample Received 19th August 199 6 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 14 0 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

17p . 68

APPENDIX 2 

### (continued)
TEST REPORT NO .9'I047cont . . . . f2h SeplembAr 1 

# 9aA

= Pafaq3etprs Result s 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 Haloform s 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 Pesticide s 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 tvsal aathion • 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_1 1 Od7cont . . . . 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 li gli <0,0 4 Bacte :iatnoi : a i Total viable coun t 1 day ,a_ 37'C per ml cfu 6 3 days Q 21'C per ml 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 * . p i5ed 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 . ezx v, that sadiun hasin ; reased . in :ne es_r,s i nci`a :e a wa :er of saa`ac- : f qualry, su ; aNe for drinking and Co m es ,= :,,- r^ses . ^ora :r .onxra :'_, lSSaCin ~~ ! DRY SERVICES Llmil=O IS~yr -- -- K SAx= Sheet 3 of 3

Aj

~~

### p . 70

NEWCOL 707SF CON C BACTERIAL MUTATION ASSAY
; ; .

,, . Sponsor Testing facili t y 

### Huntingdon Life Sciences Ltd ., P .O . Box 2 , Huntingdon, Cambridgeshire, PE18 6ES, ENGLAND . Report issued 29 May 1997 ° Page 1 of 2 2 

# 70p . 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 TABLE S 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 materials - Mutation test 2 . . . . . . . . . . . . . . . . . . . . 22

t , r ~ .~



### 2 :

`hn, l ~p . 72

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARD S The study described in this report was conducted in compliance with the following -Good Laborato ry Practice standards and I consider the data generated to be valid . Good Laboratory Practice, The United Kingdom Compliance Programme, Department of Health 1 9-89, 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, ( FIFRA), Title 40 Code of Federal Regulations Part 160, Federal Register, 29 November 1983 and subsequent amendment Federal Register 17 August 1989 . Japan Minist

ry of Agriculture, Forestry and Fisheries, 59 NohSan, Noti fication No . 3850, Agricultural Production Bureau, 10 August 1984 . United States Environmental Protection Agency, (TSCA), Title 40 Code of Federal Regulations Pa rt 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 repo rt has :, been , audited by Huntingdon Life Sciences Quality- Ass ur ance Department (Huntingdon) . The methods, practices and procedures report ed 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 Hun fingdon . 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 repo rted 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 Man agement 25 November 199 6 

## ~°i. . '_S ..'~1 .'1 . . .. . . . Rod Scammell, Dat e Audit Team Supervisor, Department of Quality Assurance,

..L 

### Huntingdon Life Sciences Ltd .

: 4 :



# ~3p . 74

RESPONSIBLE PERSONNEL
Rxcarda A ., .Gant, .B .Sc . Tong .,)r . .,,,, Study Director ; Department of Cellular Toxicology . .

5 

# ;~- -1 Yp . 7 5

- - - ._ - - - _^ 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 activi ty was seen at any dose level of NEWCOL 707SF Cone in either mutation test . The concurrent positive control compounds demonstrated the sensitivity of the as say 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
6 { INTRODUCTIO N
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 o f Salmon 4ia typhimu rium 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 to the 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 ( pKM 1 01) which introduces an error-prone repair process, resulting in increased sensitivity to mutagens . Since m any substances do not exe rt 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 typhimu rium - Reverse Mutation Assay , US Environmental Protection Agency, Method : HG-Gene Muta -,S. typhimu ri um : The Salmonella ty

phimu ri um 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 

# 76p . 77

TEST SUBSTANC E 

### Idenfity : NEWCOL 707-SF Con e Chemical nam : Information not available to Huntingdon Life Sciences Intended use : Information not available to Huntingdon Lif e Sciences Appearance : Clear viscous amber liquid Storage conditions : Room temperature in the dark 9 Batch number : 74 1



### Expiry date : 20 July 199 8 Purity : Assumed to be 100 % for testing purposes Date received : 26 July 199 6 Supplier :

t, .' . 8



# 77p . 78

EXPERIMENTAL PROCEDUR E 

### BACTERIAL STRAIN S The following strains of S . typhimurium were used in the test : S . iyph,imunum TA 1535 hisG46 rfa uvr B S. typhimunum TA 1537 hisC3076 rfa uvr B S . typhimurium TA 98 hisD3052 rfa uvrB pKM101 S . typhimurium TA 100 hisG46 rfa uvrB pKM10 1 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 mi x Identity N-Ethyl N'-nitro-1V nitrosoguanidine Supplier Sigma Batch 67F-3700 Appearance Pale yellow crystalline powder Solvent Dimethyl sulphoxid e 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 . 

# 71p . 79

I#entity 2-Nitrofluorene -- - 

### Supplier Aldrich Batch 61896 Appearance Beige powde r ° 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 sulphoxid e Concentration 2 μg/plate for strains TA 1535 and TA 1537 0 .5 μg/plate for strain TA 9 8 

### 1 μg/plate for strain TA 100 PREPARATION OF S-9 FRACTIO N Species Rat Sex Male Strain Sprague-Dawley derive d 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 . 8 0

° -- 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 O F- 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 TES T 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 concen tration 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 concen tr ation 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 PROCEDUR E The test substance was added to cultures of the four tester strains at five concentrations separated by ca half-logio interv als . 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 buff er . (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 sterili ty 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 Co unter . 

### At a later date the main test was repeated using the procedures described above with the same concentrations of test substance . STABILITY AND FORMULATION ANALYSI S 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 RESULT S 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 rv ed 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 ;

~ .~

$'

~



### 13
~ ~~p . 8 3

, _ j _`__ - . _•- - 

### RESULT S 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 0APp . 84

REFERENCE S 

### AMES, B .N ., MCCANN, J : ; and YAMASAKI, E . (1975) .N1elhods for detecting carcinogens and mutagegs with the -S almonella/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 AME S, 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 Con c

- 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 15 7 50 - 19 8 26 154 5 - 14 6 23 152 Solvent - 16 6 40 13 4 5000 + 15 4 25 131 500 + 19 4 27 164 50 + 16 6 31 13 7 5 + 22 5 39 159 Solvent + 17 5 38 152 Absenc e + Presence

~--

~ . ~ 

### 16 ' ~~p . 86



### = ', - - __ _- - - - - - TABLE 

### 2 

### Mutation test 1 NEWCOL 707SF Conc - re ve rtant 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 - Absenc e + Presenc e 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, 3 2 0 - 25 3 .1 22, 28, 26 Solvent - 22 3 .1 25, 23, 19 5000 + 28 5 .1 22, 9 9, 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, 12 9

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 - Absenc e + P re sence - SD Standard deviation

~. .



### 18p . 88

- _,- _ TABLE 3 Muta tion test 1 

### Mutability tests with 

### baeterial s 

### trains 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 - Absenc e + Presence SD Standard deviation x Too many colonies to count accurately ENNG N-Ethyl-N'-nitro-N-nitrosoguanidine 9 AC 9-Anainoacridin e NF 2-Nitrofluo rene 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 - Absenc e + Presenc e SD Standard deviation

:; .

ti 

### 20 .

~~P . 90

TABLE 4 

### (con tinued) Mntation test 2 NEWCOL 707SF Conc - revertant colony counts obtained per plate using bacterial strains TA 1535, TA 1537, TA 98 and TA 10 0

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, 2 4 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, 16 9 0 + 139 18 .1 120, 141, 156 Solvent + 137 20 .5 142, 114, 154 - Absence

- + Presen c e SD Standard deviation



### 21P . 91

TABLE 5 - : . _ . Mutation test 2 

### Mutabi li ty tests with bacte ri al 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 count s 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 - Absenc e + Presence SD Standard deviation X Too many colonies to count accurately ENNG N-Ethyl-N'-nitro-N-nitrosoguanidine 9 AC 9-Aminoacridine NF 2-Nit ro fluorene AA 2-Aminoanthracene



### :22 : - 7rp . 92



# O F

~qe q5 - 1`~ 

# 9 ~