I P .1 ~

Form Approved . O.M .B. No . 20iV-C012 . Approval Expires ;10.31-96.

U. S. ENVIRONMENTAL PROTECTION AGENCY AGENCY USE ONLY

SEPA

Date of receip t

NOTICE

r7n

FOR NEW CHEMICAL SUBSTANCES >

Company Sanitized

N

Enter the total number of page

s in the Premanufacture Notice ?a.~Iqllii3

GENERAL INSTRUCTIONS TS- 16 1131[0117 1 ~ o

• You must p rovide all information requested In this form to the extent that it is known to or reasonably ascertainable by you . Make reasonable estimates if you do

not have actual data.

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

Substances Control Act (TSCA) Information Service by calling 7 02-554-1404, or faxing 2t7Z-554-5603) .

• If a user fee has been remitted for this notice: (40 CfR 700 .45), indic a te in the boxes above the TS-user fee iden tification number you have gener ated .

Remember, your user fee ID number must also appear on your corresponding fee remittancS, which is sent to EPA, HQ Accounti ng Operations Branch (PM-266),

P.O. 360399M, Pittsburgh, PA 15251-6399, Attn . TS CA UserFee.

Put I- GENERAL INFORMATION TEST DATA AND OTHER DATA

You must provide the currently correct Ch emical Abstracts (CA) Name of the You are required to submit all test data In your possession or control

new chemical substance, even if you claim the identity as confidential. You may and to provide a description of a ll other data known to or reasonably

autho ri ze another person to submit chemical identi ty information foryou,but ascertainable by you, If these data are related to the health and

yoursubmission will not be complete and the review will not begin until EPA environmental effects of the manufacture, p roorssing, distribution

receives this Information. A letter in support of your submission should in commerce, use, or disposal of the new chemical substanc.e. Standard

reference yourTS user fee identification number. You must submit an o ri ginal Literature citations may be submitted for data In the open scientifi c

and two copies of this notice including all test data . If you claimed any literature. Complete best data (written In English) . not summaries of data

information as confiden ti al, a single sanitized copy must also be submitted . must be submitt ed If they do not appear In the open literatnm. You should

dearly identify whether test data is on the substance or on an analog . Also,

Part II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE the chemical composition of the tested material should be characterized.

Following axe examples of test data and other data . Data should b e

If there are several manufacture, procrssing, or use operations to be described in submitted aacording to the requirements of §720.50 of the Premanufacture

Part II, sections A and B of this no tice, reproduce the sections as needed . Notifi cation Rule (40 CFR Pad 720).

Part III - LIST OF ATTACTIIv>ENTS Test Data ((:heck Below any included in this no ti ce)

Attach additi onal sheets if there Is not enough space to answer a question fully.

Label each continua tion sheet with the corresponding section heading . In Part • Environmental fate data yes • Other data F'] Yes

III, list these attachments, any test data or other data and any optiona l

information Included in the notice. • Healtheffects data yes Risk assessments

OPTIONAL INFORMATION • Environmental effects data ❑ Yd Strudurdactivity relationships

You may include any Information that you want EPA to consider in evaluating • PhysicsUmemial Properties ~® Yd Test data not In the possession

the new substance. On page 11 of this form, spacehas been provided for you to or control of the submitter

describe pollution prevention and recycling Information you may have regardin g

the new substance.

'A physical and chemical properties worksheet is located on the last page

of this form.

So-ca lled "binding" boxes are Included throughout this form for you to Indicate TYPE OF NOTICE (Check Only One)

your wi ll ingness to be bound to certain statements you make in this notice, such

as use, production volume, protective equipment . .

. This option is intended to

reduce delays that routinely accompany the development of consent orders or PMN (Premanufadure Notice)

Significant New Use Rules. Except in the case of exemption applications (such as

TMEA, LVE, LORE) ) where certain information provided in such notifi cation is INTERMEDIATE PMN (submitted in sequence y~ final prod uct

binding on the submitter when the Agency approves the exemption application, PMM

checking abinding box in this notice doe-q not by itself prohibit the submitter 1 rf

from later deviating from the Information (except chemical identity) reported In ~ SNUN (Significant New Use Notice )

the form. T 17❑

TMEA (Test Marketing Exemption Applicati(AillD

CONFIDENTIALITY CLAIMS ❑ LVE (Low Volume Exemption) a 40 CFR 723:0(v(~

You may claim any Information In this notice as confidential. To assert a claim ❑

on the form, mark CO the confi dential box next to the information that you claim LOREX (Low Releise/I.ow Exposure ExempsQ)') a 7I2}~)C )

as confidential. To assert a claim in an attachment, cirde or bracket th e

information you claim as confidential. if yon dUirn information In the notice u ❑ LVE Modification ❑ LOREX I~lification

confidential . you must .~ptyvidsa l sanitired version of the notice (indudin v

attachments). For additional Instructions on claiming Information as IS THIS A CONSOLiDATED PMN7 ~ Yes

confidential, read the Instructions ManuaL

❑ Mark W(f any information in this notice Is claimed as cnnfidentlaL I of chemic•als

(P reno ti ce Communication f required, enter f on page 3 )

EPA FORlbi 7710-25 (Rev . 5-95) Replaces previous editions of EPA Form T111}25 .

/
p. 2 2

?

Public reporting burden for this collection of information is estimated to average 110 hours per response, including time for reviewing instruc ti ony

searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the coll ection of information. Send

comments regarding the burden estimate or any other aspect of this co llection of information, including suggestions for reducing this burden,

to Chief, Information Policy Branch, PM-223, U.S. Environmental Protec ti on Agency, 401 M. St., S.W.,, Washington, D .C 2 0460 ; and to the Office of

Management and Budget, Paperwork Reducti on Act (2070-0012), Washington, D .C. 2Q503 .

CERTIFICATION

I ce rtify that to the beSt of my knowledge and belief :

1 . The company named in Part I, section A, subsection la of this notice form intends to manufacture or impo rt for a

commercial purpose, other than in small quanti ti es solely for research and development, the substance identi fi ed in

Part I, Section B.

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

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

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

Additional Certification Statements:

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

statement that applies:

~ The Company named in Pa rt I, Section A has remitted the fee of $2500 specified in 40 CFR 700 .45 (b), o r

❑ The Company named in Pa rt 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 sma ll business concern under 40 CFR 700 .43 and has remitted

a fee of $100 in accordance with 4 0 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 (LoREX) application in accordance with 40 CFR 723 .50 (c) (2), check the fo ll owing

cer tification statements:

❑ The manufacturer submitting this notice intends to manufacture or impo rt the new chemical substance for

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

of 40 CFR 723 .50 .

❑ The manufactu re r is familiar with the terms of this section and will comply with those terms; and

❑ The new chemical substance for which the notice is submitted meets all app licable exemption conditions.

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

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

30 day review period.

The accuracy of the statements you make in this notice should reflect your best prediction of the anticipated facts regarding the chemica l

substance described herein . Any knowing and willful misinterpretation is subject to criminal penalty pursu ant to 18 USC 100 1

. Confidential

Signature and ti of A orized Official (Original Signature Required) Dat e

PrPS, d L~►'1 7/3I[> `U. .S

Signature of agent - (if app licable) Date

FORM EPA 7710-25 (Rev. 5-95) Page 2
p.3

1K

Part I -- GENERAL INFORMATION

Section A -- SUBMITTER IDENTIFICATION confi -

dential

Mark ( X) the "Confiden ti al" box next to any subsection you claim as confidential .

Name of authori zed official Posi ti on ,

I a .

Submitting ~b

Person 1 ,,I L

`a. k a k as 11 I

D

rQS i ~l~~ ✓

Notice Company

(in U .S.) »~ ~ ►- ~ ~ ~ . 0,8E ~ C

Mailing address ( number and street )

5s As~ sa TN sTR E iz T i4~tl~ ~I_o~) ► ~ ;

CityState, ZIP Code

~,,.y:!j)%`~'• M~i,~~~,'"M ~~ ~ ~~• , y /~, '.\, / ~ <,'~Trc~' +, ~ /~

7 1- \ (~q`, 2 ~ :.; > ~iiii> . 'i~iim•+'isi~ J

. '

>i"f%/':n y`> /

l 1 ~ V (, 7iY3y :i:;~'i 9.~'i3;~'v~~OY.!/~~ J~"'ta:~ .R;" a+~~ ~.%/ • f

b . Agent ( if Name of authorized official Positi o n

applicable)

Compan y

Mailing address ( number and street)

City, State, ZIP Code Area Code Number

Telephon

. If you are submit e c ti ng this notice as part of a joint submission, mark (X) this box

. E l

Joint Name of authori zed official Posi ti on

Submitte r

(if applicable)

Company

Mailing address (number and street)

City, State, ZIP Code i Area Code Numbe r

2 . Technic al Name Posi

Telephone t

ti o

I ~

Con tact r i/ i l "

(in U .S.) e ~ S~-h / eu- GV I ehei- F~ a-vl Gz tCompany

L1 R t μ~+

Mailing add.ress (num be r and str t )

~S E~s S~rtk Street t~ F~o~r

City, State, ZIP Code Area Code Number

Telephon e

lC p I i Cs

3. If you have had a preno ti ce communication (PC7 concerning

-D 2~- `Z ( 2 '

this notic e

I

and EPA assigned a PC Num be r to the no ti ce, enter the number

~

. Mark (X) _>

if non e

4 . If you previously submitted an exemp ti on app li cation for the chemical

substance covered by this notice, enter the exemption num ber assigned

by EPA. If you previously submitt ed a PIv(N for this substance enter the Mark (X) >

PMN number assigned by EPA (i .e. withdrawn or incomplete). if none

~

~

5 . If you have submitted a no ti ce of Bona fide intent to manufacture or Mark (X) }

import for the chemical substance covered by this notice, enter the if none

notice number assigned by EPA. >

6. Type of Notice - Mark (X) I. ❑ Manufactute ~ Import

Only OrdY

3 . ❑ Both

❑ Binding Op tion BindingOption

Mark ( x)

❑

M ark (x)

FORNt EPA 7710-25 ( Rev. 5-95) Page 3
p.4

Part I -- GENERAL INFORMATION - Continue d

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

based on the ninth Collective Index (9C) of CA nomenclature rules and conven tions.

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

Complete either item 1(Class I or 2 substances) or 2 (Polymers) as appropri ate . Complete all other items.

If another person will submit chemical identity information for you (for either item 1 or 2), mark (X) the box at the righ t.

Identify the name, company, and address of that person in a continua ti on sheet. >- El dential

1. Class 1 or 2 chemical substarfces ( for definiti ons of class 1 and class 2 substances, see the Ins tructions Manual)

a . Class of subst ance - Mark (X) 1Z Class I or 2 [:] Cl.. 2

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

For Class 1 subst ances a CA Index Name must be provided. For Cass 2 subst ances either a CA Index Name or CA Preferred Name must

be provided, whichever is appropri ate based on CA 9CI nomenclature rules and conven ions.).

Siianami ne, I, l, 1 -triethoxy-N, IN-diethyl .-

c. Please identify which method you used to develop or obtain the specified chemical inden tity information reported in this no ti ce :

(check one).

a Method 1(CAS Inventory Expe rt Service - a copy of the Identifica ti on Method 2 (Other Source)

report obtained from the CAS Inventory Expert Service must b e

submitt ed as an attachment to this no tice)

d. Molecular formula and CAS Registry Number (if a numbe r already exists for the subst ance)

C io H z sN©3 Si CAS *

3So17'1 -OD- D

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

precursor subst ances with their respective CAS Registry Numbers. (2) Describe the nature of th e reac ti on or process. (3) Indicate the

r an ge of composi ti on and the typical composition (where approp riate). (4) Provide a correct representa ti ve or partial chemical

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

CZ Hs

0

HsC.2-o-5 ;

.-N C2Hs

C2 H

0

► C2 Hs

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

FORM EPA T710-25 (Rev . 5-95) Page 4
p. 5 b

Part I -- GENERAL INFORMATION - Continue d

Section B - CHEMICAL IDFNTITY INFORMATION - Continue d

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

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

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

500 and below 1,000 absolute molecular weight of that composi ti on.

Describe the methods of measurement or the basis for your estimates: CPC F~ OtherF~ :(Spedfy)

i) lowest number average molecular weight:

ii) maximum weight % below SCO molecular weight

iii) maximum weight % below lOCO molecular weight

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

b. You must make separate confiden tiality claims for monomer or other reactant identity, composition information, and residual informa tion. Mark (X) the "Confidential" box next to any item you claim as con fi dential .

(1) - Provide the specific chemical name and CAS Registry Number (if a number e )dsts) of each monomer or other reactant used in the

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 other reactant in the polymer .

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

polymer description on the TSCA Chemical Substance Invento ry.

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

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

manufactured for commercial purposes.

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

Monomer or other reactant and CAS Registry Number Confi- Typical. Identity Confi- Maximum Confidential composition Mark (X) dential residual dential

(1) (2) (3) (4) (5) (6) (7)

% %

% %

%< w

% %

E] Mark (X) this box if you attach a continua tion sheet.

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

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

obtained from CAS Invento ry Expert Service must be submitted as

an attachment to this notice)

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

e . Provide a correct representative or par ti al chemical structure diagram, as complete as can be known, if one can be reasonably ascertained .

Not ap pl i cabl e

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

FORM EPA 7I10-25 ( Rev . 5-95) Page 5
p. 6

~

Part I - GENERAL INFORMATION - Continue d

Section B - CHEMICAL IDENTITY INFORMATION - Continued

3. Impu ri ties

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

Provide the CAS Registry Number if available. If there are unidentified impu ri ties, enter 'unidentilied."

(b) -Estimate the maximum weight ~ of each impurity . If there are unidentified impurities, estimate their total weight 9. .

Impurity and CAS Registry Number Ma)dman' Confi-

(a) percent dential

%

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

4 . Synonyms - Enter any chemical synonyms for the new chemical subst ance identifi ed in subsec tion I or 2. Confidential

DiethYl am i notri e thox ysi I qh e

❑ 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.

U - dono r

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 maximum extent possible.

Refer to the'I'SCA Chemical Substance Inventory, 1985 Edition, Appendix B for guidance on developing

generic names.

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

7 . Byproducts - Describe any byproducts resulting from the manufacture, processing, use, or disposal of the new chemical substance . Provide the CAS

Registry Number if available.

Byproduct CAS Regis try Number Confi-

(Y) (2) dential

n-Butahe I 0 6-gq - 8

Ma nesiu4 n , c hloroethox - 2K964 _ Iq - 4

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

FORM EPA 771p-25 (Rev . 5-95) Page 6
p.7 ~

Pa rt I -- GENERAL INFORMATION - Continue d

Section C - PRODUCTION, IMPORT, AND USE INFORMATION :

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

1

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

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

100% new chemical substance basis . For a Low Volume Exemption application, if you choose to have your notice revie wed at a lower produdion

volume than 20,000 kg/yr, specify the volume and mark (x) in the binding box . Ifgranted, you are bound to this volume . .

Maximum first 12 month production (kg/yr) ~~ denti al Option Maximum 12-month production ( /) Confi- Binding

(100% new chemical substance basis) (100% new chemical substance basis) M.uk rxl

2

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

devoted to each category, the formulation of the new substance, and other use information . Mark(X)the "Confidential" Box next to any item you

claim as confidential .

a

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

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

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

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

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

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

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

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

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

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

(9) - Mask (X) this column if entry(ies) in column (8) is (are) confidential business information (CBD .

Category of use (1) CBl Binding Produc- CBI

% in CBI 'Y. of substance expected per use I

Option tion Form- (8)

(by function and application i .e . a dispersive dye for ~k ~ ulation

finishing polyester fibers) Site- n- Irdus- Com- inding

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

External donor for olef in ol tnerization (ov % 100 % /v v

% qc

% %

90

%G

%

%

*If you have identified a"consumer" use, please provide on a continuation sheet a detailed description of the use(s) of this chemical

substance in consumer products. In addition include estimates of the concentration of the new chemical substance as expected in

consumer products and describe the chemical reactions by which this substance loses its identity in the consumer product .

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

b

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

use category. Read the Instructions Manual for examples of generic use descriptions.

description

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

3

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

or other information which will be provided to any person who is reasonably likely to be exposed to this substance regarding protectiv e

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

Mark this box if you attach hazard information .

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

1 6

Part II -- HUMAN EXPOSURE AND ENVIRONMENTAL RELEAS E

Section A- INDUSTRIAL SITES CONTROLLED BY THE SUB,,,IITTER Mark (X) the "Confidential" box next to any item you

claim as confidential .

Complete section A for each type of manufacture, processing, or use operation involving the new chemical substance at industri al sites you control

.

Impo rt ers do riot have to complete this section for operations outside the U .S. ; however, you may still have reporting requirements if there are

further industrial processing or use operations after import You must describe these operations. See instructions manual.

1 . Operation description F

nS

a . Identity -

Nam

Enter

e

the identity of the site at which the operation will occur. a~

Site address (number and street)

City, County, State, ZIP Code

If the same operation will occur at more than one site, enter the number of sites . Identify the r of 3 ne

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

production rates or operations, include all the information requested in this section for those

sites as attachments.

❑ Mark (X) this box if you a tt ach a continua ti on sheet

b . Type -

Mark (X) ❑ Manufacturing ❑ Processing ❑ Use

c . Amount and Duration - Complete 1 or 2 as appropri at e

Maximum kg/batch(ioo % n~++a,~td Hours/batch

I

Batches/year

•ubMtancel l

1 . Batch t

Maximum kg/day ( ioo s -< u~W ~ I Hours/day Days/year

2 . Continuous

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

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

ga llon drum, ra il car, tank truck, etc.).

(2) Provide the identity, the approximate weight (by kg/day or kg/batch on an 100% new chemical substance basis), and entry point of all star ting

materials and feedstocks (including react ants, 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 intermittant releases, to the environment of the new chemical substance .

Not appl i GGbI e ( Paqe- g and 9~

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

FORM EPA 7710-25 ( Rev . 5-95) Page 8
p.9

X6

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

Section A - INDUSTRIAL SITES CONTROLLED BY THE SUB~IfIZ-= - Continue d

2. Occupa ti onal Exposure - You must make separate con fi den ti ality claims for the description of worker activity, physical form of the new chemical subst ance, number of workers exposed, and dura tion of activity . Mark (X) the "Confidential" box next to an y item you claim as confi denti al .

(1) - Describe the ac ti vi ti es ( e .g. bag dumping, tote filling, unloading drums, sampling: cleaning, etc.) in which workers may be exposed to the subst an ce.

(2) - Mark (X) this column if entry in column ( 1) is confidenti al business informa ti on (CBI).

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

(4) and (6) - Indicate you willirlgness to have the informa ti on provided in column (3) or (5) binding.

(5) - Indicate the physical form(s) of the new chemical subst ance ( e .g . so li d : crystal, gr anule, powder, or dust) and %a new chemical substance (if part of a mix ture) at the time of exposure .

(7) - Mark (X) this column if entry in column ( 5) is confidential business informa ti on (CBl).

(8) - Estimate the maximum number of workers involved in each activity for a ll sites combined_

( 9) - Mark (X) this column if entry in column ( 8) is con fidenti al business informa ti on (CBI).

(10) and ( 11) - Estimate the maximum dura tion of the activity for an y worker in hours per day and days per year.

(12) - Mark (X) this column if entries in columns ( 10) and (11) are confidenti al business informa tion (CBI).

Worker ac tivity CBI protective Equipment/ Binding Physical form(s) Binding CBI

o f

(e .g. bag dumping, filling drums) Engineering Controls Option (e .g .solid :powder) Option Workers CBl Maximum duration CBI

,& W an d % new Mark (A Exposed Hrs/day Days/yr

4 substance

(8) 9 (10) t 12)

❑ Mark (X) this box if you attach a continua ti on sheet.

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

substance released and other release and disposal information. Mark (X) the "Confiden ti al" box next to each item you claim as con fi dential.

( 1) - Enter the number of each release point identified in the process description, part II, section A, subsection ld(3).

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

(3) - Mark (X) this column if entries in columns (1) and (2) are confi dential business informa ti on (CBI).

(4) - Identify the media of release Le . stack air, fugi tive air (opti onal-see Instruction Manual), surface water, on-site or off-site land or incineration, PQ1tiV, or other ( please specify) to which the new substance will be released from that release point.

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

For releases disposed of on l and, characterize the disposal method and state whether it is approved for disposal of RCRA hazardous waste.

On a continua ti on sheet, for each site describe any addi ti onal disposal methods that will be used and whether the waste is subject to secondary or terti ary on-site treatment. b. Estimate the amount released to the environment after control technology (in kg/day).

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

(7) - Identify the destination(s) of releases to water . Please supply NPDES (Na ti onal Po ll ut ant Discharge Elimination System) numbers for direct

dischargers or NPDES numbers of the POTW (Publidy Owned Treatment Works). Mark ()) if the POTW name or NPDES # is confidential business informa tion (CBI).

Release Amount of new subst ance • C13I Media of Control technology and efficiency (you may wish to opti ona lly attach efficiency data) CBI

Number released release

e .g . tack air 81ndta g

(1) (2a) (2b)

(3) (4) (5a) Mark (x) (5b) (6)

(7) Mark (X) the ❑ POTW provide name(s) below CBI j ❑ Navigable ❑ other _ Specify provide NPDES # CBI

destina tion(s) of waterwa y

releases to water .

❑ Mark 00 this box if you attach a continuati on sheet.

FORM EPA 7I10-25 (Rev . 5-95) Page 9
P. 10

Part II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued

Section B - INDUSTRIAL SITES CONTROLLED BY OTHER S

Complete secti on B for typical processing or use opera ti ons involving the new chemical subst ance at sites you do not control. porters do not have to

complete this sec ti on for opera ti ons outside the U.S.; however, you must report any processing or use ac ti viti es after import. See the Instruc ti ons

Manual. Complete a separate section B for each type of p ro cessing, or use operation involving the new chemical substance. If the same opera ti on is performed

at more than one site describe the typical operation common to these sites . Identify addi ti onal sites on a con ti nua ti on sheet.

1 . Operation Description - To claim informa ti on in this section as confiden ti al, circle or bracket the specific informa ti on that you claim as confiden ti al .

(1) - Diagram the major unit opera ti on steps and chemical conversions, including interim storage an d transport containers (specify- e .g . 5 ga ll on pails,

55gallon drums, rail cars, tanktrucks, etc) . On the diagram, identify by le tt er an d bri efly describe each worker ac ti vity. (2) -- Provide the iden ti ty ,

the approximate weight (by kg/day or kg/batch, on an 100% new chemical subst an ce basis), and entry point of all feedstocks (including reactan ts,

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

batch). (3) -'Identify by num ber the points of release, including sma ll or intermittent releases, to the environment of the new chemical subst an ce .

(4) Please enter the # of sites ( remem be r to identify the locations of these sites on a continua ti on sheet) :

oTTsites

Mark (X) this box if you attach a continua ti on sheet.

2 . Worker ExposurefEnvironmental Release

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

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

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

(6) - Describe physical form of exposure an d % new chemical substance (if in mix ture), and any protective equipment an d engineering controls used

to protect workers.

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

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

(10) - Estimate the amount 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 i .e. stack air, fugi tive air (opti onal-see Instructions M anual), surface water, on-site or off-site l an d or incinera tion,

PO1W, or other (specify) and control technology, if an y, that will be used to limit the release of the new substance to the environment .

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

(3), (5), ( 8), (11), (13) and (15) - Mark ( X) this column if any of the proceeding entries are con fidential business informa tion (CB O .

Letter # of CBI Duration CBI Protective Equip. / %in CBI Release Amount of CBI Media of Release CBI

of Workers of Engineering Controls/ Form- Number New & Control

Act- Exposed Exposure Physical Form and % ulation Substance Technology

ivity new substance Released

I

(1) (2) (3) (4a) (4b) (5) (6) (7) (8) (9) (1 Oa) (10b) (11 (12) (13)

I

I I

: (15I (14) - Byproducts )

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

FORIVt EPA 7I10-25 (Rev . 5-95) Page 10
4•

P. 11 ~,

OPTIONAL POLLUTION PREVENTION INFORMATIO N

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

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

efforts to reduce or minimize potential risks associated with activities surrounding

manufacturing, processing, use and disposal of the PMN substance . Please include new

information pertinent.to pollution prevention, including source reduction, recycling activities

and safer processes or products available due to the new chemical substance . Source reduction

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

modification, process and procedure modification, product reformulation, raw materials

substitution, and/or inventory control . Recycling refers to the reclamation of useful chemical

components from wastes that would otherwise be treated or released as air emissions or water

discharges, or land disposal . Descriptions of pollution prevention, source reduction and

recycling should emphasize potential risk reduction subsequent to compliance with existing

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

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 the revised Instructions Manual that includes a Pollution Prevention

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

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

performance, a decrease in the cost of produc tion and/or improved operation efficiency of the new chemical substance in

comparison to existing chemical subst ances used in similar appli cations; 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.

(S) You r,ah ~et hi h pev-1vr'M01V%G4e, of polymer When you us2 this p F-odUct .

1:1 Mark (X) this box if you attach a continuation sheet.

FORM EPA 7110.25 62av . $-95) Page 11
p.12 '

76

Part III -- LIST OF ATTACHMENT S

Attach continua ti on sheets for sec ti ons of the form and test data and other data (including physical/chemical proper ti es and

structure/activity informa ti on), and op ti onal informa ti on after this page . Clearly identify the att a chment and the secti on of the form to

which it relates, if appropriate. Number consecuti vely the pages of the attachments. In the column below, enter the inclusive page

numbers of each attachment .

Mark (X) the "Confiden ti al" box next to any attachment name you claim as confidential . Read the [nstructions Manual for

guidance on how to claim any informa tion in an attachment as confiden ti al You must include with the sani ti zed copy of the no tice

form a sanitized version of any attachment in which you claim informa ti on as confi denti al .

Attachment name Attachment Confipage number(s) dential

Material Safety Data Sheet (MSDS) /S-181

ph sic,al avid t',hei-, ► i ca l propev-ties worksh eet 1 3

Acute oral toxi c i-t in the v^at N-40

Muta enicit test by usir miuroor cuv► srr+s ~--S7

Ghromosome aber►^atior test usin cultured mammcfliah cells S9-96

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

FORM EPA 7710-25 (Rev . 5-95) Page 12
p. 13 1 ; -

~'JG

PHYSICAL AIND CHEMICAL PROPERTIES WORKSHEET

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

and include it in the no ti ce . Identify the property measured, the page of the no ti ce on which the property appe ars, the value

of the property, the units in which the property is measured (as necessa ry), and whether or not the property is claimed as

confidenti al. The-physical state of the neat substance should be provided . Th ese measured proper ties should be for the neat (1D0%

pure) chemical substance. Proper ti es that ar e measured for mixtures or formulations should be so noted (% PMN substance in _).You

are not required to submit this worksheet; however, EPA strongly recommends that you do so, as it wi ll simplify review and ensure

that con fi denti al information is properly protected. You should submit this worksheet as a supplement to your submission of test

data . This worksheet is not a substitute for submission of test data .

Property Mark (X) if Page Value or tx° Confiprovided number dential

(a) (b) (c) Mark (X)

N or E) (d)

Physical state of neat substance (s) -K- (1) (y~

Vapo

r @ T m

ess

a

ure

ture 17•

Yi q0. (7 T +~i•s• ~ °C S l G~ S0 Torr M

Density/ relative density 0-9101 g/cm3 M

Solubilit y

@ Temperature C

Solven t g/ L

Solubility in water ® Tem rature C I h so 1 N 6 I e

Melting temperature C

Boffin Boiling/sublimation tem ra ture 0 torr pressure C ~

Spectr a

Dissociation constant

Parti cle size distribution

Oct anol/water partiti on coefficient

Henry's Law constant

Volitaliza ti on from water

Volitali za tion from soil

pH concentration

Flammability

Explodabili ty

Adsorp tion/coefficien t

Other - Specify

vi s cosity Gt 2oQc IAIS C'St M

Auto- i nitioh tewperature 263 "G M

~3
p. 14

f~

Page 1 of 4 7~

First issue : 22/Jul/200 3

U-DONOR

MATERIAL SAFETY DATA SHEET

First issue : 22/Jul/2003

1 . PRODUCT AND COMPANY IDENTIFICATION

Product name : U-DONO R

General use : External donor for olefine polymerization

Manufacture r

Company name : UBE INDUSTRIES, LTD .

Address : Fine Chemicals Business Uni t

Seavans North Bldg, 1-2-1 Shibaura Minato-Ku

Tokyo 105-8449, Japa n

TEL : +81-3-5419-6175

FAX : +81-3-5419-6255

Emergency telephone number

CHEMTREC

United States : 800-424-9300 24 hours eve ryday

International : +1-703-527-3887 (Collect) 24 hours eve ryday

Nontransportation

Ube America, Inc

TEL : 212-813-8300 (9 am - 6 pm EST M-F)

UBE INDUSTRIES, LTD.

TEL : +81-3-5419-6175 (9 am - 6 pm Japan time M-F)

2. COMPOSITION / INFORMATION ON INGREDIENTS

Chemical name : Diethylamino-triethoxysilan e

Chemical formula : (C2H5)2N-Si-(OC2H5)3

Purity : min.98wt%

CAS No. : 35077-00- 0

OSHA hazardous ingredients (29 CFR 1910 . 1200) : This material is hazardous .

3 . HAZARDS IDENTIFICATIO N

Emergency overview : Colorless to pale yellow clear liquid . Combustible liquid and vapor .

Potential health effect s

Inhalation : Not available

Skin : Not available

~~
p. 15

Page 2 of 4

First issue : 22/Jul/2003

U-DONOR

Eye : Not availabl e

Ingestion : Not availabl e

Chronic toxicity / Carcinogenicity : Not available

Potential environmental effects : Not availabl e

4. FIRST AID MEASURE S

Inhalation : If adverse effects occur, remove to uncontaminated area . Give artificial respiration

if not breathing . Get immediate medical attention .

Skin : Wash skin with soap and water for at least 15 minutes while removing contaminated

clothing and shoes . Get medical attention, if needed . Thoroughly clean and dry

contaminated clothing and shoes before reuse .

Eye : Flush eyes with plenty of water for at least 15 minutes . Then get immediate medical

attention .

Ingestion : If vomiting occurs, keep head lower than hips to help prevent aspiration . Get

medical attention .

5 . FIRE FIGHTING MEASURES

Flammable properties

Flash point and method : 144F (62C) (TCC)

Flammable limits : Not available

Autoignition temperature : 505F (263C)

Specific hazards : Moderate fire hazard. Vapor/air mixtures are explosive above flash point

.

Extinguishing media : Regular dry chemical, carbon dioxide, water, regular foa m

Protective equipment : Full protective clothing and self-contained breathing apparatus .

6 . ACCIDENTAL RELEASE MEASURE S

Personal precautions : Avoid heat, flames, sparks and other sources of ignition . Stop leak if

possible without personal risk .

Environmental precautions : Prevent from contaminating soil or from entering sewage and

drainage systems and bodies of water .

Methods for cleaning up : Absorb with sand or other non-combustible material. Collect with

absorbent into suitable container.

7. HANDLING AND STORAG E

Handling : Do not breathe vapor . Avoid contact with eyes, skin and clothing .

Storage : Keep away from heat, sparks and flame . Keep containers tightly closed. Store in a

dry, cool and well-ventilated place .

/ S
p. 16

Page 3 of 4

First issue : 22/Jul/2003

U-DONOR

8 . EXPOSURE CONTROLS / PERSONAL PROTECTIO N

Engineering controls : Provide local exhaust ventilation system.

Personal protective equipment

Eye / face protection : Wear splash resistant safety goggles .

Skin protection : Wear appropriate chemical resistant clothing and gloves .

Respiratory protection : Wear suitable respiratory equipment .

Exposure guidelines : Not established

9. PHYSICAL AND CHEMICAL PROPERTIES

General information

Appearance : Colorless to pale yellow clear liquid

Odour : Slight amine odou r

Important health, safety and environmental information

Boiling point ! boiling range : 376F (191 C)

Flash point : 144F (62C) (TCC)

Vapor pressure : 5mmHg at 135 .5F (57.5C), 10mmHg at 158 .0F (70.OC), 50mmHg at

221 .9F (105.5C)

Relative density : 0 .9101 at 68F (20C)

Solubility

Water solubility : Insoluble (slight hydrolysis)

Fat solubility : Soluble in hexane, toluene

Viscosity : 1 .15cSt at 68F (20C )

Other informatio n

Auto-ignition temperature : 505F (263C)

10. STABILITY AND REACTIVITY

Stability : Stable at normal temperature and pressure.

Conditions to avoid : Avoid heat, flames, sparks and other sources of ignition .

Materials to avoid : Oxidizing agents, acids, bases and reactive agents

Hazardous decomposition products : Diethylamine, oxides of carbon, oxides of nitrogen

Hazardous polymerization : Will not polymerize

11 . TOXICOLOGICAL INFORMATIO N

Acute toxicity : LD50 500-1000mg/kg (oral rat)

Mutagenicity :

Ames test : Negative

Aa
p. 17

Page 4 of 4

First issue : 22/Jul/2003

U-DONOR

Chromosome aberration test : Negative

12. ECOLOGICAL INFORMATION

Not availabl e

13. DISPOSAL CONSIDERATION S

Comply with all federal, state and local regulations .

Do not dump this material into sewers, on the ground or into any body of water.

14. TRANSPORT INFORMATIO N

U.S. DOT 49 CFR 172 .10 1

Proper shipping name : Combustible liquid, n .o.s (U-DONOR)

ID number : NA1993

Hazard class : Combustible

Packing group : III

Maritime transport IMDG : No classification assigned

Air transport ICAO / IATA : No classification assigned

15. REGULATORY INFORMATIO N

OSHA status : This material is hazardous .

TSCA status : This product can be used solely for research and development because not

registered on TSCA inventory .

California Proposition 65 : Not listed

Please refer to any other regulations in U .S.

16. OTHER INFORMATIO N

This information is furnished without warranty, expressed or implied, except that it is accurate

to the best knowledge of UBE INDUSTRIES, LTD . The data on this sheet related only the

specific material designated herein. UBE INDUSTRIES, LTD . assumes no legal responsibility

for use or reliance upon these data .

/ ~ I
p. 18

~

,176

PAGE 1 OF 21 PAGES

SafePharm

Laboratories

DIETHYLAMIN TRIETHOXYSILAN:

ACUTE ORAL TOXICITY IN THE RAT

- ACUTE TOXIC CLASS METHOD

SPL PROJECT NUMBER: 1456/036

AUTHOR: D M Dreher

STUDY SPONSOR: TEST FACILITY:

UBE Industries, Ltd. Safepharm Laboratories Limited

Seavans North Building Shardlow Business Park

1-2-1, Shibaura London Road

Minato-ku Shardlow

TOKYO 105-8449 Derbyshire

JAPAN DE72 2GD

Telephone: +44 (0) 1332 792896

Facsimile: +44 (0) 1332 799018

1456-036.doclJS

i 9
P. 19

Lo

~

SPL PROJECT NUMBER : 1456/036 PAGE2

QUALITY ASSURANCE REPORT

This study type is classed as sho rt-term. The standard test method for this study type ("General

Study Plan" in OECD terminology) was reviewed for compliance once only on initial production .

Inspection of the routine and repetitive procedures that constitute the study is car ried out as a

continuous process designed to encompass the major phases at or about the time this study was in

progress .

This report has been audited by Safepharm Quality Assurance Unit, and is considered to be an

accurate account of the data generated and of the procedures followed .

In each case, the outcome of QA evaluation is reported to the Study Director and Management on

the day of evaluation . Audits of study documentation, and process inspections approp riate to the

type and schedule of this study were as follows :

07 June 2002 Standard Test Method Compli ance Audit

17 October 2002 Test Material Preparation

24 October 2002 Animal Preparation

08 October 2002 Dosing

17 October 2002 Assessment of Response

23 October 2002 Necropsy

§ 03 December 2002 Draft Repo rt Audit

§ Date of QA Signature Final Repo rt Audit

§ Evaluation specific to this study

DEC `. . .

. ~~~~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DATE : . . .7 .

.C ~QO~

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

For Safepharm Quality Assurance Unit*

*Authorised QA Signatures :

Head of Department: JR Pateman CBiol MIBiol DipRQA FRQA

Deputy Head of Department: JM Crowther MIScT MRQA

Senior Audit Staff: JV Johnson BSc MRQA; G Wren ONC MRQ A

1 q
p. 20

2

fib

SPL PROJECT NUMBER: 1456/036 PAGE3

GLP COMPLIANCE STATEMENT

The work described was performed in compliance with UK GLP standards (Schedule 1, Good

Laboratory Practice Regulations 1999 (SI 1999/3106)). These Regulations are in accordance with

GLP- standards published as OECD Principles on Good Laboratory Practice (revised 1997,

ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of

Directives 87/18/EEC (as amended by Directive 1999/11/EC) and 88/320/EEC (as amended by

Directive 1999/12/EC) .

These international standards are acceptable to the Regulatory agencies of the following

countries : Australia, Austria, Belgium, Canada, the Czech Republic, Denmark, Finland, France,

Germany, Greece, Hungary, Iceland, Ireland, Israel, Italy, Japan, Republic of Korea,

Luxembourg, Mexico, The Netherlands, New Zealand, Norway, Poland, Portugal, Slovenia,

Spain, Sweden, Switzerland, Turkey, the United Kingdom, and the United States of America.

This report fully and accurately reflects the procedures used and data generated .

1 1 DEC ^c 00%'

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DATE : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D M Dreher BSc (Hons)

Study Director

4020
p. 21

SPL PROJECT NUMBER: 1456/036 PAGE 4

CONTENTS

QUALITY ASSURANCE REPORT 2

GLP COMPLIANCE STATEMENT 3

CONTENTS 4

SUMMARY 5

1. INTRODUCTION 7

2. TEST MATERIAL AND EXPERIMENTAL PREPARATION 7

2 .0 Description, Identification and Storage Conditions 7

2.0 Preparation of Test Material 7

3. METHODS 8

3 .0 Animals and Animal Husbandry 8

3 .0 Procedure 8

3 .0 Evaluation of Data 9

4. ARCHIVES 10

1 . RESULTS 11

1 .1 Mortality Data 11

1 .2 Clinical Observations 11

1 .3 Bodyweight 11

1 .4 Necropsy 11

1 . CONCLUSION 12

Table 1 Mortality Data 13

Table 2 Individual Clinical Observations - 300 mg/kg 14

Table 3 Individual Clinical Observations - 2000 mg/kg 15

Table 4 Individual Bodyweights and Weekly Bodyweight Changes - 300 mg/kg 16

Table 5 Individual Bodyweights and Weekly Bodyweight Changes - 2000 mg/kg 17

Table 6 Individual Necropsy Findings - 300 mg/kg 18

Table 7 Individual Necropsy Findings - 2000 mg/kg 19

Appendix 1 Test Procedure with a Starting Dose of 300 mg/kg Bodyweight 20

Appendix 2 Statement of GLP Compliance in Accordance with Directive 88/320/EEC 21
p. 22

A

SPL PROJECT NUMBER: 1456/036 PAGE 5 716

TRIETHOXYSILAN :

ACUTE ORAL TOXICITY IN THE RAT

- ACUTE TOXIC CLASS METHOD

SUMMARY

Introduction. The study was performed to assess the acute oral toxicity of the test material

following a single oral administration in the Sprague-Dawley CD strain rat. The method was

designed to meet the requirements of the following :

■ OECD Guidelines for the Testing of Chemicals No . 423 "Acute Oral Toxicity - Acute Toxic

Class Method" (adopted 17 December 2001 )

Method. A group of three fasted females was treated with the test material at a dose level of

300 mg/kg bodyweight. Based on the results from this dose level further groups of fasted females

were treated at dose levels of 300 and 2000 mg/kg bodyweight. Dosing was performed

sequentially.

The test material was administered orally undiluted at a dose level of 2000 mg/kg and as a

solution in arachis oil BP at a dose level of 300 mg/kg . Clinical signs and bodyweight

development were monitored during the study . All animals were subjected to gross necropsy .

Mortality. Two animals treated with 2000 mg/kg were found dead one day after dosing . There

were no deaths noted at a dose level of 300 mg/kg .

Clinical Observations. Signs of systemic toxicity noted in animals treated with 2000 mg/kg were

hunched posture, lethargy, ataxia, decreased respiratory rate, diarrhoea, ptosis and pilo-erection.

There were no signs of systemic toxicity noted at a dose level of 300 mg/kg .

Bodyweight. The surviving animals showed expected gains in bodyweight over the study period .

Necropsy. Abnormalities noted at necropsy of animals that died during the study were

haemorrhagic lungs, dark liver, dark kidneys, haemorrhage of the gastric mucosa, non-glandular

epithelium of the stomach and small intestine. Stomach adhering to the liver was noted in the

animal treated with 2000 mg/kg that was killed at the end of the study . No abnormalities were

noted at necropsy of animals treated with 300 mg/kg .

~
p. 23

z

~d

SPL PROJECT NUMBER : 1456/036 PAGE 6

Conclusion. The acute oral median lethal dose (LD50) of the test mate rial in the female

Sprague-Dawley CD strain rat was estimated to be in the range of 500 - 1000 mg/kg bodyweight.

The test material was classified as HARMFUL and the symbol "Xn" and risk phrase

R 22 "HARMFUL IF SWALLOWED" are required according to EU labelling regulations

Commission Directive 2001/59/EC.

~3
p. 24

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SPL PROJECT NUMBER : 1456/036 PAGE7

DIETHYLAMIN TRIETHOXYSILAN :

ACUTE ORAL TOXICITY IN THE RAT

- ACUTE TOXIC CLASS METHO D

1 . INTRODUCTION

The study was performed to assess the acute oral toxicity of the test material following a single

oral administration in the Sprague-Dawley CD strain rat. The method was designed to meet the

requirements of the following :

■ OECD Guidelines for the Testing of Chemicals No . 423 "Acute Oral Toxicity - Acute Toxic

Class Method" (adopted 17 December 2001 )

The rat was selected for this study as it is a readily available rodent species, historically used in

safety evaluation studies, and is acceptable to appropriate regulatory authorities. The oral route

was selected as the most appropriate route of exposure and the results are believed to be of value

in predicting the likely toxicity of the test material to man .

The study was performed between 10 October 2002 and 04 November 2002 .

2. TEST MATERIAL AND EXPERIMENTAL PREPARATION

2.1 Description, Identification and Storage Conditions

Sponsor's identification : DEATES

Description : clear colourless liquid

Batch number : U020920

Date received : 27 September 2002

Storage conditions : room temperature in the dark

Data relating to the identity, purity and stability of the test material are the responsibility of the

Sponsor.

2.2 Preparation of Test Material

For the purpose of the 2000 mg/kg dose level the test material was used as supplied . The specific

gravity was determined and used to calculate the appropriate dose volume for the required dose

level. For the purpose of the 300 mg/kg dose level the test material was freshly prepared, as

required, as a solution at the appropriate concentration in arachis oil BP . Arachis oil BP was used

because the test material did not dissolve in distilled water .
p. 25

~h

SPL PROJECT NUMBER: 1456/036 PAGE8

Determination by analysis of the concentration, homogeneity and stability of the test material

preparations was not appropriate because it was not specified in the Study Plan and is not a

requirement of the Test Guideline .

3 . METHODS

3.1 Animals and Animal Husbandry

Female Sprague-Dawley CD (Cr1 : CD® (SD) IGS BR) strain rats were supplied by Charles River

(UK) Ltd, Margate, Kent, UK . On receipt the animals were randomly allocated to cages . The

animals were nulliparous and non-pregnant . After an acclimatisation period of at least five days

the animals were selected at random and given a number unique within the study by indelible

ink-marking on the tail and a number written on a cage card . At the start of the study the animals

were eight to twelve weeks of age . The bodyweights fell within an interval of + 20% of the mean

initial bodyweight of the first treated group .

The animals were housed in groups of three in suspended solid-floor polypropylene cages

furnished with woodflakes . With the exception of an overnight fast immediately before dosing

and for approximately three to four hours after dosing, free access to mains drinking water and

food (Certified Rat and Mouse Diet (Code 5LF2) supplied by International Product Supplies

Limited, Wellingborough, Northants, UK) was allowed throughout the study . The diet, drinking

water and bedding were routinely analysed and were considered not to contain any contaminants

that would reasonably be expected to affect the purpose or integrity of the study .

The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70%

respectively. Any occasional deviations from these targets were considered not to have affected

the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per

hour and the lighting was controlled by a time switch to give twelve hours continuous light (06 :00

to 18 :00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to

contain any contaminant of a level that might have affected the purpose or integrity of the study .

3.2 Procedure

In the absence of data regarding the toxicity of the test material, 300 mg/kg was chosen as the

starting dose .

4y 5
p. 26

2-7

~b

SPL PROJECT NUMBER : 1456/036 PAGE 9

Groups of fasted animals were treated as follows :

Dose Level Concentration Specific Dose Volume Number of Rats

(mg/kg) (mg/ml) Gravity (mUkg) Female

300 30 - 10 3

2000 - 0.905 2.21 3

300 30 - 10 3

All animals were dosed once only by gavage, using a metal cannula attached to a graduated

syringe. The volume administered to each animal was calculated according to the fasted

bodyweight at the time of dosing. Treatment of animals was sequential. Sufficient time was

allowed between each group and each dose level to confirm the survival of the previously dosed

animals.

The animals were observed for deaths or overt signs of toxicity '/2, 1, 2 and 4 hours after dosing

and subsequently once daily for up to fourteen days.

Individual bodyweights were recorded prior to dosing and seven and fourteen days after treatment

or at death.

At the end of the observation period the surviving animals were killed by cervical dislocation . All

animals were subjected to gross pathological examination . This consisted of an external

examination and opening of the abdominal and thoracic cavities for examination of major organs .

The appearance of any macroscopic abnormalities was recorded . No tissues were retained .

The sequence of dosing may not always follow the Test Guideline as shown in the schematic

diagram in Appendix 1 . It is Company Policy to minimise the number of animals used on each

study in accordance with UK Government Home Office guidelines . The sequence of testing does

not affect the final classification of the test material .

3.3 Evaluation of Data

Data evaluations included the relationship, if any, between the exposure of the animal to the test

material and the incidence and severity of all abnormalities including behavioural and clinical

observations, gross lesions, bodyweight changes, mortality and any other toxicological effects .

Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the

test material was made as shown in the schematic diagram in Appendix 1 .

Z~
p. 27

28

6

SPL PROJECT NUMBER: 1456/036 PAGE 10

The results were evaluated according to Commission Directive 2001/59/EC for classification and

labelling of dangerous substances and preparations .

4. ARCHIVES

Unless instructed otherwise by the Sponsor, all original data and the final report will be retained

in the Safepharm archives for five years, after which instructions will be sought as to further

retention or disposal.

27
p. 28

2 ~

SPL PROJECT NUMBER: 1456/036 PAGE 11

5. RESULTS

5.1 Mortality Data

Individual mortality data are given in Table 1 .

Two animals treated with 2000 mg/kg were found dead one day after dosing . There were no

deaths noted at a dose level of 300 mg/kg .

5.2 Clinical Observations

Individual clinical observations are given in Table 2 and Table 3 .

Signs of systemic toxicity noted in animals treated with 2000 mg/kg were hunched posture,

lethargy, ataxia, decreased respiratory rate, diarrhoea, ptosis and pilo-erection . There were no

signs of systemic toxicity noted at a dose level of 300 mg/kg .

5.3 Bodyweight

Individual bodyweights and weekly bodyweight changes are given in Table 4 and Table 5 .

The surviving animals showed expected gains in bodyweight over the study period .

5.4 Necropsy

Individual necropsy findings are given in Table 6 and Table 7 .

Abnormalities noted at necropsy of animals that died during the study were haemorrhagic lungs,

dark liver, dark kidneys, haemorrhage of the gastric mucosa, non-glandular epithelium of the

stomach and small intestine. Stomach adhering to the liver was noted in the animal treated with

2000 mg/kg that was killed at the end of the study. No abnormalities were noted at necropsy of

animals treated with 300 mg/kg .

t~
p. 29

3 76

SPL PROJECT NUMBER : 1456/036 PAGE 12

6. CONCLUSION

The acute oral median lethal dose (LD50) of the test material in the female Sprague-Dawley CD

strain rat was estimated to be in the r ange of 500 - 1000 mg/kg bodyweight .

The test material was classified as HARMFUL and the symbol "Xn" and risk phrase

R 22 "HARMFUL IF SWALLOWED" are required according to EU labelling regulations

Commission Directive 2001/59/EC.

~
p. 30

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p. 38

SPL PROJECT NUMBER: 1456/036 PAGE 21

Appendix 2 Statement of GLP Compliance in Accordance with Directive 88/320/EEC

4 a

o z

AE J► v

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT

OF THE UNITED KINGDOM

GOOD LABORATORY PRACTiCE

srATEMENT OF COMPLIANCE

IN ACCORDANCE WITH DIRECTIVE 88l320 EEC

LABORATORY TEST TYPE

S:ifePl Srm Ltiboratories Ltd Aealy~ Chemistry

Sbardlow Business Park Eavironmental Fate

London Road ravireumental Toxicity

Shardlow MataFaicity

Derbyshire Pttys/Cbem Tats

DE72 2GD Toxicology

DATE OF INSPECTION

28 February 2000

A general inspection for compliance with the Principles of Good Laboratory Practice

was carri ed out at the above laboratory as pan of UK GLP Compliance Programme .

At the time of the inspection no deviations were found of sufficient magnitude to affect

the validity of non-clinical studies performed at these facilities.

1(5~-

2ts~tf~e o

Dr. Roger G. Alexander

Head . UK GLP Monitoring Authority

3b
p. 39

4

9b

SAFEPHARM LABORATORIES LTD

DIETHYLAMIN TRIETHOXYSILAN :

ACUTE ORAL TOXICITY IN THE RAT

- ACUTE TOXIC CLASS METHOD

SPL PROJECT NUMBER: 1456/036

I verify that this is an exact copy of the original report which is located in the Archives of

Safepharm Laboratories Ltd., Derby, UK.

~. . . . . . . . . . . . . . . DATE: . . . . . . . . . . . . . . 1

. .9.

.DEC 20OZ. . . . . . . . . . . . . . . . . . . . . .

D M Dreher BSc (Hons)

Study Director

37
p. 40

41

16

Mutagenicity Test o f

Diethyla minotriethoxysilane

by using Microorganisms

September 30, 2002

UBE SCIENTIFIC ANALYSIS LABORATORY, INC.

_ ~I-/
p. 41

42/

USA-R-02133

PURPOSE

Purpose of this test is to evaluate the mutagenicity of diethylaminotriethoxysilane by the

• microbial mutagenicity test using Salmonella typhimurium and Escherichia coli.

MATERIALS & METHODS

Test substance ;

Diethylaminotriethoxysilane was supplied by UBE INDUSTRIES, LTD .

Bacterial strains;

Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli

WP2uvrA were supplied by Dr . T. Matsushima, Japan Bioassay Laboratory, Japan Industrial Safety And Health Association, Hadano-city, Kanagawa .

Bacterial cultures were freshly prepared in Oxoid nutrient broth #2, by inoculating

bacteria from frozen stock cultures (kept at -80°C). 30μL stock solution was inoculated

into the 15mL broth and incubated for 10 hours at 37°C and 120rpm using rotary shaker .

S9 and S9 mix;

S9, post-mitochondrial supernatant of rat-liver homogenate, was obtained from Oriental

Yeast Corporation, Tokyo (Japan). The S9 was prepared by a slight modification of the

method described by Ames, McCann, and Yamazaki; 5,6-Benzoflavone and phenobarbital

were used as an inducer of drug-metabolizing enzyme system .

The S9 mix contained 4mM NADPH, 4mM NADH, 5mM Glucose-6-Phosphate, 8mM

MgCl,, 33mM KC1, 100mM sodium phosphate buffer(pH7 .4) and 10% S9(50μL S9 per

plate).

Mutagenicity assay ;

The mutagenicity was assayed from a maximum level of 5000μg test substance/plate by

the preincubation method (37°C, 20min) [3] with or without metabolic activation system .

When the test substance showed toxicity, the highest dose was the one where a toxicity was

observed.

A compound is regarded as mutagenic when it induced the revertant colonies dosedependently and the number of revertants is more than twice that of the control .

RESULTS

Mutation test result of diethylaminotriethoxysilane are shown in Appendix 1 and 2 .

The tested compound did not increase revertant colonies when tested up to 5000μg/plate

with or without metabolic activation system.

CONCLUSION

Diethylaminotriethoxysilane did not show mutagenicity in Salmonella typhimurium strains

TA98, TA100, TA1535 and TA1537 , and Escherichia coli . WP2uvrA by the preincubation

method with or without metabolic activation system .

1

~1
p. 42

46

USA-R-02133

REFERENCES

[1] Ames, B. N ., McCann, J. and Yamazaki, E. (1975). Method for detecting carcinogens

and mutagens with the Salmonella /mammalian microsome mutagenicity test . Mutation

Res., 31, 347-364.

[2] Matsushima, T., Sawamura, M ., Hara, K. and Sugimura, T. A safe substitute for polychlorinated biphenyls as an inducer of metabolic activation system .

In: de Serres, F. J., Fouts., J. R., Bend, J.R. and Philpot, R. M. (Eds), (1976). In vitro Metabolic Activation in Mutagenesis Testing, Elsevier, Amsterdam, pp . 85-88

[3] Matsushima, T., Sugimura, T., Nagano, M ., Yahagi, T., Shirai, A. and Sawamura,M .

Factors modulating mutagenicity in microbial test, in: Norpoth, K. H. and Garner, R. C.

(Eds), (1980). Short-Term Test System for Detecting Carcinogens, Springer-Verlag, Berlin,

pp. 273-285

2

11Z
p.43

USA-R-02133

76

Report of Mutagenicity Test using Microorganis m

1. General Items

(1) Test substance

Name of the new chemical sub- Diethylaminotriethoxysilane

stance

Other name DEATES

Structural formula or rational for- (C2H5)2NSi(OC2H5 )3 mula (Manufacturing process) C1 oH25NO3Si

Supplier of the new chemical sub- UBE INDUSTRIES, LTD .

stance tested

Lot No. (Batch No.) DEATES-1

Purity of the new chemical sub- 97.6%

stance tested

Name and concentration of impuri- Tetraethoxysilane 1 .6%

ties unknownimpurities 0.8%

Physicochemical properties of the Molecular weight 235.40

new chemical substance Appearance at ordinary colorless clear liquid

temperature

Stability stable under

nitrogen atmosphere

in shade

Melting point

Boiling point 75°C/0.5mmHg

Vapor pressur e

Partition coefficient -

Water decomposed

Degree of with water

solubility DMSO Insolubl e

Acetone -

(2) Solvent control substanc e

Name Obtained from (Lot No.) -Purity etc.

Dimethylsulfoxide KANTO CHEMICAL CO ., for UV Spectroscope

(DMSO) INC. 311F1104 99.7%

(3) Positive control substanc e

Name Obtainedfrom (Lot No.) Purityetc.

2-(2-Furyl)-3-(5-nitoro-2- WAKO PURE CHEMICAL INDUSTRIES, LTD 99.Owt%

fur 1 acrylamide (AF-2) SEL1402

Sodium azide NaN3 NACALAI TESQUE CO ., INC. 5F9579 99.9wt%

9-Aminoacridine 9-AA SIGMA CHEMICAL COMPANY 106F06681 97 .Owt%

2-Aminoanthracen 2-AA NACALAI TESQUE CO., INC. (M4M9899) 96.7wt%

3

107,7)
p.44

96

USA-R-0213 3

2. Tester Strains

(1) P ro curement

Name of tested Date obtained Obtained from

strains

TA98 June 29, 1995 Dr. T. Matsushima, Japan Bioassay Laboratory,

TA100 June 29, 1995 Japan Industrial Safety And Association, HadanoTA1535 June 29, 1995 City, Kanagawa.

TA1537 June 29, 1995

WP2uvrA June 29, 1995

(2) Storage

Storage temperature -80°C

Bacteria] suspension 0.8mi

Composition DMSO 0.07m1

Others( ~

3. S9mix

(1) Source of S9

Manufacturer ORIENTAL YEAST CO., LTD.

Purchase Prepared on June 7, 2002

Purchased August 29, 2002

Lot No. 02060703

(2) Storage of S9

Storage temperature 80°C Storage apparatus SANYO MEDICAL

MDF192AT

(3) Pre aration of S9

Animal used Inducing substance

Species, Strain Rat, Sprague- Dawley Name Phenobarbital(PB)and

5,6-Benzoflavone BF

Sex Male Injection Intra eritoneall

Age 7 weeks Amount PB 4 time 30-60

Body weight 217.8 ± 9.7g (mg/kg-body weight) BF 1 time 80

Mean .4 S.D.

(4) Composition of S9mix

Constituents Amount in Constituents Amount in lml S9mix

S9 0

lml S9mix

.1m1 NADPH 4 mol

MgC12 8 mol NADH 4 mol

KCL 33 mol Na-phosphate 100μmo1

Glucose-6-phosphate 5 mol Buffer (pH7 .4)

4

~
p.45

~

USA-R-0213 3

4. Preparation of the Solution of the Test Substance

Solvent used DMSO

Preparation Test substance was suspended in anhydrous DMSO.

5. Conditions of Pre-cultu re

Name of nutrient broth OXOID, Nutrientbroth #2

and manufacturer

Period of pre-culture 10 hours

6. Minimum Glucose Agar Plate

Purchase Manufacturer ORIENTAL YEAST CO ., LTD.

Prepared on Lot No.) May 14, 2002 ANI290ER

7. Sterility Test

Bacterial growth

Test substance solution No

S9mix No

8. Test Method

(1) Test method

Preincubation method

(2) Test condition

Preincubation method

Bacterialsuspension 0.1m]

Test substance solution 0.05m1

Composition Na-phosphate buffer or S9mix(in case 0 .5m1

of metabolic activation method)

Top agarsolution 2,0m]

Others

Pre- Temperature 37'C

incubation Time 20min

Incubation Temperature 370C

Time 48hours

5

~-S
p.46

USA-R-02133 ~

9. Test results

(1) Test results are reported on the appendix 1 and 2 .

(2) Judgement of the results

Judgement Negative

Reason for judgement and referential matters:

Diethylaminotriethoxysilane was tested on Salmonella typhimurium TA98, TA100,

TA1535 and TA1537, and E.coli WP2uvrA with or without metabolic activation by the preincubation method.

Diethylaminotriethoxysilane did not increase revertant colonies up to 5000μg/plate with or

without metabolic activation.

10. Storage and Retrieval of Records and Samples

The all records (master plan, protocol, and raw-data etc .) and test substance are kept for 10

years.

11. Others

Name UBE Scientific Analysis Laboratory, Inc

Testing

Institution Address 1978-6, Aza-okinoyama Ooaza-kogushi, Ube-city

Yamaguchi-pref., Japan

Tel 0836 (31) 5809

Test period From September 3, 2002 to September 30, 2002

6

_ ~ ~i
p.47

~b

USA-R-02133

12. Confirmation

This test (Study code number USA-R-02133) was conducted according to "Standards to be

observed by "Mutagenicity Testing Institutions " of "Notification of Ministry of the Labor, Japan, No.13, March 29, 2000" and "Standards to be observed by "Mutagenicity Test Procedures

(Using Microorganisms) " of Notification of Ministry of the Labor, Japan, No.77, September 1, 1988 and No.67 (revised), June 2, 1997. "

We, the undersigned, hereby declare that this report provides an accurate and faithful record

of the results obtained, and a summary of protocol .

Study Director ~ _9Z30/200Z

Jun-ichi Hashimoto

7

q7
p. 48

4ry`

USA-R-0213 3

Appendix 1

Test Results(Dose-determination test)

Name of Test Substance : DEATES

Test period From September 4, 2002 to September 6, 2002

With(+) or Test substance Number of revertants(Number of colonies/plate )

Without(-) concentration Base-pair substitution type Frame-shift type

S9 mix (f.c glplate) TA100 TA1535 Wp2uvrA TA98 TA1537

Solvent control 93 (87) 8 (10) 39 (37) 16 (15) 11 (11)

81 11 34 13 10

4 .9 110 (110) 13 (11) 41 (39) 19 (17) 9 (7)

109 9 36 14 5

19 .5 105 (110) 9 (7) 40 (39) 14 (15) 5 (5)

114 4 37 16 5

-S9 mix 78.1 76 (93) 13 (11) 21 (23) 24 (16) 8 (9)

109 8 25 7 9

313 99 (92) 10 (12) 36 (34) 13 (14) 7 (11)

85 13 31 14 14

1250 85 (85) 15 (15) 39 (42) 19 (15) 11 (8)

84 14 44 10 5

5000 108 (102) 7 (7) 42 (42) 21 (21) 9 (9)

95 7 42 20 8

Solvent control 128 (118) 9 (12) 35 (33) 31 (32) 14 (14)

107 14 31 32 13

4 .9 131 (134) 15 (15) 41 (38) 28 (26) 10 (12)

137 15 35 24 14

19.5 125 (124) 4 (8) 34 (33) 29 (27) 14 (14)

122 11 31 25 1 3

+S9 mix 78.1 130 (123) 9 (10) 42 (39) 30 (24) 9 (9)

116 10 35 18 9

313 122 (124) 10 (12) 40 (33) 19 (28) 19 (15)

126 14 26 36 11

1250 107 (111) 8 (12) 35 (35) 28 (29) 14 (13)

114 15 35 30 1 1

5000 117 (122) 16 (14) 28 (31) 32 (26) 11 (13)

126 11 34 20 15

Positive Name AF-2 1) NaN3 2) AF-2 AF-2 9-AA 3)

control not concentration

requiri ng ( ,u 9/plate) 0 .01 0 .5 0.01 0.1 80. 0

S9 mix Number of 751 (770) 348 (339) 239 (240) 632 (632) 338 (335)

colonies/plate 788 330 241 632 331

Positive Name 2-AA 4) 2-AA 2-AA 2-AA 2-AA

control concentration

requiring ( /[ 9/plate) 1 .0 2 .0 10.0 0 .5 2. 0

S9 mix Number of 1491 (1488) 182 (174) 959 (1028) 678 (674) 376 (371)

colonies/plate 1485 166 1096 670 365

1)AF-2 :2-(2-Furyl)-3-(5-n i tro-2-furyl)acryl amide

2)NaN3 : Sodiumazide

3)9-AA : 9-Aminoacridine

4)2-AA : 2-Aminoanthracene

[Notes]

1 . When toxicity was obse rved, "*" was placed to the right of the number of the revertants .

2. When precipitation was obse rved, "t" was placed to the right of the test substance concentration .

3 . The average number of revertants in each concentration was showed in the () .

qS
p. 49

7G

USA-R-0313 3

Appendix 2

Test Results (Mutagenicity test)

Name of Test Substance : DEATES

Test period FromSeptember 10, 2002 to September 12, 2002

With(+) or Test substance Number of revertants(Number of colonies/plate)

Without(-) concentration Base-pair substitution type Frame-shift type

S9 mix (μ g/plate) TA100 TA1535 WP2uvrA TA98 TA1537

Solvent control 80 (91) 8 (7) 35 (33) 13 (16) 8 (11)

101 5 31 19 13

156 100 (93) 3 (3) 25 (31) 18 (14) 14 (11)

86 3 36 9 8

313 118 (118) 8 (6) 30 (27) 24 (25) 7 (9)

118 4 24 25 11

-S9 mix 625 104 (96) 9 (8) 35 (40) 18 (19) 7 (8)

88 7 45 20 8

1250 91 (94) 8 (9) 40 (37) 18 (15) 5 (5)

96 10 33 11 4

2500 93 (93) 10 (10) 37 (36) 16 (17) 4 (4)

92 9 34 18 4

5000 98 (92) 8 (10) 51 (48) 12 (15) 14 (9)

86 11 44 18 3

Solvent control 109 (107) 10 (11) 36 (40) 20 (19) 11 (12)

104 11 44 18 13

156 102 (100) 13 (11) 33 (30) 23 (25) 9 (10)

98 8 26 26 11

313 114 (107) 8 (8) 49 (43) 25 (28) 7 (9)

100 8 36 30 10

+S9 mix 625 135 (123) 9 (9) 41 (41) 23 (27) 10 (11)

110 9 41 31 11

1250 113 (111) 13 (11) 37 (39) 20 (25) 13 (11)

109 9 41 29 8

2500 106 (106) 8 (9) 47 (48) 29 (25) 13 (13)

106 9 48 20 1 2

5000 108 (112) 9 (7) 37 (42) 29 (29) 13 (14)

115 5 47 29 15

Positive Name AF-2 1) NaN3 2) AF-2 AF-2 9-AA 3)

control not concentration

requiring (u gfplate) 0 .01 0.5 0.01 0 .1 80 . 0

S9 mix Number of 784 (780) 289 (298) 245 (246) 628 (632) 364 (381)

colonies/plate 775 307 246 635 398

Positive Name 2-AA 4) 2-AA 2-AA. 2-AA 2-AA

control concentration

requiring ((L g/plate) 1 .0 2.0 10.0 0 .5 2 . 0

S9 mix Number of 1496 (1496) 191 (183) 936 (972) 587 (597) 352 (357)

colonies/plate 1496 175 1008 607 361

1)AF-2 :2-(2-Furyl)-3-(5-nitro-2-furyl)acryl amid e

2)NaN3 :Sodiumazide

3)9-AA :9-Aminoacridine

4)2-AA :2-Aminoanthracene

[Notes ]

1 . When toxicity was observed, was placed to the right of the number of the revertants.

2 . When precipitation was observed, "t" was placed to the right of the test substance concentration.

3 . The average number of revertants in each concentration was showed in the O .

LK
p.50

96

USA-R-0213 3

Dose-response curves (Dose-determination test)

TA100 -S9 mix

250

200

0. 150

E 100

50

0

0 4.9 19.5 78.1 313 1250 5000

Dose( μ g/plate)

TA100 +S9 mix

250

200

1S0

E 100

cx

50

0

0 4.9 19.5 78.1 313 1250 5000

Dose( μ g/plate)

100 TA1535 -S9 mix

8 0

60

o

t 40

t

a:

20

0

0 4.9 19.5 78.1 313 1250 5000

Dose( u g/plate)

100 TA1535 +S9 mix

80

60

~

40

tx

20

0

0 4.9 19.5 78.1 313 1250 5000

Dose( μ g/plate)

S0
p. 51

USA-R-02133

Dose-response curves (Dose-determination test )

100 WP2uvrA -S9 mix

80

60

40

C4

20

0

0 4.9 19.5 78.1 313 1250 5000

Dose( ,u g/plate)

100 WP2uvrA +S9 mix

8 0

N 60

40

v

a

20

0

0 4.9 19.5 78.1 313 1250 5000

Dose( μ g/plate )

100 TA98 -S9 mix

80

~

m

60

e

40

20

0

0 4.9 19.5 78.1 313 1250 5000

Dose( μ g/plate)

100 TA98 +S9 mix

80

~

° 60

a

~

i 40

20

0

0 4.9 19.5 78.1 313 1250 5000

Dose( u g/plate)

vI
p. 52

s.~

USA-R-02133

Dose-response curves (Dose-determination test)

TA1537 -S9 mix

100

80

~

60

~

d 40

20

0

0 4.9 19.5 78.1 313 1250 5000

Dose( μ g/plate)

100 TA1537 +S9 mix

80

~

°7 60

JN

40

20

0

0 4.9 19.5 78.1 313 1250 5000

Dose( μ g/plate)

S2
p. 53

S.f~ .

USA-R-0313 3

Dose-response curves (Mutagenicity test)

250 TA100 -S9 mix

200

0. 150

ai 100

c4

50

0

0 156 313 625 1250 2500 5000

Dose( μ g/plate)

250 TA100 +S9 mix

200

150

100

5 0

0

0 156 313 625 1250 2500 5000

Dose( u g/plate)

100 TA1535 -S9 mix

80

60

40

2 0

0

0 156 313 625 1250 2500 5000

Dose( μ g/plate)

100 TA1535 +S9 mix

8 0

60

40

20

0

0 156 313 625 1250 2500 5000

Dose( ,u g/plate)

53
p.54 5/

USA-R-03133 ~6

Dose-response curves (Mutagenicity test)

100 WP2uvrA -S9 mix

8 0

m

°• 60

~

~ 40

20

0

0 156 313 625 1250 2500 5000

Dose( ,u g/plate )

100 WP2uvrA +S9 mix

8 0

R

60

40

20

0

0 156 313 625 1250 2500 5000

Dose( m g/plate)

100 TA98 -S9 mix

8 0

60

m

40

c4

20

0

0 156 313 625 1250 2500 5000

Dose( μ g/plate)

100 TA98 +S9 mix

80

~

° 60

R

d 40

2 0

0

0 156 313 625 1250 2500 5000

Dose( g g/plate)

45V
p. 55

USA-R-03133 76

Dose-response curves (Mutagenicity test)

100 TA1537 -S9 mix

8 0

Sz 60

u 40

20

0

0 156 313 625 1250 2500 5000

Dose( g g/plate)

100 TA1537 +S9 mix

8 0

°- 60

"U 40

2 0

0

0 156 313 625 1250 2500 5000

Dose( μ g/plate)

~5
p. 56

Quality Assurance Certificate

1 . Kind of test

Mutagenicity Test using Microorganisms (study code No .USA-R-02133)

2.Name of the test substanc e

Diethylaminotriethoxysilane

I certify that the methods and procedures used in the test are described precisely in the final

report, that test was precisely performed according to the protocol and standards of procedure,

and that the reported results reflect the raw-data accurately.

The circumstances of audits and inspections are as follows.

Account

Date Conducted of Object Date Reported to Date Reported to

audits or the Study Director the Administrator

inspections

September3, 2002 Protocol September3, 2002 September3, 2002

September 6, 2002 Circumstances of September 6, 2002 September 6, 2002

performance of the

test (Observation)

September 10, 2002 Circumstances of September 10, 2002 September 10, 2002

performance of the

test (Test Procedure)

September25, 2002 Final Report (draft) September25, 2002 September2S, 2002

September30, 2002 Final Report September30, 2002 September30, 2002

Individual responsible for quality assurance

Organizational assignment UBE SCIENTIFIC ANALYSIS

LABORATORY, INC.

Title Quality Assurance Unit

Name

*'fc~~i ~o~t~c~

Date

a / Zoo
p. 57

7

Chromosome Aberration Test Of

Diethylaminotriethoxysilane Using Cultured

Mammalian Cells

November 22, 200 2

UBE SCIENTIFIC ANALYSIS LABORATORY, INC.

57
p. 58

GU STATEMENT

UBE SCIENTIFIC ANALYSIS LABORATORY, INC.

1978-6, Okinoyama, Kogushi, Ube, Yamaguchi 755-8633

TITLE

Chromosome aberration test of Diethylaminotriethoxysilane using cultured mammalian

cells

SPONSOR

UBE INDUSTRIES, LTD chemicals & Plastics

8-1,Goi-Minamikaigan, Ichihara, Chiba 290-0045

STUDY CODE NUMBER

USA-R-02131

I, the undersigned, hereby declare that this study was conducted in compliance with

"Standards to be observed by Testing Institutions for Toxicity Investigations" (Japan's

Ministry of Labor, No .13, March 29, 2000), and "Concerning Testing Facilities Stipulated in

Article 4 of the Order Prescribing the Items of the Test Related to the New Chemical

Substances and of the Toxicity Investigations Related to the Designated Chemical

Substances" (Notification No . 268 of the Planning and Coordination Bureau, Environmental

Agency (EA), No . 41 of the Environmental Health Bureau, Ministry of Health and Welfare

(MHW) & No .1 (2000 • 02 • 14) of the Basic Industries Bureau, Ministry of International Trade

and Industry (MITI), revised on March 1, 2000) .

Study Directo r

Date 2- 2- 00Z,

1.-~
p.59 6

'77,

Quality Assurance Certificate

1 . Kind of test

Chromosomal aberration test using cultured mammalian cells

(study code No . USA-R-02131 )

2.Name of the test substance

Diethylaminotriethoxysilane

I certify that the methods and procedures used in the test are described precisely in the fina l

report, that test was precisely performed according to the protocol and standards of procedure,

and that the reported results reflect the raw-data accurately.

The circumstances of audits and inspections are as follows .

Account

Date Conducted Object Date Reported to Date Reported to

of audits or the Study Director the Administrator

inspections

August 20, 2002 Protocol August 20, 2002 August 20, 2002

September 17, 2002 Circumstances of September 17, 2002 September 17, 2002

performance of th e

test (Test Procedure)

October 9, 2002 Circumstances of October 9, 2002 October 9, 2002

performance of th e

test (Observation)

October 22, 2002 Circumstances of October 22, 2002 October 22, 2002

performance of th e

test (Observation )

November2l, 2002 Final Report (draft) November2l, 2002 November2l, 2002

November22, 2002 Final Report November22, 2002 November22, 2002

Individual responsible for quality assuranc e

Organizational assignment UBE SCIENTIFIC ANALYSIS

LABORATORY, INC.

Title Quality Assurance Unit

Name

Date

A-1 /Z6o Z

~~ ~y
p.60

USA-R-0213 1

TITLE

Chromosome aberration test of Diethylaminotriethoxysilane using cultured mammalian

cells

STUDY CODE NUMBER

USA-R-02131

PURPOSE OF STUDY

The purpose of study is to identify agents that cause structural chromosome aberrations in

Chinese hamster lung fibroblasts (CHL) cells.

TESTING METHO D

This study was conducted in accordance with "Standard for Chromosome aberration test

using cultured mammalian cells" (Notification No . 652 of Labour Standard Bureau,

Ministry of Labour (MOL), September 29, 1997), and "Notification on Partial Revision of

Testing Methods Relating to the New Chemical Substances" (Notification No. 287 of the

Planning and Coordination Bureau, Environmental Agency (EA), No. 127 of

Environmental Health Bureau, Ministry of Health and Welfare (MHW) & No.2 (1997• 10 -

31) of the Basic Industries Bureau, Ministry of International Trade and Industry (MITI),

October 31, 1997).

SPONSOR

UBE INDUSTRIES, LTD.

Seavans North Bldg, 1 -2-1,Shibaura,Minato-Ku,Tokyo 105-8449

TESTING FACILITY

UBE SCIENTIFIC ANALYSIS LABORATORY, INC.

1978-6, Okinoyama, Kogushi, Ube, Yamaguchi 755-8633

PERIOD OF STUDY

Commencement of Study: August 20, 2002

Initiation of Preliminary Test: September 17, 2002

Completion of Observation : November 12, 2002

Completion of Study : November 22, 2002

GLP COMPLIANCE

This study was conducted in compliance with "Standards to be observed by Testing

Institutions for Toxicity Investigations" (Japan's Ministry of Labor, No.13, March 29,

2000) and "Concerning Testing Facilities Stipulated in Article 4 of the Order Prescribing

the Items of the Test Related to the New Chemical Substances and of the Toxicity
p. 61 62

USA-R-02131 7

Investigations Related to the Designated Chemical Substances" (Notification No . 268 of

the Planning and Coordination Bureau, EA, No . 41 of the Environmental Health Bureau,

MHW & No .1 (2000•02•14)of the Basic Industries Bureau, MITI, revised on March 1,

2000) .

LOCATION AND PERIOD FOR RETENTION OF RAW DATA

Data, specimens and the test substance will be retained in the archives and test substance

storage room, respectively, of Ube Scientific Analysis Laboratory for 10 years the date of

notification specified under Item 1 of Article 57-2 of Industrial Safety and Health Low .

After termination of the retention period, any measures taken will be done so with the

approval of the sponsor. However, specimens that are liable to deteriorate markedly will

be retained only for the period the quality of specimens permits evaluation, and the

abandonment of them will be taken with approval of the sponsor .

AUTHOR AND PERSONS CONCERNED WITH STUDY

Study Director:

November 22, 2002

Person in charge of Storage : Kiyomi Okimoto

`E~I
p. 62 ov

USA-R-0213 1

TABLE OF CONTENTS

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2

1 . TEST SUBSTANCE AND POSITIVE CONTROL SUBSTANCES . . . . . . . . . . . . . . . . . . . . . . . . . . .2

2. PREPARATION OF THE TEST SUBSTANCE AND THE POSITIVE CONTROL

SUBSTANCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

3 . CELLS AND MEDIUM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

4 . S9 MIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

5 . CELL GROWTH INHIBITION TEST (DOSE FINDING) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

6. CHROMOSOME ABERRATION TEST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

7. OBSERVATION AND SCORING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

8. EVALUATION OF RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL. . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

1 . CELL GROWTH INHIBITION TEST(Table 1 and 2, Fig . 1 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

2. CHROMOSOME ABERRATION TEST(Table 3 and 4, Fig . 2 and 3) . . . . . . . . . . . . . . . . . . . . . . . . 7

CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
p.63

USA-R-02131 7

SUMMARY

Chinese hamster lung fibroblasts (CHL) cells were exposed to Diethylaminotriethoxysilane

both with and without metabolic activation . As a result , the test substance did not induce

structural chromosome aberrations . Numerical aberration was also not increased . On the

other hand, in positive control groups, a significant induction of chromosomal aberration

was observed following treatment with mitomycin C and Benzo(a)pyrene . Based on the

above results, Diethylaminotriethoxysilane did not induced chromosome aberrations under

the present test conditions.

-1-

03
p.64

USA-R-02131 ~

MATERIALS AND METHOD S

1 . TEST SUBSTANCE AND POSITIVE CONTROL SUBSTANCES

1 .1 Test Substance (Information Provided by the Sponsor)

1) Name Diethylaminotriethoxysilane

Other name : DEATES

2) Structural Formula

(C2H5)2NSl(OC2H5)3

3) Purity 97.6%

4) Name and concentration of impurities

Tetraethoxysilane 1 .6%

unknown impurities 0 .8%

5) Lot No. DEATES- 1

6) Supplier UBE INDUSTRIES, LTD .

7) Physicochemical Properties

Molecular Weight : 235 .40

Melting Point : -

Boiling Point : 75°C/0.5mmHg

Vapor Pressure : -

Stability : stable under nitrogen atmosphere in shade

Appearance at Ordinary Temperature : a transparent liquid

Degree of Solubility : Water : decomposed with wate r

DMSO : <235mg/ml

Acetone : 235mg/ml<

Storage Conditions : Stored under the condition of nitrogen gas-purge at cold and

dark place .

1 .2 Negative (solvent) Control Substances

Acetone

Manufacturer : Wako pure chemical industries, Ltd.

Lot No.: KSQ3371

Purity : ? 99.5%

1 .3 Positive Control Substances

1) Mitomycin C (MMC )

Manufacturer : Kyowa Hakko Kogyo Co., Ltd .

Lot No. : 339AJH

Composition : 2 mg titer of J .P. MMC and 48 mg of J .P. sodium chloride in

one vial

-2-

W
p.65 6/

USA-R-02131 `Ib

2) Benzo(a)pyrene (BP)

Manufacturer : Wako Pure Chemical Industries, Ltd .

Lot No. : ELE2013

Purity : _> 99 .8%

2 . PREPARATION OF THE TEST SUBSTANCE AND THE POSITIVE

CONTROL SUBSTANCES

2 .1 Preparation of Test Substance

1) Preparation method

Acetone was selected as a solvent, because degree of solubility of the test substance

was less than 235 mg/ml in DMSO, more than 235 mg/mi in acetone . In addition,

the acetone was dehydrated and used, because the test substance was decomposed

with water.

2) Preparation time

The test substance solutions were prepared immediately before use, and used within

2 hours .

3) Stability of test solution

Denaturation of test solution including change in a color and exothermic reaction

was not observed until 2 hours after preparation.

2.2 Amount of Test Substance Solutio n

The test substance solution was added to the medium at the ratio of 1% .

2.3 Positive Control Substance s

1) Preparation method

MMC was dissolved in distilled water (Otsuka Pharmaceutical Factory Co., Ltd.)

and Saline (Otsuka pharmaceutical factory Co ., Ltd .). BP was dissolved in DMSO

(Dojindo Laboratory Co ., Ltd.)

2) Storage Conditions and Handling

MMC and BP was stored in a freezer under -20°C . They did not reuse after thawing.

3 . CELLS AND MEDIU M

3.1 Cell Line and Reason for Selectio n

Chinese hamster lung fibroblasts (CHL cells, Lot No.113092) were supplied by Health

Sciences Research Resources Bank of the Japan Health Sciences Foundation on

December 11, 1996 were used . The modal number of chromosomes was 25 per cell,

and the doubling time was about 15 hours.

The passage number of received cells was 5, and stock cultures was 7 . Cells were

propagated from stock cultures that they were checked the absence of mycoplasma

contamination, and sub-cultured at 3-5 days interval . The number of passages were

-3-

vGJ5
p.66

6

USA-R-02131 1176

from 5 to 14 in this study .

There are chosen because that they have high sensitivity to inducers of chromosome

aberrations and available data have been accumulated .

3.2 Medium

Eagle's minimum essential medium (Lot No . 32K24497, 53K2367 ; Sigma-Aldrich Co .

Ltd.) was supplemented with Bovine calf serum (Lot No . 7G2148; JRH Biosciences) to

the extent of 10 v/v%.

3 .3 CELL CULTUR E

Five ml of medium containing 4,000 cells/ml was seeded on a dish . Petri dish (Becton

Deckinson Co ., Ltd .) with 60 mm diameter was used. Three days after seeding, cells in

logarithmic growth phase were used for the test substance treatment .

The culture was carried out in a C02-incubator maintained at 37°C and at a relative

humidity of substantially 100% and in 5% CO2 .

4 . S9 MI X

4.1 Rat Liver S9 (Oriental Yeast Co., Ltd . )

Induction Method : Male Sprague-Dawley rats (weight:217.4±8 .5 g) aged 7-weeks old

were intraperitoneally administered phenobarbital (30mg/kg X 1

time, 60mg/kg X 3 times) and 5,6-benzoflavone (80mg/kg X 1 time) .

Lot No. : 02041201 (manufactured on April 12, 2002)

Storage condition: S9 was stored in an ultra-deep freezer (MDF-192AT, SANYO Co .,

Ltd.) at -80°C .

4.2 Composition of S9 mi x

One ml of S9 mix comprised 4 μmol HEPES(pH 7.2), 5 μmol MgCl2, 33 μmol KC1,

5 μmol G-6-P, 4 μmol NADP, and 0 .3 ml of S9 .

4 .3 S9 mix/ME M

S9 mix/MEM was prepared by adding 0 .5 ml of S9 mix to 2 .5 ml of medium .

5 . CELL GROWTH INHIBITION TEST (DOSE FINDING)

In the cell growth inhibition test, the concentration of test substance causing 50% growth

inhibition was determined . The highest concentration was determined as the maximum

concentration at which the chromosomes was observed.

5 .1 Concentrations

In the first test of the short and continuous treatment method, the seven concentrations

were used which they were separated by a factor 2 . Maximum concentration was 2 .35

-4-

6(0
p.67

USA-R-02131 q6

were used which they were separated by a factor 2 . Maximum concentration was 2 .35

mg/ml (=10mM).

5.2 Short treatment metho d

Five ml of medium containing 4,000 cells/ml was seeded on a dish . Three days after

seeding, the cells were treated with 3 ml of medium (the group without S9 mix) or S9

mix/MEM (the group with S9 mix) containing the test substance for 6 hours. After

treatment, the medium was removed, and 5 ml of flesh medium was added to the cells.

Then, incubation was continued further for 18 hours .

After the completion of the culture, the medium was removed and treated cells were

detached from the dish by treatment with 2 ml of 0.25% trypsin . The number of live

cells was counted with a hemocytometer using a portion of the treated cells, and the

cell growth rate were calculated .

Chromosome specimens were prepared using the treated cells in the cell growth

inhibition test to examine the presence or absence of mitotic cells .

5.3 Continuous treatment method

Five ml of medium containing 4,000 cells/ml was seeded on a dish . Three days after

seeding, the cells were treated with 5 ml of medium containing the test substance for

24 and 48 hours. After the completion of the culture, the number of live cells was

counted.

6. CHROMOSOME ABERRATION TEST

6.1 Concentrations

Test Substance :The highest concentration to be used was determined from the results

of the cell growth inhibition test. Three concentrations were prepared by diluting the

maximum concentration using a geometric progressions.

Positive Controls: Based on our historical data, 0 .04 μg/ml of MMC for the continuous

treatment method, 0 .07μg/ml of MMC for the absence of a metabolic activation system

in the short treatment method, and 0 .02mg/ml of BP for the presence of a metabolic

activation system in the short treatment method were applied .

6.2 Methods

Cells were cultivated and treated using the same procedure as in the cell growth

inhibition test, subsequently specimens were prepared .

A solvent-treated group for negative control and MMC and BP-treated groups for

positive control were used. Two dishes were used for each group .

6.3 PREPARATION OF MICROSCOPE SLIDES

Cells were treated with 0 .2 μg/ml of colcemid for 2 hours, and after treatment with 2

'7 .
p.68

USA-R-0213 1

nil of 0.25% trypsin. Then, 0.075 M KCl was added to the tube, and hypotonic

treatment was carried out at 37°C for 15 minutes . Calnoy's solution (ethanol:acetic

acid=3 : 1) was then poured into the tube to fix the cells and make a slightly cloudy cell

suspension, which was then dropped onto a microscope slide and spread . The cells on

each slide were finally dried and stained with 2% Giemsa's solution (at pH6.8).

7. OBSERVATION AND SCORIN G

All slides were independently coded before microscopic analysis. 200 well-spread

metaphases were scored per concentration and control equally divided amongst the

duplicates. The cells were scored contain a number of centromeres equal to 25±2 for all

cell types. The types of aberrations were classified into 6 groups : chromatid breaks (ctb),

chromatid exchanges (cte), chromosome breaks (csb), chromosome exchanges (cse),

others (fragmentation, etc.), and gap. The gap was defined a non chromosomal segment

on the same axis as a chromatid, smaller than the width of the chromatid . Gaps were

recorded separately and reported but not included in the total aberration frequency . A

cell with any aberration was counted as an aberrant cell . Polyploidy and

endoreduplication were recorded .

8 . EVALUATION OF RESULT S

For the evaluation of frequencies of structural aberrations and polyploidy, the following

criteria which were usually used for chromosome aberration testing with CHL were

employed.

Negative (-) : less than 5 %

Equivocal (±) : from 5% to less than 10%

Positive (+) 10% or more

The test substance was judged to be positive for the induction of chromosomal

aberrations was dose-related or reproducible, and the concentration at which some

degree of aberration was observed in 20% of cells (D21 Value) was calculated . Further, it

was determined that if the test result was suspect positive, or dose dependency of the

appearance of aberrant cells was not observed, the test should be carried out again .

UNEXPECTED SITUATIONS AND DEVIATIONS FROM

PROTOCOL

There were neither unforeseeable circumstances that might cast doubts on the reliability of

the study nor deviation from the protocol .

RESULTS

The values for duplicate dishes were not markedly different, the incidence of cells with any

aberration did not exceed 5% in the negative controls, the incidence of cells with structura l

-6-

6=~g
p

.69 710V

USA-R-02131 7

'aberrations was 10% or more in the positive group, and there were no fluctuations in the

test conditions . The test results were therefore considered to be valid .

1 . CELL GROWTH INHIBITION TEST(Table 1 and 2, Fig . 1 )

In the short treatment method, the test substance was not toxic . However, in the

continuous treatment method, Cytotoxity was greater than 50% at the highest

concentration (2 .35 mg/ml) .

Based on these results, the three concentrations were used in each system for the

chromosome aberration test.

Short treatment method ;

In the absence a metabolic activation system : 0 .589, 1 .18, 2.35mg/ml

In the presence a metabolic activation system : 0.589, 1 .18, 2.35mg/ml

Continuous treatment method ;

24 h treatment: 0.613, 0.858, 1 .20mg/ml

48 h treatment: 0.429, 0.601, 0.841, 1 .18mg/ml

2 . CHROMOSOME ABERRATION TEST(Table 3 and 4, Fig . 2 and 3)

In the present test conditions the test substance did not induce structural chromosome

aberrations . Numerical aberration was also not increased . On the other hand, the

incidence of chromosome aberrations in solvent control was within the normal range,

whereas the incidence in the positive control showed a clearly induced.

CONCLUSION

In the chromosomal aberration tests of Diethylaminotriethoxysilane using CHL Cells, the

test substance did not induce chromosomal aberration and numerical aberration under the

present test conditions. On the other hand, the incidence of chromosome aberrations in

solvent control was within the normal range, whereas the incidence in the positive control

showed a clearly induced.

REFERENCES

1 . Ministry of Labor ( 1991) Test guideline and GLP, Mutagenicity test for Industrial Safety

and Health Law published by Japan Industrial Safety and Health Association .

2. Japan Environmental Mutagenicity Society/Mammalian Mutagenicity Study Group (ed .)

( 1988) Atlas of chromosomal aberration induced with chemical substance (in Japanese).

Asakura Publishing Co ., Tokyo .

3 . Sofuni, T. (ed.) (1999) Revised edition 1998, Data book of chromosomal aberration test

in vitro. L.I.C . Inc ., Tokyo .

4. Kikuchi, Y. and Miyake, Y(1992) Mutagenicity test Question & Answer (in

Japanese). Scientist Co., Tokyo.

-7-

67
p.70 ry~

USA-R-02131 76

Table 1' Dose range finding test of DEATES by short treatment metho d

Without metabolic activation (6-18h) With metabolic activation (6-18h)

Concentration (mg/ml) Growth rate (%) Concentration (mg/ml) Growth rate (% )

0 100 - 0 100 -

0.0368 114 - 0.0368 85 -

0.0736 90 - 0.0736 117 -

0.147 124 - 0.147 92 -

0 .294 107 + 0.294 99 -

0 .589 115 + 0.589 106 +

1 .18 93 + 1.18 92 +

2.35 t 86 ++ 2.35 ~ 110 +

Table 2 Dose range finding test of DEATES by continuous treatment method

Without metabolic activation (24-0h) Without metabolic activation (48-0h)

Concentration (mg/ml) Growth rate (%) Concentration (mg/ml) Growth rate (%)

0 100 - 0 100 -

0.0368 108 - 0.0368 110 -

0 .0736 140 - 0.0736 111 -

0 .147 94 - 0.147 141 -

0 .294 99 - 0.294 114 -

0 .589 90 + 0.589 125 -

1 .18 55 +++ 1.18 56 +++

2.35 4++++ 2.35 j- 2++++

(6-18h) : treatment time-recovery tim e

t : precipitation of test substance

Observation of cell growth by phase contrast microscop

: none, + : cell form chage a little, ++ : cell death -:5 30%, +++ : 30%< cell death _< 60%,

++++ : 60% < cell death -:5 90%, TOX : 90% < cell deat h

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USA-R-0213 1

140

120

° 100 ><

~~ ~ ~ • `~ ~ -0 - without S9 mix 8 0

60 -A- with S9 mix

v 20 ~

4 0

0

0 0.0368 0.0736 0.147 0.294 0.589 1.18 2.35

Concentration of the test substance (mg/ml )

Short treatment method

150 - -- -

A

8-0

100

0 -0- 24-0h

50 -A- 48-Oh

0

0 0.0368 0.0736 0.147 0.294 0.589 1.18 2.35

Concentration of the test substance ( mg/ml)

Continuous treatment metho d

Fig. 1 Results of the cell growth inhibition test of DEATES

7~~
p.74

USA-R-0213 1

~ •

60 - - 120

50 - ♦----~ ~\ 1000

E 40 - ~ 80 -0 -Structial aberrations

U O

30 60 -Numerical aberrations

`' 20 40 *-Growth rat e

° cz 10 ~ 20 C7

0 0

0 0.589 1.18 2.35

Concentration of the test substance ( mg/ml )

Without S9 mix

60 120

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~ 40 80 ~ -~- Stractial aberrations

E

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40 3 -~- Growth rate

10 20

O

0"

0 A ,_2__j 0

0 0.589 1.18 2.35

Concentration of the test substance ( mg/ml)

With S9 mix

Fig. 2 Results of chromosome aberration test of DEATES by short treatment metho d

7~
p. 75

USA-R-02131

9

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60 120

0 IU 50 ♦-- -_~ 100

U o~ 30 - \

40 80 Structial aberrations

♦ - 60 -Numerical aberrations

U

;, 20 - 40 -Growth rate

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0 0.613 0.858 1.20

Concentration of the test substance (mg/ml )

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L) -0 40 ~\ 1 80 -0 -Structial aberrations

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0 __J 0

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Concentration of the test substance (mg/ml )

48 h-treatmen t

Fig. 3 Results of chromosome aberration test of DEATES by continuous treatment method

~✓