TS~CA NON-CONFIDENTIAL BUSINESS INFORMATION

DOCUMENT DESCRIPTION DOCUMENT CONTROL NUMBER DATE RECEIVED

p- - ". Sl0 Iv 

COMMENTS: DECLASS

DOIES NOT CONTAIN CII
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON D.C., 20460

OFFICE OF

~ PROI~CHEMICAL SAFETY AND

POLLUTION PREVENTIr"

Notice of Change in CBI Chemical Identity Status

Pre-Manufacture Notice (PMN) P-03-0354

Notice of commencement (NOC) received: 8/30/2004

Please be advised that the Confidential Business Information (CBI) claim for the

above referenced substance was dropped by the information submitter in the

NOC.

The identified non-confidential chemical name for this substance as provided in

the NOC is:

1-PENTEN-3-ONE, 1-(4-CHLOROPHENYL) -4,4-DIMETHYL-*

The current TSCA Inventory name is

1 -Penten-3-one, 1 -(4-ch lo rop henyl)-4,4-d im ethylCAS RN: 1577-03-3

This notice as submitted to the NOIC files on 9/1/2011

Name of staffer: Jeffrey Santacroce

Position: Asst. NOC Coordinator

Branch: RDMB/IMD/OPPT/OCSPP

CONTAINS NO CBI
Form Approved. 0.MB. No. 2070-0012. Approval Expires 10-31-96.

U.S. ENVIRONMENTAL PROTECTION AGENCY AGENCY USE ONLY; Date of receipt

PREMANUFACTURE

NOTICE q~pA FOR NEW CHEMICAL SUBSTANCES

~~ ~DOCUMENT CONTROL OFFICER "-

OFFICE OF POLLUTION PREVENTION CompanySanitlzed

AND TOXIC SUBSTANCES. 7407

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

WASHINGTON, D.C. 20460j

Enter the total number of pages 90 ?5at E a bp in the Premanufacture Notice 70i; -"

GENERAL INSTRUCTIONS TS - M 1C1 013 6 2

* You must provide all information requested in this form to the extent that it is known to or reasonably ascertainable by you. Make reasonable estimates if you do

not have actual data.

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

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

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

your user fee ID number must also appear on your corresponding fee remittance, which is sent to EPA, HQ Accounting Operations Branch (PM-226), P0O

360399M, Pittsburgh, PA 1525 1-6399, Attn. TSCA User fee.I E "

Part I - GENERAL INFORMATION PS UaU AC CO'

You must provide the currently correct Chemical Abstracts (CA) Name of the new You are required to submit all test data in your possession or control and to provide

chemical substance, even if you claim the identity as confidential You may authorize a description of all other data known to or reasonably ascertainable by you, if these

another person to submit chemical identity information for you, but your submission will data are related to the health and environmental effects on the manufacture. not be complete and the review will not begin until EPA receives this information. A processing, distribution in commerce, use, or disposal of the new chemical

letter in support of your subission should reference your TS user fee identification substance. Standard literature citations may be submitted for data in the open number You must submit an original and two copies of this notice including all test data

If you claimed any information as confidential, a single sanitized copy must also be scientific literature. Complete test data (written in English), not summanies of data,

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

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

composition of the tested material should be characterized. Following are

Part It - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE examples of test data aisd other data. Data should be submitted according to the

requirements of §720.50 of the Premanufacture Notification Rule (40 CFR Part

If there are several manufacture, processing, or use operations to be described in Part 11, 720).

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

Part III - LIST OF ATTACHMENTS Test Data (Check Below any included in this notice)

Attach additional sheets if there is not enough space to answer a question fully. Label *Environmental fate data 0 Yes *Other data 0 Yes

each continuation sheet with the corresponding section heading In Part 111, list these

attachments, any test data or other data and any optional information included in the *Health effects data 0 Yes Risk assessments

notice

*Environmental effects data 0 Yes Structure/activity relationships

*Physical/Chemical Properties* 0 Yes Test data not in the possession

OPTIONAL INFORMATION or control of the submitter

You may include any information that you want EPA io consider in evaluating the new *A physical and chiemical properties worksheet is located on the last page of this form

substance On page I I of this form, space has been provided for you to described

pollution prevention and recycling information you may have regarding the new TYPE OF NOTICE (Check Only One)

substance

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

willingness to be bound to certain statements you make in this section. such as use,

production volume, protective equipment , This option is intended to reduce delays UINTERMEDIATE PMN (submitted in sequence with final product PMN)

that routinely accompany the development of consent orders or Significant New Use

Rules Except in the case of exemption applications (such as TMEA. LYE, LOREX) r

where centain information provided in such notification is binding on the submitter when USNUN (Significant New Use Notice)

the Agency approves the exemption application, checking a binding box in this notice -1

does not by itself prohibit the submitter from later deviating from the information (except El TMEA (Test Marketing Exemption Application) 5

chemical identity) reported in the form. Cz~~~

El LYE (Low Volume Exemption) @ 40 CFR 723 50(c)(I)

CONFIDENTIALITY CLAIMS 1

You may claim any information in this notice as confidential, To assert a claim on the LOREX (Law Release/Low Exposure Exemption) @ 4IFR 723,SO( (2)

form, mark (X) the confidential box next to the information that you claim as -ri r- confidential. To assert a claim in an attachment, circle or bracket the information you ULYE Modification Li LOREX Mo-dUication -

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

also provide a sanitized version of the notice, (including attachments). For additional IS THIS A CONSOLIDATED PMN? Yes

instructions on claiming information as confidential, read the Instructions Manual

# of chemicals ___________

Mark (x) if any information in this notice is claimed as confidential (Prenotice Communication # required, enter # on page 3)

EPA FORM 7710-25 (Rev. 5-95) 6 7 qS Replaces previous editions of EPA Form 77 10-25.
Public reporting burden for this collection of information is estimated to average 110 hours per response, including time for reviewing

instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of

information. Send comments regarding the burden estimate or any other aspect of this collection of information, including suggestions for

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

20460; and to the Office of Management and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503.

CERTIFICATION

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

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

commercial purpose, other than in small quantities solely for research and development, the substance identified in Part 1,

Section B.

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

3. 1 am submitting with this notice all test data in my possession or control and a description of all other data known to or

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

Additional Certification Statements:

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

statement that applies:

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

Dj The Company named in Part 1, 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

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

in accordance with 40 CFR 700.45(b).

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

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

statements:

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

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

D- The manufacturer is familiar with the terms of this section and will comply with those terms; and

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

DIf this application is for an LVE in accordance with 40 CFR 723.50(c)(1), the manufacturer intends to commence

manufacture of the exempted substance for commercial purposes within I year of the date of the fkVd

Treview period. PUBLIC

Teaccuracy of the statements you make in this notice should reflect your best prediction of the anticipated facts regarding the chemical substance

described herein. Any knowing and willful misinterpretation is subject to criminal penalty pursuant to 18 usc 1001. Confidential

Signature and title of Authorized Official (Original Signature Required) Date

x

Signature of agent - (if applicable) Date

x

EPA FORM 77 10-25 (Rev. 5-95) Page 2
Part I -- GENERAL INFORMATION

Section A -- SUBMMfER ]IDENTIFICATION ConfiMark () the "Confidential" box next to any subsection you claim as confidential -dential

I a. Person Name of authorized official Position

Submitting x

Notice (in U.S.)

Company

b. Agent (if Name of authorized official Psto

applicable)

c. If you are submitting this notice as part of a joint submission, mark (X) this box--

Joint Submitter (if Name of authorized official Psto

applicable)

2. Technical Name of authorized official Position

Contact (in

U.S.) _____________________

3 If you have had a prenotice communication (PC) concerning this notice

and EPA assigned a PC Numher to the notice, enter the numberMak()0 EX

if none

4 If you previously submitted an exemption application for the chemical

substance covered by this notice, enter the exemption number assigned by Mark (X) - EPA If you previously submitted a PMN for this substance enter the E

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

5 If you have submitted a notice of Bona fide intent to manufaciure or impori

for the chemiucal substance covered hy this notice, enter the notice number Mark (X - * 0

assigned by EPA - Fif none E

I 1 Manufacture 2. E] Import 3Bt

6. Type of Notice -Mark (X) Only Only3.Bt

D Binding Option D Binding Option

Mark (X) IMark (X)

EPA FORM 7710-25 (Rev. 5-95) Page 3
Part I -- GENERAL INFORMATION -- Continued

Section B -- CHEMICAL ]IDENTITY 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 conventions.

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

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

If another person will submit chemical identity information for you (for either Item I or 2), mark (X) the box at the right. ConfiIdentify the name, company, and address of that person in a continuation sheet. 0 EJ -dential

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

a. Class of substance - Mark (X) 1I E Class 1 or 2 E] Class 2 X

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

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

be provided, which ever is appropriate based on CA 9C1 nomenclature rules and conventions).

e. FoPleass 1de substanhcetpovde ao comete dlo corectai h chemical structure cf diara.oFr rmcass2stncped (1) in is te mm : ediat e

prcuso sutances with Intry Espet CSeistracoy Numbes ()destfcrtibeen atr of ethraton or rOces Sore niaether 

ran eopotioand frthe pCA compostony (wher eaproite). (4)t Prvdbeorcersnaierprilceiatutr

darmscbmie as an henoni oeicae raoa seie

D dak.Xt boxua ifoulattc an ContRiuton shmeet. ube led xit o hesbtne

EPA ORM77 1-25(Rev 5-5) Pge
D.Poyesc(Frib th denthods of plmeseenrthe astrcios orayusates: GPCOter (Secfy

a. Indica loest numberaverage ih ftels molecular weightcmoiino h oye yuitn omnfcue

ii) maximium weight % below 500 molecular weightiii) maximum weight % below 1000 molecular weight: _________

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

b. You must make separate confidentiality claims for monomer or other reactant identity, composition information, and residual information. Mark (X) the

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

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

the polymer.

(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 Inventory.

(5) - Mark (X) this colun 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)

N/A%%

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

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

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

obtained from CAS inventory Expert Service must be submitted as

as attachment to this notice)

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

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

EPA FORM 7710-25 (Rev. 5-95) Page 5
EB 13 28003 14:24 FR BRYER CURPURI N 412 777 7484 TO 912025648955 P.03/03

Mark~ (.i boxRA iNORATO you aCchntoiinntonshet ar IX 

Sectyonym - EntCAr a]Dny r mINFOMAIO sy-n- orhenwCoia ntiedinsbeinIor2

(.Tad Identif c i urit y ta a e r thc n eabl santce idb pent in sube cheion l su-m or 2auatrdfr.omrilprpx.Poieh

CAS a Rk gistr Nofyumaerach avconiua.Ion thee acuietfetmuiis ne Uietfe.

(.benri cEsmate name maimyum weaigh %hec i pti y . s con eruidentiied yoimtp riie en tiae foer yor taeig hat reval

thespcicch mic puienity ond CAe egistry Nubser Maxmu th axmm et osil.Ree t h

SenAtachdl

7. Synrodncms 

El Mak -

(X) Eneri 

this box 

any 

if you bmca 

atach 

yonycs 

a continuation 

rhn rmtemnfcee 

sheet. 

rcsiguco 

I i 

ipslo 

1(' the new chemical 

C

subsnfec rfled thc CASc licgisorry

Cl) (2)dnial

SEe Aak( hed bxi o taha otnainse

Mark X) this box i you attach a continuation qheet,

6.A GeOrc 7710-25a (R e -I3) camcemclieniya cof t a gc yo 6utpoieagnrcnm o orsbtneta eel

thespecific ~ oftenw*cn*lusa ~ ~ ~ ~ otemaiu chemica ~ identity ~ xetpsil.Rfrt ~ ~ * h
Part I -- GENERAL INFORMATION -- Continued

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

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

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

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

substance basis. For a Low Volume Exemption application, if you choose to have your notice reviewed at a lower production volume than 10,000

kglyr, specify the volume and mark (x) in the binding box. If granted, you are bound to this volume_________

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

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

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 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 (CBI).

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

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

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

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

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

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

Category of use (1) CBtI Binding Produc- CBI % in CBI % of substance expected per use CBI Option tion % Form- _________ (8)

(by function and application i.e. a dispersive dye Mark (x) ulation Site- Con-* Indus- Coin- Binding

for finishing po1yester fibers) (2) (3) (4) (5) (6) (7) limited sumer trial mercial Option (9)

Intermediate X X X

*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 descrnbe the chemical reactions by which this

substance loses its identity in the consumer product.

D 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 category. Read the

use Instructions Manual for examples of generic use descriptions.

description

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

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

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

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

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

EPA FORM 7710-25 (Rev. 5-95) Page 7
Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

Section A -- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER 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 industrial sites you

control. Importers do not have to complete this section for operations outside the U.S.; however, you may still have reporting requirements if

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

1. Operation description Confia. Identity -- Enter the identity of the site at which the operation will occur. dential

Name x

Site address (number and street)

City. County, State, ZIP code

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

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

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

sites as attachments.

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

b. Type -- X

Mark (X) ElManufacturing EProcessing D]Use _____ c. Amount and Duration -- Complete I or 2 as appropriate

Maximum kg/batch (100% new chemical Hours/batch IBatches/yearx

substance)

1. Batch

Maximum kg/batch mxt% new chemical i Hours/batch iBatches/year

substance) x

2. Continuous

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

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

drum, rail car, tank truck, etc ).

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

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

used daily or per batch.).

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

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

EPA FORM 77 10-25 (Rev. 5-95) Page 8
Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued

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

2. Occupational Exposure -- You must make separate confidentiality claims for the descniption of worker activity, physical form of the new chemical substance,

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

(1) -- Describe the activities (i.e. bag dumping, tote filling, unloading drums, sampling, cleaning, etc.) in which workers may be exposed to the substance.

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

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

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

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

at the time of exposure.

(7) -Mark (X) this column if entry in column (5) is confidential business information (C131).

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

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

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

(12) --Mark (X) this column if entries in columns (10) and (11) are confidential business information (C131). __ ___ ___

Worker activity Cl~t Protective Equipmnent/ Binding Physical formns(s) Binding C131 # of CBI Maximun duration CBI

mn

(i.e., bag dumping, filling drums) Engineering Controls Option~ and % new Opion Workers Hrs/day Daysfyr

Mark (x) substance Mark (x) Exposed

()(2) (3) (4) (5) (6) (7) (8) (9) (t0) (tt) (12)

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

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

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

(1) -Enter the number of each release point identified in the process description, part 11, section A, subsection I d(3).

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

(3) -Mark (X this column if entries in columns (1) and (2) are confidential business information (CBIf).

(4) -- Identify the media (stack air, fugitive air (optional-see Instruction Manual), surface water, on-site or off-site land or incineration. POTW, or other (specify)) to

which the new substance will be released from that release point.

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

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

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

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

(6) -Mark (X) this column if entries in columns (4) and (5) are confidential business information (C131).

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

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

Release Amount of new substance CBI Media of Control technology and efficiency (you may wish to optionally attach efficiency data) B

Number released release

() (2a) (2b) e.g stackau. Binding

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

x x

(7) Mark (X) the EiPOTW provide name(s) below: CBI LIINavigable ElOther - Specify Iprovide NPDES # CBI

destination(s) of aera X 

releases to water.

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

EPA FORM 77 10-25 (Rev. 5-95) Page 9
Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued

Section B -- INDUSTRIAL SITES CONTROLLED BY OTHERS

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

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

section Bfor each tpie of processing, or use operation involviing the new chemical substance. If the same operation is performed at more than one site describe the

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

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

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

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

(by kg/day or kg/batch, on an 100% new chemical substance basis), and entry point of all feedstocks (including reactants, solvents and catalysts, etc) and all

products, recycle streams, and wastes. Include cleaning chemicals (note frequency if not used daily or per batch). (3) -- Identify by number the points of release,

including small or intermittent releases, to the environment of the new chemical substance. (4) Please enter the # of sites (remember to identify the locations of

these sites on a continuation sheet):

2

# of sites

The material made in will go to two (2) different "tollers" that we will use to evaluate capabilities and cost issues. I am

told this would be done via intermodal containers. We may choose to return the tolled product in these same

containers. This is not decided yet. In future years we have not determined how it will be shipped and who will clean the

container. We are considering returning the in the same containers used to ship the

.The toilers would probable 'flush" the railcars with the as the flush solvent. No other "firm" data at

this time.

Information on how the toiler will handle the material has not been determined yet. It should be readily available once we

make decision on who to send it to.

The will then be returned to for conversion to in the

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

2. Worker Exposure/Environmental Release

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

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

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

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

protect workers.

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

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

(10) -- Estimate the amount of the new substance released (a) directly to the environment or (b) into control technology to the environment (in kg/day or kg/batch). (1 2) -- Describe media of release i.e. stack air, fugitive air (optional-see Instructions Manual), surface water, on-site or off-site land or incineration, POTW. or other

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

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

(3), (5), (8). 11I), (13) and (15) -- Mark (X) this colun if any of the proceeding entries are confidential business information (CBI). _______

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 nwsbtceReleased

(1) (2) (3) (4a) I (4b) (5) (6) (7) (8) (9) (10a) (0b) (11) (12) (13)

(14)- Byproducts: (15)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

alternatives may be provided.

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

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

examples.

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

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

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

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

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

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

Normally we would have sent the for .This material was then

returned to the . Our method will prevent the need to ship these chemicals transatlantic

with corresponding reduction in risk to the environment and humans.

The currently used process in uses in the of

.Our process will not use a solvent. The current method recycles the

solvent but there is some wastage involved.

Also, we will have a any unreacted to be recycled and re-used in the

reaction step.

The will most likely be shipped in .This is a reduction in

the shipments and containers being transported.

The company will realize significant in the

compared with shipping to for conversion to

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

EPA FORM 7710-25 (Rev. 5-95) Page I11
Part III -- LIST OF ATTACHMENTS

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

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

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

numbers of each attachment.

Mark (X) the "Confidential" box next to any attachment name you claim as confidential. Read the Instructions Manual for guidance on

how to claim any information in an attachment as confidential. You must include with the sanitized copy of the notice form a sanitized

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

Attachment name Attachment Confipage number(s) dential

x

x

x

x

x

x

Ames-Test 22-69

Summary of Acute Oral, Acute Dermal, Acute Inhalation, Irritation and Cauterisation of Rabbit skin and

eye

U1

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

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

PAGES 13-21

/3
-: Report No

* Report*Date:

SPECIAL STUDY

AMES-TEST SCREENING

Study No..

Prior to publication, the findings tot'his report

may only be used with the apprva o Further reproduction of all or part of this repor is no permitted.

page 1 of 48
4,

This page is intentionally left blank for the purpose of sub-'-

mitting administrative information that is required by regulations promulgated by various countries.

Z2

- -. A5
1. Signatures 5 -*~

2. Summary

3. introduction 7

4. Material and Methods 9

4.1 Substances- 9

4.1.1-Test Substance 9

4.1.2 Positive Controls 10

4.2 Indicator Organisms 11

4.2.1 Description of Test Strains 11

4.2.2 origin of Strains 12

4.2.3 Production of Stock Cultures 12

4.2.4-Checking of Genotype 14

4.2.4.1 Histidine Requirement 14

4.2.4.2 Ampicillin Resistance (pKM 101) 14

4.2.4.3 Tetracycline Resistance (pAQ1) 14

4.2.4.4 Crystal-Violet Sensitivity (deep-rough) 14

4.2.4.5 UV Sensitivity (uvrB) 1

4.2.5 Stock Batches .15

4.3 S9 Mix 15

7 -4.4-Test Design 17-

r4.5 Assessment Criteria 21

4.6 Study Identification and Responsibilities 21

4.6.1 Type of Test and Study Number 21

4.6.2 Responsibilities 21*

P UBLIC0 COPY,

3
oh

5. Results 22

6. Assessment 2

7. References 24

8. Historical Controls 27

8.1 Plate Incorporation Method 27

8.2 Preincubation Method 34

Tables 1 -10 39
1. Signatures

Study Director

Section Head

PUBLW CoPY

S
2. Summary

(~as initially screened with one plate per dose

4 4 *.. amnel/irsm plate incorporation test 'for

point mutagenic effects in doses of up to and including

5000 jig per plate on five Salmonella typhimurium LT2 mutants.

These comprised the histidine-auxotrophic strains TA 1535,

TA 100, TA 1537, TA 98 and TA 102. The independent repeat was

performed as preincubation for 20 minutes at 370C. Other conditions remained unchanged.

16 jig per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of

growth was observed. At higher doses, the substance had a

strain-specific bacteriotoxic effect, so that this range

could only be used to a limited extent for assessment purposes. At 500 jig per plate, the substance started to precipitate.

Evidence of mutagenic activity of s not seen.

No biologically relevant increase in the mutant count, in

comparison with the negative controls, was observed.

The positive controls sodium azide, nitrofurantoin, 4-nitro1,2-phenylene diamine, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a

biologically relevant increase in mutant colonies compared to

the corresponding negative controls.

Therefore , as considered to be non-mutagenic without anVwit 9 Wmix in the plate incorporation as well as

in the preincubation modification of the Salmonella/microsome

test.

6 PUBLI CopY

C 7

19
3. Introduction

The mutagenicity evaluation was performed using a screening

version (one plate per dose and strain) of the Salmonella/microsome test, also termed the Ames Test, as described by Ames

et al. (1973a, 1975) and Maron and Ames (1983).

The Salmonella/microsome test is a screening method which detects point mutation caused by chemical agents in vitro.

Auxotrophic mutants of Salmonella typhimurium are used to

demonstrate this effect. For this purpose, the rate of reversion to prototrophy is evaluated in negative control and

treated groups. A mutagenic effect is assumed if this rate

increases sufficiently in the treated groups.

Mammalian metabolism, which is of great significance in chemical mutagenesis, is simulated in this test by the 9000 g

fraction of homogenized mammalian livers. Together with cofactors, this forms the "IS9 mix"l which represents the metabolic model in this test.

The method itself is considered to be very sensitive (Herbold

et al. , 1976; Herbold, 1978) and is well suited for -fast,

screening. Available literature indicates a high correlation

between the positive and negative responses of the Ames assay

and the carcinogenic activity of the tested substances

(McCann et al., 1975a, 1976; Purchase et al., 1976, 1978). In

addition, the test represents a good screening system for potential carcinogenic effects, although the results should not

be overrated, as this high correlation may not apply to all

substance groups (Ames, 1979; Andrews et al., 1978; Clayson,

1980; Glatt et al., 1979 and Rinkus and Legator, 1979; Zeiger, 1987).

PUBIC Opy

7 LI U

:~Io
Eprmnare fmledion Dtearhvsote

StudPULy at COPYt

8
4. Material and Methods

4.1 Substances

4.1.-1 Test Substance

name of

test substance

manufacturer

article number

batch number

content '

approved______

appearance

storage

chemical name

structure

9
molecular weight

molecular formula IV1

CAS No.

intended use

The batch used was analysed prior to study initiation and approved for use during the test period.

4.1.2 Positive Controls

Sodium azide (Na-azide, SERVA), order no. 30175 (Control:D) ,

a direct-acting mutagen used as specific positive control forTA 1535.

Nitrofurantoin (NF, SIGMA), order no. N-7878, lot no.

67F0765, a direct-acting mutagen used as specific positive

control for TA 100.

4-nitro-1,2-phenylene diamine (4-NPDA, Fluka), order no.

73630, analysis no. 247722/1 194, a direct-acting mutagen

used as specific positive control for TA 1537 and TA 98.

Cumene hydroperoxide (Cumene, Sigma), order no. C-0524, lot.

no.-35H1056; a direct-acting mutagen used as specific positive control for TA 102.

2-aminoanthracene (2-AA, Aldrich), order no. A3,880-0, batch

no. 'S2234-024, a promutagen which reverts all the strains and

serves as a control for the activating effect of the S9 mix.

The positive controls sodium azide, nitrofurantoin, 4-nitro1,2-phenylene diamine and cumene hydroperoxide were only used

without S9 mix; the positive control 2-aminoanthracene was

only used with S9 mix.

10
4.2 Indicator Organisms

4.2.1 Description of Test Strains

Histidine-deficient mutants of Salmonella tvphimurium LT2

served as indicators to demonstrate point mutagenic.,effects. ~ These'strains were selected specifically for thei Saimonela

microsome test. Since point mutations can be divided-intol two

basic classes, base-pair substitutions and frameshift mutations, several strains were used which cover both types.

These included the strains selected by Ames et al. (1973b) ,

Salmonella typhimurium TA 1535 and TA 1537, as well as Salmonella typhimurium TA 100, TA 98 and TA 102 developed by McCann et al. (1975b) and Levin et al. (1982), respectively.

TA 1535 and TA 100 bear the base-pair substitution, his G 46,-

and TA 100 additionally contains the plasmid pKM 101. This R

factor, also contained in TA 98 and TA 102, codes for an ampicillin resistance and should raise the sensitivity of -the

strains. TA 102 carries the ochre mutation his G 428 on the

multicopy plasmid pAQl, which codes in addition for tetracycline resistance. TA 1537 and TA 98 bear frameshift markers.

TA 1537 exhibits the +1 mutant, his C 3076, while TA 98 bears

the +2 type, his D 3052.

Furthermore, the strains have other properties, which should

increase their sensitivity. They are all deep rough, i.-e.

partly deficient in lipopolysaccharide side chains in .their

cell walls, enabling larger molecules to penetrate the bacterial cell wall and produce mutations. With the exception of

TA 102, all strains have reduced capability to repair DNA-damage which increases the likelihood that such damage results

in mutations.

Whereas TA 1535 was used in addition to TA 100, TA 1538 is

not, normally used in addition to TA 98. This has two reasons:
a) There is no relevant increase in the spontaneous mutant

counts of TA 98, compared to the spontaneous range of

TA 1538. Special differences in sensitivity existing between

TA 1535 and TA 100, and attributed to the relatively high

spontane 'ous rate of TA 100 (10 times that of TA 1535),' do not

exist between TA 1538 and TA 98.' b) An 'international 4_general>_

inquiry had shown, that using TA 1538 'in addition to' anyof

the test strains in this study' would not 'provide further, 'nformation of biological relevance (Herbold, 1983).

This is in agreement with international guidelines, as pub--,-

lished by OECD, EEC, or EPA. Strain TA 1538 was either deleted in these guidelines, or never introduced at all. Maron'

and Ames (1983) also reported: "Although TA 1538 is useful

for the detection of particular aromatic frameshift mutagens

such as 4 -nitro -o -phenylene diamine, we decided to drop thestrain because it overlaps considerably with TA 98".

TA 1538, which differs from TA 98 in lacking the plasmid

pKM 101, is used in spite of these considerations, if questionable TA 98-results need clarification. This was not the

case in the present investigation, however.

4.2.2 Origin of Strains

The original.._strz~s_~eQ~~ ~e

arrived at

4.2.3 Production of Stock Cultures'

Immediately-upon receipt, the samples were inoculated on nutrient agar plates, to which ampicillin had been added for

the -TA 100, TA 98 and TA 102 strains. The plates for TA 102

contained additionally tetracycline., These plates were incubated at 37 0 C for approximately 24 -hours. Samples were taken

from individual colonies with a sterile inoculation loop, and

transferred to nutrient broth. In the case of TA 100, TA 98

and TA 102 ampicillin had also been added to this broth. The

broth for TA 102 contained additionally tetracycline. The

samples were again incubated overnight at 370C. New samples

of these cultures were inoculated onto nutrient agar plates, which had again been provided with ampicillin for TA 100,

TA 98 and TA 102 and in addition with tetracycline for

TA 102.

123

CL)Py

.ZSt
After an incubation period of approximately 24 hours at 370C,

new samples of individual colonies f rom these plates were

transferred to flasks containing approximately 20 ml normal

nutrient broth. T his inoculum was incubated overnight at

370C, after which a small sample was taken tocheckthe genotype., At the same time, the remaining cultures 1were treated

with DMSO to protect against the effects 'of'freezing, and im-,

mediately frozen at -80 0C in-i ml portions (Ames et al.,,

1973b; McCann et al., 1975b). No ampicillin--or tetracyclineresistance test was done on the samples used for testing

genotype since the cultures had already been sufficiently selected by ampicillin and tetracycline.

In addition to the test for crystal-violet sensitivity (deep

rough character), a test was done for UJV sensitivity (uvrB)

where appropriate. The crystal-violet and UV sensitivity

tests are described below. The frozen cultures which did not

produce satisfactory results here were discarded. Remaining cultures were stored for future tests. In addition, frozen

cultures of batches with unsatisfactory negative and/or positive control results in the definitive tests were also discarded.

Whenever new stock cultures needed to be produced, individual

cultures grown on nutrient agar were used, to which ampicillin had been added for the TA 100, TA 98 strains and to which

ampicillin and tetracyline was added for strain TA 102. Samples of these individual colonies were then transferred to

approximately 20 ml nutrient broth, incubated, :divided up, and checked for crystal-violet and UV sensitivity, if appro- priate.

One 1 ml-portion was thawed for each test and strain, and

quantities of 0. 2 ml of the thawed culture were added to 10

ml nutrient broth. This culture was incubated overnight at

37 0C and used only on the same day. A new, small stock culture, which had been checked for its properties directly before freezing, was thus available for each individual test.

In general this obviated any need to re-check the genotype for each Salmonella/microsome test. This procedure is in accordance with the methods described by Ames et al. (1975) and

Maron and Ames (1983).

13G
4.2.4 Checking of Genotype

4.2.4.1 Histidine Requirement

In each individual test, histidine dependence of the cul'tures,

was automatically checked by the accompanying -negati.ve con-'

trols. The number of mutants per individual plate is liste'd

in the Tables 1 to 10.

4.2.4.2 Ampicillin Resistance (pKM 101)

A special test for ampicillin resistance was not necessary

since strains TA 100, TA 98 and TA 102 were incubated on ampicillin containing nutrient agar and formed individual colonies. Consequently surviving bacteria were ampicillin resistant.

4.2.4.3 Tetracycline Resistance (pAQi)

A special test for tetracycline resistance was not necessary

since TA 102 was incubated on nutrient agar containing in addition to ampicillin tetracycline and formed individual colonies. Consequently surviving bacteria were also tetracycline

resistant.

4.2.4.4 Crystal-Violet Sensitivity (deep rough)

A quantity of 0.1 ml was taken from the samples of individual

stocks and spread onto nutrient agar, using four plates per strain. After a few minutes, filter papers, to which 10 A1 of

an aqueous, crystal-violet solution had been added at a concentration of 1 mg/ml, were placed in the middle of the

plates. The plates were then incubated overnight at 370C. The

diameters of the inhibition zones that had formed were then

measured. The inhibition zones of all stock batches used indicated an adequate sensitivity to crystal-violet.

14
4.2.4.5 UV Sensitivity (uvrB)

As described under 4.2.4.4, samples were spread onto nutrient

agar plates. One half of each plate was covered -with aluminiurn foil and irradiated without a lid for ,six seconds -(TA 1535

and 'TA 153 7), or ,eight -seconds -(TA 100 and -TA 9 8) with :.Lw

light of a wavelength of 254 nm at' a -distance',of *33 cm.,The

irradiated plates' were incubated as described under'4.2.4A.4

and checked. To demonstrate adequate sensitivity in' this

test, cultures had to show an inhibition of growth over half

their area, i.e. no bacteria should have grown on the irradiated half. This was the case with all the stock batches used.

For TA 102 no such test is needed.

4.2.5 Stock Batches

Stock Batches Used in Tables Strain

1-5 6-10

07.05.98/2 07.05.98/3 TA 1535

07.05.98/1 07.05.98/3 TA 100

07.05.98/2 07.05.98/4 TA 1537

07.05.'98/3 07.05.98/4 TA 98

17.07.98/1 17.07.98/2 TA 102

4.3 S9 Mix

S9 mix was used to simulate the mammalian metabolism of the

test substance. It was made from the livers of at-least six

adult male Sprague Dawley rats, of approximately 200 to 300 g

in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in

corn oil,- at a dose of 500 mg/kg body weight, five days prior

to sacrifice. The animals were prepared unfasted, following the directions of Ames et al. (1975) and Maron and Ames

(1983).

The rats were terminated. Livers were removed under sterile

conditions immediately after sacrifice and kept at 40C until

all animals had been prepared. All the remaining steps were

carried out under sterile conditions at 40C.

~~i~4 L

lots~
The livers were washed with cold (4 W), 0. 15 M KCl solution

(approximately 1 ml KCl per 1 g liver), and then homogenized

in fresh, cold W4C), 0.15 M KCl (approximately 3 ml KCl per

1 g liver). The homogenate was then centrifuged in a cooling

centrifuge at 40C arnd 9000 g for 10 minutes. The supernatant

(the S9 fraction) was stored at -800W in small portions,._-

These portions were slowly thawed before use. The S9 mix-was

freshly prepared (Ames et al., 1973a) and used only- on' the

same day. It was placed in a vessel with a double glass wall

until used. The hollow wall was f illed with ice to keep the

S9 mix cold.

seventy ml of cofactor solution are composed as follows:

MgCl 2 x 6 H2 0 162.6 mg

KCl 246.0 mg

glucose-6-phosphate, disodium salt 179.1 mg

NADP, disodium salt 315.0 mg

phosphate buffer 100.0 mm

S9 mix consists of this cofactor solution, S9 fraction and,

if needed, 0.15 M KCl. The amount of S9 fraction in S9 mix is

indicated in Tables 1 to 10 in percent. The S9 mix comprised. the amount of S9 fraction (x%) indicated in Tables 1, to .10,'

70k cofactor solution and (30-x)% 0.15 M KC1. The S9 fraction',

was derived from the preparation dated June 9, *1998 (protein.

content 28.3 mg per ml).- Prior to first use, each batch -was

checked for its metabolizing capacity by using reference

mutagen(s); appropriate activity was demonstrated. -'At'..-the

beginning of each experiment 4 aliquots of the S9 mix-were

plated (0.5 ml per plate) in order to assess its sterility. This was repeated after completion of test tube plating. The

sterility control plates were then incubated for 48 hours at

370C. No indication of contamination of S9 mix was found.
4.4 Test Design

The initial plate incorporation test followed the directions

of Ames et al. (1973a, 1975) and Maron and Ames (1983).

bacterial colonies

0.1 ad compound

0.1 ml bacteria . *

0.5 ml S-9 mix or * * b~uffer

2.0 ml soft agar00

45 degrees C

in waterbath petri dish with solid agar colony count using

max. 30 sec. automatic counter

mixing ircubation at

37 degree C

for 48 hous

For the mutant count, one plate was used, both with and without S9 mix, f or each strain and dose. One plate, f illed with

the solvent minus the test substance, comprised the negative

control. Each positive control also contained one plate per

strain.

The amount of solvent for the test substance and for the controls was 0.1 mi/plate.

The doses for the first trial were routinely determined on

the basis of a standard protocol: if not limited by solubility 5000 jug or 5 Al per plate were used as the highest dose.

At least five additional doses were routinely used. If less

than three doses were used for assessment, at least two repeats were performed. The results of the first experiment

were then considered as a pre-test for toxicity. However, in

case of a positive response or if at least three doses could

be used for assessment, the first trial was included in the

assessment. If the second test confirmed the results of the

first, no additional repeat was performed. Doses of repeats

were chosen on the basis of the results obtained in the first

experiment.

41'I ~/d W

Soo
The independent repeat was performed as preincubation in a

water bath at 370 C for 20 minutes. At the end of the preincubation period 2 ml of molten sof t agar were added to the

tubes, the content mixed and plated.

2 ml soft Do

bateral ckm

01 lt bactons

01I ml compound sdubat* - 0.5 i $9 mix or butffer

pitri d~ In t

orseeraon adtioi of#WACdue to egolnlt toxicity Slo co ~ute

PCORIClbbto f f 'i rctbstlal fit

37'C for4two.

For the mutant count, one plate was used for each strain and

dose. One plate, filled with the solvent minus the test sub-'

stance, comprised the negative control. Each positive control

also contained one plate per strain. In experiments without,

S9 mix buffer was used as replacement.,

The doses of this trial were determined on the basis of theresults of the plate incorporation assay. Doses are given as

ptg/tube for better separation of-plate incorporation and preincubation trials, despite the fact that ptg/plate and pLg/tube

could be used synonymously.

18
The toxicity of the substance was assessed in three ways.

The first method was a gross appraisal of background growth

on the plates for mutant determination. If a reduction in

background growth was observed, it was indicated in the 'tables by, the letter "b" after the mutant ,count. Where only. a

single" "b", - Without any other values, 'is noted for 'a',concen-.,

tration, this "b" represents three plates with reduced background'Ngrowth. (The same applies to the signs ".1,1'VI if" Dpi,

fin" or I'V, which may also be used in the tables.) Secondly,

a toxic effect of the substance was assumed when there was a

marked and dose-dependent reduction in the mutant count per

plate, compared to the negative controls. Thirdly, the titer

was determined. Total bacterial counts were taken on two

plates for each concentration studied with S9 mix. However,

if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.

The bacterial suspensions were obtained from 17-hour cultures

in nutrient broth, which had been incubated at 37 0C and

90 rpm. These suspensions were used for the determination of

mutant counts. No standardized procedure was employed to set

the bacterial suspensions at a defined density of viable

cells per milliliter, since the chosen method of incubation

normally produces the desired density. 'However, the numbers

of viable cells were established in a parallel procedure by

determining the titers. The results of these determinations

may be seen in the negative controls in Tables 1 to 10.

The dilution of bacterial suspensions used for the determination of titers was 1:1,000,000. Titers were determined under

the same conditions as were the mutations, except' that -the

histidine concentration in the soft agar was increased fivefold to permit the complete growth of bacteria.

The tests were performed both with and without S9 mix. Full

details are given in the Tables 1 to 10.

The count was made after the plates had been incubated for

48 hours at 370C. If no immediate count was possible, plates

were temporarily stored in a refrigerator.

19 COB

31a
The following criteria determined the acceptance of an assay:

a) The negative controls had to be within the expected range,

as defined by published data (e.g. Maron and Ames, 1983) and/

or the laboratories' own historical data (see Chapter 8).''

b) The 'positive controls had to show suf ficient ef fects, as

defined by the"laboratories' experience (see Chapter 8).

c) Titer' determinations had to demonstrate sufficient bacterial density in the suspension.

Only trials which complied with all three of the above criteria were accepted for assessment. Even if the criteria for

points (b) and (c) were not met, a trial was accepted if it

showed mutagenic activity of the test compound. Furthermore,

an unacceptable trial would have been repeated.

The following doses per plate were evaluated:

.zg per plate

1. Negative control 0

5000

158.

50

16

8.-Posi ive'control, Na-azide 10 (only TA 1535)

9.",Positive control, NF 0.2 (only TA 100)'

10. Positive control, 4-NPDA 10 (only TA 1537)-,

11. Positive control, 4-NFDA 0.5 (only TA 98)

12.* Positive control, Cumene 50 (only TA 102)

13-."Positive control, 2-AA 3

waas dissolved in DMSO and formed a clear color-

.100 0siulion. The positive controls were dissolved in DMSO.

The solvent used was chosen out of the following solvents, in

the order given: water, DMSO, methanol, ethanol, acetone,

ethylene glycol dimethylether, and DMF according to information given by the internal sponsor. The order of these solvents is based on their bacteriotoxic effects in preincuba- tion experiments.

20

33
No "untreated" negative control was set up for the used solvent, since sufficient evidence was available in the literature (e.g. Maron and Ames, 1983) and from our own experience

(see Chapter 8), indicating that this solvent had no -influence on the spontaneous mutant counts of the used strains.

4.5 Assessment Criteria

A reproducible and dose-related increase in mutant counts of

at least one strain is considered 'to'be a positive result.

For TA 1535, TA 100 and TA 98 this increase should be about

twice that of negative controls, whereas for TA 1537, at

least a threefold increase should be reached. For TA 102 an

increase of about 150 mutants should be reached. Otherwise,

the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.

In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation

is possible.

4.6 Study Identification and Responsibilities

4.6.1 Type of Test and Study Number

Ames-Test Screening:

4.6.2 Responsibilities

Head of Toxicology

Senior ExpertGenotoxicity

Study Director

Senior Technician

Head of Archives7

21

-311
5. Results

The colony number of each plate are listed for each dose in.K 4~&

Tables 1 to 10'. As ma. was no indicatio of4&§

bacteriotoxic effect of-Jt 16 jug, per plate. Th>~e

total bacteria counts~c en y produced results "cmarble to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. -Higher

doses had a strain-specific bacteriotoxic effect. Therefore

they could only partly be used for assessment purposes. At

500 Atg per plate, the substance started to precipitate.

None of the five strains concerned showed in the plate incorporation test a dose-related and biologically relevant increase in mutant counts over those of the negative controls.

This applied both to the tests with and without S9 mix (Tables'1 to 5) and was confirmed by the results of the preincubation screen (Tables 6 to 10).

ofmmary of ts with

in the Sa/n l~Micosome Test

S9 mix TA 1535 TA 100 TA 1537 TA 98 TA 102f

without -ye -ye -ye -ye -ye

with -ye -ye -ye -ye -ye

-ve =negative

The positive controls sodium azide, nitrofurantoin, 4-nitro1,2-phenylene diamine, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the

negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

22
%. * " '- ~ I V-,, --

6. Assessment

The Ames scre§ .~~yng doses of up to 5000 jig per

plate, showe o tt produce bacteriotoxic effects at

50 A~g-per plat~ above.y Substance precipitation occurred

at 500 Ag per-plate and above."

Evaluation *of individual -dose 1groups, with respect to relevant assessment parameters (dose effect, reproducibility),

revealed no biologically relevant variations from the respective negative controls.

In spite of the low doses used, positive controls increased

the mutant counts to well over those of the negative controls, and thus demonstrated the system's high sensitivity.

Despite his nsitivity, no indications of mutagenic effects

of oucold be f ound at assessable doses of up to

500 per plate in any of the Salmonella typhimurium

strains used.

Due to these result has to be regarded as nonmutagenic.

23
7. References

Identifying environmental chemicals causing mutation's'

and cancer

Science 204, 587-593, 1979

2Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee:

carcinogens are mutagens: A simple test system

combining liver homogenates for activation and

bacteria for detection

Proc. nat. Acad. Sci. (USA) 70, 2281-2285, 1973a

3. Ames, B.N., F.D. Lee and W.E. Durston:

An improved bacterial test system for the detection

and classification of mutagens and carcinogens

Proc. nat. Acad. Sci. (USA) 70, 782-786, 1973b

4. Ames, B.N., J. McCann and E. Yamasaki:

Methods for detecting carcinogens and mutagens with

the Salmonella/mammalian-microsome mutagenicity test

Mutation Res. 31, 347-364, 1975

5. Andrews, A.W., L.H. Thibault and W. Lijinsky:

The relationship between carcinogenicity andmutagenicity of some polynuclear hydrocarbons.

Mutation Res. 51, 311-318, 1978

6. Clayson, D.B.:

ICPEMC working paper 2/1

Comparison between in vitro and in vivo tests for

carcinogenicity

An overview

Mutation Res. 75, 205-213, 1980

1 7 T$W1

24
7. Glatt, H.R., H. Schwind, F. Zajdela, A. Croisy,

P.C.v,.acquignon-and F.-.6schi:

Mutagenicity of 43-structurally related heterocyclic ~

compounds''and its relationship to their carcinogenicity ,4

Mutation Res. 66, 307-328, 1979

8. Herbold, B., W. Buselmaier and G. Rt~hrborn:

Kritische Betrachtung zum Lebermikrosomen-Test,

einem System zum Nachweis von Punktmutationen

Arzneim. -Forsch. 26, 1705-1707, 1976

9. Herbold, B.:

Mutagenitatsuntersuchungel mit dem Lebermikrosomen-Test

Biol. Zbl. 97, 137-152, 1978

10. Herbold, B.A.:

Preliminary results of an international survey on

sensitivity of S. typhimurium strains in the Ames

-test

Toxicology Letters 15, 89-93, 1983

11.'Levin, D.E., M. Hollstein, M.F. Christman,

E.'Schwiers and B.N. Ames

A new 'Salmonella tester strain (TA 102) with A-T

base pairs at' the site of mutation detects oxidative mutagens

Proc. ,Natl. Acad. Sci. (USA) 79, 7445-7449, 1982

12. Maron, D.M. and B.N. Ames:

Revised methods for the Salmonella mutagenicity test

Mutation Res. 113, 173-21S, 1983

13. McCann, J., E. Choi, E. Yamasaki and B.N. Ames:

Detection of carcinogens as mutagens in the Salmonella/

microsome Test: I. Assay of 300 chemicals.

Proc. nat. Acad. Sci. (USA) 72, 5135-5139, 1975a

25 UB1CCOPY

32-
14. McCann 4 . N.E..Spingarn,-'J. Kobori-and B.N. Ame s:i

Detection of.'carcinogens as mutagens::Bacterial .tester 1

strains'with R factor plasi.

Po.nt'ca.Si (USA) 72, 979-983,'1975b -

15. McCann, J. and B.N. Ames:

Detection of carcinogens as mutagens in the Salmonella!.

microsome test: Assay of 300 chemicals: Discussion.

Proc. nat. Acad. Sci. (USA) 73, 950-954, 1976

16. Purchase, I.F.H., E. Longstaff, J. Sahib, J.A. Styles,

D. Anderson, P.A. Lefevre and F.R. Westwood:

Evaluation of six short term tests for detecting organic

chemical carcinogens and recommendations for their use.

Nature (London) 264, 624-627, 1976

17. Purchase, I.F.H., E. Longstaff, J. Sahib, J.A. Styles,

D. Anderson, P.A. Lefevre and F.R. Westwood:

An evaluation of-six short term tests for detecting

organic chemical carcinogens.

Brit. J. Cancer 37, 873-959, 1978

18.- Rinkus, S.J. and M.S. Legator:

Chemical characterization of 465 known or suspected

carcinogens and their correlation with mutagenic

-activity in the Salmonella-typhimurium system

Cancer Res. 39, 3289-3318, 1979

19. Zeiger, E.:

Carcinogenicity of mutagens: Predictive

capability of the Salmonella mutagenesis

assay for rodent carcinogenicity

Cancer Res. 47, 1287-1296, 1987

PUBLCcopy

26
,j,

8. Historical Controls

8.1 Plate Incorporation Method

Summary of historical .negatiiejand positive

controlso ex ri~entifedNrmf rom N~'

using, ean, ,v spren'da -

medas and ~ t

-Compound ~ Strain -

and S9 TA*1535 T100 A13 )T~9< ~

MixZ, QR Z QR 'Z Q Z~Q~

water - 16 2 9311 9 2 3 4

DMSO - 14 81 9 9 1: 3' 4

DMF 13 4 68 441 1

ethanol-- 15 4 91 15 7 2 ~25 2

acetone- 13 2 59 -12 7 1 1

.EGDE2 - 17 69 7 14

Na-azid -660 147

NF -285 65

4-NPDA -52 7 74 13

30%

water' +: 23 .4 124 18 _12 1- 31 6-

DMO + 22 5 120 20 12 2 3 5

DF + 18 2 :89 5 _11 1 27 1

~hanol + '5145: 9 3

'ctneg. 17 3' "79, 9- 8 2'~ 4

.ED2 + 19 1,19, 84 41

.'-A +151,, 17 669 208 6213 ,382'11

10%f

water + 20 4 118 17 12 3 33 5

DMSO + 17 3 111 15 10 2 3

DF + 17 87 9 34

ethanol + 24 4 98 5 9 2 3

actn+ 15 3 69 7 12 6 29 5

EGDE2 + 11 48 11 - 22

2-A). + 1159 35 11148 332 1246 21 J1126 292

2Ethylene glycol dimethylether27
4- 

4",

Summary of historical negative and positive

controls ried from

--using mean ,,aus resented'as

-Compound Strain

and S9 TA 1535 TA 100 TA 1537 TA-98 -"'

Mix Z QR Z QR Z QR Z -_QR-"

water - 13 2 66 8 11 2 28 4

DMS0 14 2 61 7 10 1 26 5

methanol- 11 - 64 - 7 - 30 -

ethanol - 11 1 57 4 8 2 27 5

acetone - 12 - 60 - 10 - 20 -

EGDE2 - 13 1 54 13 8 1 23 2

Na-azid - 729 108

NF - 251 27

4-NPDA - 52 5 84 16

30%~

water + 21 - 108 - 12 - 34 -

DMSO + 18 1 96 13 11 1 30 2

ethanol + 25 - 84, -- 12'- 4

Acetone+ 1 86 - 15 - 2.f

EGDE2 + 22 - 68 - 11 - 30

2-AA + 152 45 491 114 80. 16 5 38 34

water + 19 3 79 16 12 2 38 6

DMS0' + 18 5 85 12 13 3 '38 6

methanol+ 20 - 78 - 10 - 42 -

ethanol + 19 - 93 - 13 - 40 -

acetone + 19 - 83 - 11 - 33 -

EGDE2 - 14 4 71 20 10 5 34 2

2-AA + 179 65 1978 220 1254 53 11129 226

2) Ethylene glycol dimethylether:

28

VON*& ~UBUC C
Summary of historical negative and positive

cotosof ed from

using mean va "ues presrent)

medians (Z) and semi-Q range (QR)

Compound Strain

and S9 TA 1535 TA 100 TA 1537 TA 98

mix Z QR Z QR Z QR Z QR

water - 13 2 96 13 10 3 25 4

DMSO - 11. 2 80 9 8 1 22 3

DMF - 10 - 70 - 11 - 16 -

ethanol - 10 1 77 - 10 - 26 -

acetone - 14 1 88 11 9 1 25 3

EGDEI - 14 2 95 14

Na-azid - 683 86

NF -337 52

4-NPDA - 57 9 82 15

30%t

water + 17 - 116 - 11 - 36 -

DMSO + 15 2 111 7 9 2 30 5

ethanol + 28 - 135 - 10 - 38 -

EGDE2 + 36 - 137 - 14 - 45 -

2-AA + 157 35 571 190 82 25 494 76

*10%~

water +I 18 4 118 9 10 3 39 5

DMS0 + 14 2 101 13 9 1 31 3

DMF + 13 - 87 - 10 - 23 -

ethanol + 29 - 113 - 13 - 45 -

acetone + 16 3 114 5 11 1 43 7

EGDE2 + 16 - 118 9 10 - 36 -

2-AA + 154 34 11177 246 1225 100 11236 195

2) Ethylene glycol dimethylether

29 PUBLIC COPY
summary of historical negative and positive

r-~re from

uigmean au

medians (Z) and semi-Q range (QR)

Compound Strain

and S9 TA 1535 TA 100 TA 1537 TA 98'

Mix Z QR Z QR Z QR Z QR

water - 11 2 82 21 7 3 21 2

DMSO - 11 2 76 15 8 2 19 2

ethanol - 17 - 121 - 8 - 27 -

acetone - 16 - 92 - 9 - 20 -

EGDE2 - 17 3 95 20 9 2 18 5

Na-azid - 643 145

NF -284 74

4-NPDA - 65 11 58 9

3 01

water + 16 - 127 - 6 - 26 -

DMSO + 18 4 105 16 10 3 37 9

EGDE2 + 15 - 140 - 10 - 27 -

2-AA + 220 23 448 203 88 21 478 86,

10%

water + 12 4 100 19 8 2 27 7

DMSO + 14 3 98 21 10 2 29 6

ethanol + 25 - 125 - 13 - 47 -

acetone + 15 - 113 - 10 - 32 -

EGDE2 + 18 - 118 11 12 1 29 3

2-AA + 145 - 39 812 342 164 40 11127 223

2) Ethylene glycol dimethylether

30PULCCP
Summary of historical negative and positive

contro d from

Using mean values presen

medians (Z) and semi-Q range (QR)

Compound Strain

and S9 TA 1535 TA 100 TA 1537 TA 98

mix Z QR Z QR Z QR Z QR

water - 10 2 89 14 8 2 22 3

DMS0 - 9 2 82 9 9 2 21 4

DMF - 12 - 94 - 9 - 25 -

methanol- 12 - 95 - 11 - 15 -

ethanol - 7 2 56 10 8 2 24 11

acetone - 7 3 79 12 6 2 18 3

EGDE2 - 11 3 79 10 8 2 16 6

Na-azid - 706 98

NF - 263 33

4-NPDA - 105 35 138 23

30%~

water + 13 - 98 - 7 - 29 -

DMSO + 11 - 96 - 5 - 22 -

EGDE 2 + 10 - 115 - 7 - 22 -

2-AA + 152 24 594 140 72 29 489 93

10%-

water + 13 2 112 15 8 2 30 7

DMS0 + 11 2 105 15 8 2 27 5

DMF + 13 - 106 - 9 - 36 -

methanol+ 20 - 119 - 11 - 32 -

ethanol + 10 3 84 11 4 2 28 4

acetone + 10 - 106 11 6 3 28 2

EGDE2 + 14 3 114 18 8 2 29 10

2-AA + 171 49 11111 330 1103 73 11301 2 08-

2) Ethylene glycol dimethylether

31PULCC y

43
Summary of historical negative and positive

cotop.2~, f rom

usnmean vaues preset

medians (Z) and semi-Q range -(QR)

Compound Strain

and S9 TA 1535 TA 100 TA 1537 TA 98 TA 102

mix Z OR Z QR Z QR Z QR Z OR

water - 9 1 86 10 9 2 22 4 301 24

DMSO - 9 2 81 13 8 2 20 4 292 21

ethanol - 9 - 67 -- 8 - 24 ----

EGDE2 - 9 - 74 -- 9 - 24 - 237 -

Na-azid - 739 71

NF - 253 25

4-NPDA - 139 16 158 18

Cumene - 624 103

10%~

water + 11 1 107 11 10 2 30 3 361 37

DMSO + 11 2 103 16 10 3 30 5 340 20

ethanol + 10 - 86 -- 9 - 42 ----

EGDE2 + 10 - 97 -- 12 - 30 - 267 -

2-AA + 204 48 1540 228 281 77 1461 163 829 147

2) Ethylene glycol dimethylether

PUBrLIC COPYO

32
Summary of historical negative and positive

coto d from

uigmean auspen

medians (Z) and semi-Q range '(QR)

Compound Strain

and S9 TA 1535 TA 100 TA 1537 TA 98 TA 102

mix Z QR Z QR Z QR Z QR Z QR

water - 9 2 88 12 8 1 23 4 228 31

DMSO - 9 2 70 8 7 1 19 3 220 30

EGDE2 - 12 2 79 9 7 1 18 5 214 21

Na-azid - 741 80

NF - 224 22

4-NPDA - 118 17 155 20

Cumene - 430 61

10%O

water + 11 1 103 19 9 2 35 4 287 30

DM50 + 10 2 89 7 8 2 27 6 288 28

EGDE2 + 11 2 95 12 7 1 29 10 305 34

2-AA + 150 36 1161 208 222 62 1343 220 555 93

2) Ethylene glycol dimethylether

PUBLJC COPY

33
8.2 Preincubation Method

Summary of historical negative and positive

cotosoe iensefre from

using mean va ues present

medians (Z) and semi-Q range (QR)

Compound Strain

and S9 TA 1535 TA 100 TA 1537 TA 98

mix Z QR Z QR Z QR Z QR

water - 15 2 129 10 13 2 34 5

DMS0 - 13 3 94 8 10 1 26 4

acetone - 15 - 123 - 13 - 27 -

Na-azid - 689 106

NF -475 67

4-NPDA - 60 10 101 22

10901

water +~ 20 4 140 10 12 2 45 4

DM50 + 16 3 114 11 10 1 34 5

acetone + 16 - 141 - 114 - 33 -

2-AA + 1166 20 11176 149 1255 114 1196 206

34
Summary of historical negative and positive

controls o med from

using mean v e rsn

medians (Z) and semi-Q range (QR)

Compound Strain

and S9 TA 1535 TA 100 TA 1537 TA 98

mix Z QR Z QR Z QR Z QR

water - 13 2 128 13 10 1 22 1

DMS0 - 15 4 120 21 9 2 24 3

ethanol - 10 - 168 - 7 - 26 -

acetone - 15 - 108 - 11 - 24 -

EGDE2 - 14 1 126 20 11 - 20 2

Na-azid - 754 162

NF - 443 56

4-NPDA - 65 13 73 8

10%~

water + 15 2 153 25 9 1 26 3

DMSO + 15 2 129 20 11 1 34 5

ethanol + 19 - 131 - 10 - 28 -

acetone + 19 - 147 - 12 - 36 -

EGDE2 + 16 2 152 23 12 3 30 8

2-AA + 167 25 11188 138 1226 44 11100 198

2) Ethylene glycol dimethylether

35
Summary of historical negative and positive

coto Wf-O c from

medians (Z) and semi-Q range (QR)

Compound Strain

and S9 TA 1535 TA 100 TA 1537 TA 98

mix Z QR Z QR Z QR Z QR

water - 11 2 131 18 8 2 19 6

DMS0 - 10 2 99 10 8 2 22 5

methanol- 12 - 139 - 6 - 18 -

ethanol - 9 2 103 10 10 4 20 4

acetone - 11 - 113 - 8 - 23 -

EGDE2 - 13 2 105 22 10 2 20 5

Na-azid - 787 116

NF -402 41

4-NPDA - 98 33 137 26

10!k

water + 13 2 163 25 9 2 29 7

DMSO + 11 3 115 13 8 2 29 6

methanol+ 20 - 139 - 7 - 24 -

ethanol + 14 2 118 9 9 3 24 6

acetone + 14 - 175 - 7 - 31 -

EGDE 2 + 13 2 142 19 10 2 33 7

2-AA + 1185 35 j1357 166 j178 55 1268 208

2) Ethylene glycol dimethylether

36
Summary of historical negative and positive

contro ex ed from

uigmean values present

medians (Z) and semi-Q range (QR)

Compound Strain

and S9 TA 1535 TA 100 TA 1537 TA 98 TA 102

mix Z QR Z QR Z QR Z QR Z QR

water - 11 2 118 17 11 2 24 6 326 20

DMS0 - 9 1 91 9 9 2 24 5 303 34

ethanol - 9 - 114 -- 9 - 19 ----

EGDE2 - 12 - 113 -- 9 - 23 1 304 --

Na-azid - 735 69

NF - 390 47

4-NPDA - 143 15 172 24

Cumene - 708 116

10k

water + 13 2 136 26 11 2 33 5 378 29

DMS0 + 12 2 109 13 9 2 31 4 348 34

ethanol + 11 - 137 -- 8 - 29 ----

EGDE2 + 13 2 142 14 13 - 36 - 350 --

2-AA + 1218 4311626 152 1265 5011306 1911 676 95

2) Ethylene glycol dimethylether

37

49,
Summary of historical negative and positive

contolsof exnerimnt-erformed from

uslanvauVes presen

medians (Z) and semi-Q range (QR)

Compound Strain

and S9 TA 1535 TA 100 TA 1537 TA 98 TA 102,

mix Z QR Z QR Z QR Z QR Z QR

water - 11 3 100 19 9 2 31 9 252 32

DMS0 - 9 2 80 9 8 1 22 7 271 23

EGDE2 - 12 1 122 16 8 1 28 3 276 13

Na-azid - 747 71

NF - 278 30

4-NPDA - 127 21 183 27

Cumene - 602 97

10%k

water + 13 2 117 22 10 2 35 8 331 45

DMS0 + 11 2 94 9 8 2 28 8 337 33

EGDE2 + 14 2 139 24 8 2 39 5 400 62

2-AA + 172 39 1318 154 164 69 1239 143 601 93

2) Ethylene glycol dimethylether

38
Table 1 Study N~.~e

study Director Technician

Date

Strain.;

REVERT'ANT'S PER PLATE TITER QWOTIEW~

Dose/Plate 10!k Dilution per ml

(Qug/Plate) -S9 M SD +S9 M SD 10-6 10+8 -S9 +59

rMO6 6 / I 11 / 129 12.9 1.0 1.0

16 8 8 / 13 13 / 113 11.3 1.3 1.2

50 7 7 / 12 12 / 131 13.1 1.2 1.1

158 4 4 / 9 9 / 47 4.7** 0.7 0.8

500 7 P 7 / 12 P 12 / 3 0.3** 1.2 1.1.

1581 8 P 8 / 9P 9 / 8 0.8** 1.3 0.8

5000 8 P 8 / lP 0 10 I P / 1.3 0.9

Na-azide 607 607 // / 84 8.4** 101.2* /

10

2-PA / 200 200 / 25 2.5* I 18.2*

3

*mutagenic effect **: bacteriotoxic effect

%not tested P: Precipitation

M: Mean SD: Standard-Deviation

-S9: without S9 Mix +S9: with S9 Mix

39I

51
Table 2 Study Number

REVERTANTS PER PLATE TTR QUOTIENT

Dose/Plate 1O1; Dilution per ml

(jig/Plate) -S9 M SD +S9 M SD 10-6 10+8 ..g9 +S9

£E4SO 62 62 / 96 96 / 128 12.8 1.0 1.0

16 64 64 / 113 113 I 85 8.5 1.0 1.2

50 56 56 / 67 67 / 44 4.4,** 0.9 0.7

158 52 52 / 77 77 / 4 0.4** 0.8 0.8

500 41 P 41 66 6B 66 / 0 < 0.l** 0.7 0.7

1581 31 P 31 / 46 P 46 / P/ 0.5 0.5

5000 42 P 42 / 36 P 36 / P /0.7 0.4

NF 250 250 // / 103 10.3 4.0* /

0.2

2-AA 1065 1065 / 44 4.4** /

3 7

*: ntagenic effect *:bacteriotoxic effect

%not tested B: Background lawn reduced P: Precipitation

M: Mean -SD: Standard-Deviation

-S9: without S9 Mix +S9: with S9 Mix

40
Table :3 suyNr

Study Director

REVERI'ANI'S PER PLATE TITER QflIET

Dose/Plate 10%; Dilution per ml

(jhg/Plate) -S9 M SD +S9 M SD 10 6 10+8 -S9 +S9

UvJSO 5 5 / 7 7 / 77 7.7 1.0 1.0

16 6 6 / 10 10 / 85 8.5 1.2 1.4

s0 5 5 / 7 7 / 60 6.0 1.0 1.0

158 2 B 2 / 5 5 / 51 5.1 0.4 0.7

500 2 P 2 2 2P 2 / 30 3.O** 0.4 0.3

1581. 2 P 2 3 3P 3 / 12 1.2** 0.4 0.4

5000 3 P 3 6 6P 6 / 15 1.5** 0.6 0.9

4-NPDA~ 120 120 // / 70 7.0 24.0* /

10

2-AA 278 278 / 59 5.9 / 39.7*

3

*:mutagenic effect **: bacteriotoxic effect

%not tested B: Background lawn reduced P: Precipitation

M: Mean SD: Standard-Deviation

-S9: without S9 Mix +S9: with S9 Mix

41
Table :4 SuyMStudy Director

Dose/Plate 1echDilu in r

(j~g/P~ate) M -9 SD+S9 M Da1te 1+ 9 ,s

Do0e7Plat 170 17uto 95 pe 950.l.

158 7 7 / 13 13 / 33 3.3** 0.5 0.7

5000 7 P 7 / 14 P 14 / 14 1.4** 0.5 0.8

4-NPDA 160 160 // / 108 10.8 12.3* /

0.5

2-AA I1; 1401 1401 / 90 9.0 / 77.8*

3

*: utagenic effect *:bacteriotocic effect

k: not tested P: Precipitation

M: Mean SD: Standard-Deviation

-S9: without S9 Mix +S9: with S9 Mix

42
Table :5 study Nunter

study Director

REVERTANT'S PER PLATIE TITE,;R

Dose/Plate 10% Dilution prm

Q.Ag/Plate) -S9 M SD +S9 M SD 10-6 10+8m -59 +S9

UISO 136 136 / 227 227 / 115 11.5 1.0 1.0

16 150 150 / 252 252 / 102 10.2 1.1 1.1

50 180 180 / 250 250 / 68 6.8 1.3 1.1

158 163 163 / 254 254 / 26 2.6** 1.2 1.1

500 141 P 142.1 147 147 / 32 3.2** 1.0 0.6

1581 108 P 108 / 129 P 129 / 36 3.6** 0.8 0.6

5000 115 P 115 l US P 115 / P I0.8 0.5

Cunene 371 371 /% / / 96 9.6 2.7* /

s0

2 -AA % k 790 790 / 86 8.6 / 3.5*

3

*: nutageaic effect **: bacteriotoxic effect

!k: not tested P: Precipitation

M: Mean SD: Standard-Deviation

-S9: without S9 Mix +S9: with S9 Mix

43
Table :6 study Nme

DilutIo Study Direcor

Technicia

DateTIR

DoseTube1006Diluionper ml (jig/Tube) -S9 M SD +S9 M SD 10-6 10T8 -59 +S9

rMO7 7 / 11 11 45 4.5 1.01 1.0-

16 10 10 / 7 7 / 19 1.9 1.4 0.-.6

50 9 9 / 8 8 / 26 2.6 1.3 0.7

158 8 8 / 7 7 / 5 0.5* 1.1 0.6

500 9 P 9 10 lP 10 I 0 <0. 1** 1.3 0.9

1581 lop P 10 4 P 4 / 0 < 0.l1** 1.4 0.4

5000 li P 11 / 7P 7 / p/ 1.6 0.6

Na-azide 588 588 I/ / 30 3.0 84.0*/

10

2-AA / / 119 119 / 18 1.8 I10.8*

3

*: nutagenic effect **: bacteriotaxic effect

%k: not tested P: Precipitation

M: Mean SD: Standard-Deviation

-S9: without S9 Mix +S9: with S9 Mix

44
D~se3 le 70 siludyo pmer m

(jig/Tube)~~~sud S 0. DirMector 1+ -9 5

106' I 06 W 97 T 6.6 .0>1.

5000vbe 8P 86 /) 79 75 D 10- 1V 0.8 0.8

----------------------- 7------------------------------------- -------- EM 10ua6ni e 1fec 97: 97fj effect

M: 93a SD 90 0 6 6.t0. 0.

tete not50 86 8664 6. 07 .

S9 t15t S90 Mix 949 wit S9 Mix08 .

500 78 61 7 61 01** 0.7 0.

158178 5 P78 P 5 P 07 0.

5000 86 P 86 5 7 . .

NF431 3. 8 83579
Table 8 Studyr Number

JIM Dlutio er

(~Lg/ube) 59 M D +S M SD 10-6........+5 5 5 / 6 6 83 a'd .3'~ 1z0~,-1. 

1581ue 3P9 M /D P M /D 1- 0.1r0.6

50 3P 3 P 86 8. 1.4 0.6 8

4-NaIA 152 152 // / 130 13.0 30.4* /

10

2-AA I k 157 157 / 107 10.7 I 26.2k

3

*:mutagenic effect **: bact-priotaxic effect

not tested B: Backgrcund lawn recliced P: Precipitation

M: Mean SD: Standard-Deviatian

-S9: withcut S9 Mix +S9: with S9 Mix

46

4,7
Table 9 study Numbier,

DoeStudy DiS9ctor

(ag/Tue) M -S9 SD S9 M S 10.6 0i -

UvTS w13 13 1 .17 7 / 29 12.9 1.Date~

158b 15 15- 18+ 18q / 5 6 ** 12 .

500 16 16 / 7 1 7 185 /'. 11 . 0. 1.1

1581 4 P 4 / 13 P 13 / 18 P 1.8** 0.3 0.8

5000 4 P 4 10 lp io / PI 0.3 0.6

4-NPDA 152 152 / /- 52 15.2 11.7* /

0.5

2-M / 1226 12:26 / 130 13.0 /.7.

*? nutagenic effect *.bacteriotc~cic effect

%not tested P: Precipitatioi

M: Y9am SD: Standard-Deviatiori

-S9: without S9 Mix +S9: with S9 Mix

47
Table 10 St~udy Nmber

Diuyirecpr 

D~ t

16 4824 / 23723 /33 33 1 0'F

50 215 215 / 227 22 / 33e 33.7i.9i0.

158~~~~ 17.13..23.33..75 2 . 0.7 .8 500 35 / P135 18 188 / 17 167 0.a0.

1581 38 P138 199 199St22aPi .6 0.6 0.

------------------------------------------ ------------ --- --- --

-S:w -~tS9 M -4SD wihS9 Mix 06 IY-8 _9''s

16 48 48237 23 33 3.4 1 .0 0.8

50 15 15227 22 33 337 .948.

158 23 73 73 227 275 0. 0.
By

Unti tme as the results in this report are published, they may only be used with the permission of

Nocopying of this report whether in its totality or as an extract or extracts, is permitted.

705
Table of Contents

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

2. INTROUCTION .................................................... 2

3. MATERIALS AND METHODS ..................................... 3

3.1 Substance tested.................................................. 3

3.2 Experimental animals and living environment...................... 3

3.3 Experimental methods ............................................ 3

3.3. 1. Observational period, conclusions and autopsy .................... 4

3.3.2 Acute oral toxicity .............................................. 4

3.3.3. Acute dermal toxicity.............................................4

3.3.4. Acute inhalation toxicity (powder) .............................. 4

3.3.4. 1. Analysis of test atmosphere.................................... 5

3.3.4.2 Particle analysis in test atmosphere ............................ 5

3.3.5. Examination of irritation and cauterisation of rabbit skin

3.3.5. 1. Procedure adopted ............................................ 5

3.3.5.2. Results ...................................................... 6

3.3.5.3. Evaluation criteria ............................................ 6

3.3.6. Test for irritation and cauterisation of the eye

3.3.6. 1. Procedure adopted ............................................ 6

3.3.6.2. Results ...................................................... 6

3.3.6.3. Evaluation, criteria............................................ 7

3.3.7. LC5O.LD5O - calculation ....................................... 7

3.3.8. Quality control (GLP)...........................................7

4. RESULTS ........................................................ 8

4. 1. Acute oral toxicity ............................................. 8

4.2. Acute dermal toxicity .......................................... 8

4.3. Acute inhalation toxicity (powder)..............................9

4.4. Examination of irritation and cauterisation of rabbit skin ........ 10

4.5. Examination of irritation and cauterisation of rabbit eye ......... 10

5. EVALUATION ................................................... 12

APPENDIX.......................................................... 13

Analysis of particle distribution ....................................... 13

APPENDICES ....................................................... 15

Appendix 1: Evaluation table - skin.................................... 15

Appendix la: Evaluation criteria - skin................................15

Appendix 2: Evaluation table - eye.................................... 16

Appendix 2a: Evaluation criteria - eye ................................ 17

Bibliography......................................................... 18
STAGE I EXPERIMENTS IN INDUSTRIAL TOXICOLOGY

1. SUMMARY

The methodology adopted was in accordance with the relevant OECD guidelines.

LD50-ora

Rat (fasted) :bigger than 5000 mg/kg bodyweight.

LD50-derma

Rat (exposure 24hr): bigger than 5000 mg/kgy bodyweight.

LD5O-inhalation (powder)

Rat (exposure 4 hr) bigger than 1267mg/rn air

(maximum technical ly producible concentration)

Local irritation (rabbit)

Skin: the experimental subject can be considered not to exhibit irritation of the skin. (exposure 4 hr, flank).

~the experimental subject can be considered not toe

Evaluation: the experiments indicate Itha xhibited no

evidence of oral, dermal or inhaled toxicity under te prevailing "experimental conitT nso significant

toxicological potential to skin or eye could be established.

The experiments indicate that proper use of the product presents no potential for acute risk to the health of

human beings.
- STAGE I EXPERIMENTS IN INDUSTRIAL TOXICOLOGY

2. LIM TION onfidential

On!" , Ihandled txct N~dbetr=no y ansiAm was

tested on rabbits.

These experiments were carried out in order to assess the possibility of any risk to health for people using

the product for industrial purposes.

_________________________exhibits the following structural formula:

[refer to original]

Studies no.:

Acute oral toxicity:

Acute dermal toxicity:

Acute inhalation (in powder fo

Local Irritation (rabbits)-
PAGE 05/20

STAGE 1 EXPERIME UiFSTRIAL TOXICOLOGY

3. MATERIALS AND METHODS Cniet

Propertyv

3.1. Substanggg5ted

3.2. Exnerimental animals and living environment

Young adult Wistar rats Bor: WISW (SPF-Cpb) with a body weight of approx. 160 - 200 g from the

breeder WINKELMANN, Borchen in Paderborn.

The animals were kept in a conventional manner in Makrolon cages, type 111, 5 animals per cage in an

experimental animal laboratory: lab. Temperature 19-25* Celsius, relative humidity of the air 40-60%

light/dark rhythm 12 hours. As cage litter wood granules with a low dust content were used.

The animals were fed on "Altromin 1324 - animal food for rats and mice" (producer Altromin GmnbH,

Lage) and unlimited water from the domestic supply.

Animals were identified by their colour and cage.

Animals were settled into their environment for at least 5 days before the commencement of the

experiment.

All animals were randomly allocated to the experimental groups.

Adult Albino rabbits, pedigree HC:NZW (breeder: Hacking & Churchill Ltd., Huntingdon, UK). They were

fed a standard diet ("Ssniff K4 from Versuchstierdi~ten GmbH, Soest, Westfalen) and unlimited water. The

rabbits were kept separately in individual metal wire cages (type III, high). For further living conditions see

rats.

3.3. Exnjerimental Methods

The methodology adopted was in accordance with the OECD "Guidelines for Testing of Chemicals; section

4: Health Effects.
STAGE 1 EXPERIMENTS IN INDUSTRIAL TOXICOLOGY

Con

3.3. 1. Observational period, conclusions and autopsy.

The body weight of the rats was recorded before application/exposure and after that at weekly intervals,

their behaviour and appearance was recorded several times on the day of application/exposure and after

that twice daily. During the 4-hour head/nose exposure no observations were recorded.

In the case of the experiments for acute oral, dermal and inhalation toxicity the observational period was 14

days. After that the animals were put down using ethyl ether (oral and dermal application) or with Evipan (inhalation) and were given a pathological anatomical examination. -- 4.

In the tables the column 'Toxicological Results" the following abbreviations were used:

I S number = number of animals which died.

2nd number =number of animals with symptoms

3rd number =number of animals used in the experiment.

3.3.2. Acute oral toxicity

The test sample was dissolved in demineralised water using Tylose (0.5%) and was administered at a

volume of l0mi per kg bodyweight for 16 hours to fasted male and female animals (5 rats per dose and per gender); this procedure was carried out once using a gastric feeding tube. Two hours after this application

the animals were offered food.

3.3.3.Acute dermal toxicity

The experiments were carried out on male and female rats using the foil body-wrap method of NOAKES

and SANDERSON (Brit. J. Ind. Med. 26,59, 1969).

The dose was individually weighed out, dissolved in physiological cooking salt, and applied to the shaved

skin of the back of the rats (5 animals of each gender in each dose group). The application was covered

with aluminium foil and fixed to the skin using bandages. The exposure time was 24 hours. After the

removal of the bandages the skin areas treated were cleaned with soap and water.

3.3.4. Acute inhalation toxicology (powder)

The experimental material was administered in the form of a powder inhalant kept in dynamic condition in

a 20 litre inhalation chamber. The inhalation was carried out at intervals with a dispersion pressure of

approx. 50 kPa, the duration of each pulse being 0.5 seconds

PUBLIC copy
with a pause of 3 seconds between each pulse. During the exposure the rats were kept stationary in tubes

(head/nose exposure) 5 male and 5 female rats were exposed to the powdered inhalant at each session of

exposure.

3.3.4. 1. Analysis of the test atmospher

The concentration of the inhalant was gravimetrically determined on a membrane B-acetate filter. The

sample extraction was carried out in the breathing field of the'experimen tal animals at a'suction velocity ... ,

which enables the analysis of the entire particle spect m (suction velo'city ap~rox125 cm p ;'rsecod).' N .: _z__

The concentrations recorded refer to mg of per m3 of air.

3.3.4.2. Particle analysis in the test atmosphere

The sample extraction from the inhalation chamber was carried out under conditions comparable to those in

3.3.4. 1. Analysis of test atmosphere may be found below.

The particle analysis was carried out in relation to its mass with the use of an ANDERSEN CASCADE

IMPACTOR, the specifications of which may be found in the appendix, page 13.

The particles impacted on the impactor gradations were measured on a gravimetric basis.

The calculation of the distribution-specific p-maraintewas carr~giwpawith.an anmnoriate computer

programme on an HP 3000 computer of tiI

The parameters calculated were: the MMAD (Mass Median Aerodynamic Diameter, the NMAD (Nurber

Median Aerodynamic Diameter) and the geometric standard deviation. The alveolar mass was taken as the

relative mass proportion: this shows a MMAD smaller than 5

3.3.5.Examination of irritation and cauterisation of rabbit skin

3.3.5. 1. Procedure adopted

On the day befor e application of the test sample the skin on the flanks on both sides of 3 adult rabbits was

shaved over an area of 6x6 cm with an electric hair cutter. Only animals with undamaged skin were used.

PUBLIC COPY

-74
-STAGE I EXPERIMENTS IN INDUSTRIAL TOXICOLO Y

Confidential -

Each animal was administered 500 mg of the test sample made into a paste by the addition of water and

spread onto a cellulose dressing (approx. 2.5 x 2.5 cm). A second dressing was dampened with water and

placed on the opposite flank of the animal. Both dressings were then fixed in place with an elastic adhesive

plaster. After 4 hours the dressings were removed and the exposed area of skin was washedw wiA water.

3.3.5.2. Results

The results of the tests at various times after exposure are given in table 4. The extent of

erythema/encrustation formation, i.e. oedema formation was recorded in accordance with DRAIZE (see

appendix 1,* OECD Guidelines); all other noticeable findings were also recorded.

The part of the skin not treated served as a control.

3.3.5.3 Evaluation Criteria

Assessment of erythema/encrustation formation on the basis of the DRAIZE scale of measurement was

made by calculating the primary skin irritation values (see appendix Ila) at intervals of 24, 48 and 72 hours

and by averaging over the respective periods of time. Further details of the assessment criteria are given in

appendix Ia.

3.3.6.Test for irritation and cauterisation of the eve

3.3.6. 1. Procedure adopted

3 adult rabbits of each gender were treated with 100 -- of the test sample in powder form; this corresponds

to a weight of approx. 60 mg, applied to the conjunctiva tissue of the eyelid.

After application the eyelids were held together for approx. I second. The eye on the opposite side was not

treated and served as a control. A wash of physiological NaCI solution was applied to the eye which was

treated after a period of 24 hours.

3.3.6.2. Results

Results of the application were recorded at the intervals indicated in table 5. Assessment is made of the

state of the cornea (degree of clouding and the area affected), the iris (hyperaemia, reaction to light) and

conjunctiva (erythema and chemosis) in accordance with DRAIZE (see appendix 2, i.e. OECD Guidelines).

PUBL ,IC CoP

77
STAGE I EXERIMENTS IN INDUSTRIAL TOXICOLOGY

The flow (outflow) of tears is also measured. All other findings not covered by the DRAIZE scale were

also recorded. Cornea and iris findings are taken with the use of optical instruments. To determine the

area of cornea affected after a period of 24 hours I drop of a 1% fluorescein solution is applied to the

cornea of the eye concerned: in the case of positive results this is also carried out for any finding recorded

after the 24 hour period. After that a wash of physiological NaCI is applied to the eye. UV light is used to

determine the extent of cornea results..

3.3.6.3. Evaluation Criteria

In the assessment of results only those findings which persist for 24 hours or longer are used. The

assessment criteria and the mode of calculation for eye irritation values are given in appendix 2a.

3.3.7. LC50ILD5O Calculation

Wherever a calculation of average lethal dose/concentration (LD5OILC5O) is possible, it is carried out on

the computer (HP 3000) in accordance with A.P. ROSIELLO, J.M. ESSIGMANN and G.N. WOGAN (J.

Tox. And Environ. Health 3, 797-809 (1977)).

This procedure is based on the Maximum Likelihood Method of C.I. Bliss (QJ. Pharm. Pharmocol. 11,

192-2 16 (1938).

3.3.8. Quality control (GLP)

The experiments were carried out in accordance with OECD Principles of Good Laboratory Practice (GLP)

(Bundesanzeiger 35 3-16 (2nd March 1983)).

In accordance wijhiib~cenrinciples the experiments were recorded i nerimental plans which are

archived in tq(Archive) together with all other

papers relating to-theseexperim-ens.

(pqY
4. RESULTS

4. 1. Acute Oral Toxicity

The results of the experiments on fasted rats are listed in the following table.

Table 1: Acute Oral Toxicity - rat fasted

Group Dose Toxicol. Duration of Time of Mortality

(mg/kg result symptom death(%

bodyweight)

rat

15000 01015 -- 0

rat

15000 0/0/5 - 0

LD50 greater than 5000 mg/kg bodyweight.

Symptoms: the treatment was tolerated without any symptoms.

Autopsy results

Animals killed at the end of the observation period: there was no evidence of any substance-induced

macroscopically recognisable organ damnage.

4.2.Acute dermal toxicity

The results of the experiments on rats are listed in the following table.

Table 2: Acute dermal toxicity - exposure: 24hr

Group Dose Toxicol. Duration of Time of Mortality

(mg/kg result symptom death(%

bodyweight)

rat

15000 0/0/5 -- 0

rat

1 5000 010/5 - -0

LD50 greater than 5000 mg/kg bodyweight.

Fa!BL! COPY'a

'70W
-TGE I EXPERIMENTS IN INDUSTRIAL TOXICOLOGY

SYMptoms

The treatment was tolerated without any clinical symptoms.

Localised results on the area of treatment were not found.

Autopsy results

Animals killed at the end of the observation period: there was no evidence of any substance-induced

macroscopically recognisable damage to the visceral organs.

4.3.Acute Inhalation Toxicity (powder)

The results of the test for acute toxicity of the test sample powder with a period of exposure of 4 hours are

summarised in the following table:

Table 3:Acute Inhalation Toxicity -powder (exposure 4 hours)

Group Concentration Toxicol. Duration of Time of Mortality

(mg/kg result symptom death M%

bodyweight)

rat

1267 0/0/5 - 0

rat

1267 010/5 - -0

(maximum concentration technically possible to manufacture)

LD50 greater than 5000 mg/kg bodyweight.

The analysis of particle distribution gave a relative alveolar mass of 56% (MMAD 4.6- geometric

standard deviation 1.8). The individual results of the particle analysis are given in the form of graph and

table in the appendix (see page 13, 14).

Symptoms: The exposure was tolerated without any symptoms.

Autonsv - Animals killed at the end of the observation period: there was no evidence of any

substance-induced macroscopically recognisable damage to the organs.

?UBLI' C CPY

7'o
11 r6 AGE I EXPERIMENTS IN INDUSTRIAL TOXICOLOG

4.4. Examination of irritation and cauterisation of rabbit skin

The individual skin reactions are summarized in the following table 4.

Table 4: test for skin irritation (exposure 4 hours)

Animal Body DRAIZE-scale after irritation value

No. weight lhr 24hr 48hr 72hr 7d 14d

EOEOEOEOEOEO E 0

E91 (3.7kg)OO00O00O00O00O0- - 0 0

E90 (3.4k ) 00 00 00 0 00 0- - 0 0

E99 (3.3k-.) 00 0 00 0 00 00- 0 0

Evaluation see appendix I

E-- erythema

0 oedema

-=not tested

The test sample is therefore considered "not irritating to the skin" (Evaluation see Appendix Ila).

PUBI ICloPY
STAGE 1 EXPERIMENTS IN INDUSTRIAL TOXICOLOGY

4.5.Examination of irritation and cauterisation of rabbit eThe individual results at the various times of evaluation are summarized in the table below.

Table 5: Test for irritation of the eye (exposure 24 hr)

Animal no. organ symptoms DRAIZE SCALE Irritation

(Body weight) examined lhr 24hr 48hr 72hr 7d -14d 21d value

D52 cornea-clouding, 0 0 0 0 0 - - 0.0

(3.l1kg) -area 0 0 0 0 0 -

iris 0 0 0 0 0 - - 0.0

conjunctiva R I I I 1 0 - - 1.0

S 2 0 0 0 0 - - 0.0

T 2 0 0 00-

D66 cornea-clouding, 0 0 0 0 0 - 0.0

(3.5kg) -area 0 0 0 0 0 - -

iris 0 0 0 0 0 - - 0.0

conjunctiva R I I I 1 0 - - 1.0

S 2 0 0 0 0 - 0.0

T 3 0 0 0 0-

D80 cornea-clouding 0 0 0 0 0 - - 0.0

(3.0kg) -area 0 0 0 0 0 - -

iris 0 0 0 0 0 - - 0.0

conjunctiva R 1 1 0 0 0 - - 0.3

S 10 00 0- 0.0

T 10 00 0-

Evaluation: see appendix 2

R=reddening, S--swelling, T=tear flow

- -not tested

The low degree of hyperaemia of the conjunctiva is seen as the result of mechanical

irritation (caused by the physical composition of the test material).

Therefore the test material is considered "not irritant to the eye" (Evaluation see

appendix 2a).

PI!uc OPY
)TAGE I EXPERIMENTS IN INDUSTRIAL TOXICOLOGY

o tiail

xhbtdno acute oral, dermal or inhalation toxicity on

There was no significant potential for toxicity in respect of the skin or eye.

Given appropriate use of the product there is therefore no potentially acute risk to health for human beings.

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

7C py
APPENDIX

Analysis of Rarticle distribution

N Impactor Cut Off Mass Relative Cumulative degree diameter

(absolute) Mass M% Mass (%)

1 0.01-0.4 0.01 .00 .00 .00

2 0.4'-0.7 0.4 .00 .00 .00

3 0.7 -1.1 0.7 .10 1.20 .00

4 1.1 -2.1 1.1 .60 7.23 1.20

5 2.1 -3.3 2.1 1.70 20.48 8.43

6 3.3 -4.7 3.3 1.70 20.48 28.92

7 4.7 -5.8 4.7 .90 10.84 49.40

8 5.8 -9.0 5.8 2.40 28.92 60.24

9 9.0 20.0 9.0 .90 10.84 89.16

- - --- - - - -- --- --- - - -- - - - - ---------------- -------

Mass Median Diameter (MMAD): 4.62 --

Geometrical Standard Deviation: 1.84

Number Median Diameter (NMAD): 2.86 --

Surface Median Diameter (SMAD): 3.94 --

System: ANDERSEN IMPACTOR

Air propulsion rate 4.00 litres/minute

Duration of use 1200.00 seconds

Alveolar proportion (small than 5 -

1. relating to mass: 56% (measured)

2. relating to no. of particles: 82% (extrapolated)

EFFECTIVE CUT-OFF DIAMETER (ECD): the cumulative distribution is related to the Effective Cut-off

Diameter. The ECD represents a constant

factor in the use of the impactor.
TSI NUTRILTXCLG

MMAD= 4.62

Sigma-- 1.83

Norm. rel. Mass concentration

Aerodynamic diameter (mcm)

1. Mass- 2. surface- 3.particle distribution

IL Ar -4

Ilb
% proportion smaller than ECD

Aerodynamic diameter (mcm)

177
APPENDICES

Appendix 1: Evaluation-table - skin

Ervthema - reddening/encrustationl of skin

-no reddening o0

-slight, hiardly perceptible reddening

-easily perceptible reddening 2

-moderate to strong reddening 3

deep reddening to slight encrustation

(changes a ffecting deep into the skin) 4

Oedema - swellin2

-no swelling 0

-very slight, hardly perceptible swelling I

-slight swelling, edges well defined 2

-moderate swelling (edges approx. 1mm

raised) 3

marked swelling (edges raised more than

I1mm, all exposed area affected) 4

Appendix I a: Evaluation criteria - skin

Evaluation Primary skin irritation value

-no primary irritation 0 to 0.99

-slight primary irritation 1.0 to 1.99

-moderate primary irritation 2.00 to 2.99

-strong primary irritation 3.00 to 4.00 (maximum value)

- cauterization: deep wounds such ulceration, necrosis, formation of scars on removal of the dressing or

within the observation period.

Calculation of the skin irritation values:

In calculating the values the DRAIZE scale was used approx. 24, 48 and 72 hours after application. The

DRAIZE values of erythema! scar formation as well as the degree of swelling are calculated separately. If

more than 3 animals are used, the irritation values are calculated for all animals and all the

above-mentioned evaluation periods. If the number of animals used is 3, the irritation value for each animal

is calculated individually. The evaluation is then carried out according to the individual irritation values

and the irritation values of the two most sensitive animals was recorded. If delayed reactions occur or if no

irritation values can be calculated (due to coloration of the test sample), then deviating evaluation criteria

are used.
'SAG I$ EXEIET -I NUTIA OIO

Appendix 2: Evaluation table 

- ee

A) ComeLa -no clouding 

0

- cloudiicg in individual, scattered

areas, fine structure of the iris

clearly visible 

-

- easily observable, translucent areas of

clouding, details of iris slightly darkened 

2

-mother of pearl type clouding. No

details of iris recognizable. Size of

pupils hardly distinguishable. 

3

-complete clouding of cornea. Iris

no longer visi ble. 

4

In the case of patchy cornea clouding

the following additional evaluation is used:

Area of cornea affected

-44 or less but not 0 (zero) 

I

-more than 1/ but less than V2 

2

-more than V2 but less than 34 

3

-from 34 to whole area 

4

B) Iris

-normal 

0

-clear, deep folds, hyperemia

swelling, circumcorneal vascular

dilation (one or all of these symptoms

or combinations). Iris still reacts to,

light (slow reaction to light is taken

as positive). 

1

-no reaction to light, bleeding, coarse

destruction of the iris (one or all of

these symptoms 

2

C) Conjunctiva

Reddening of the conjunctiva on the

Lids and eyeball only on cornea and

Iris

-Blood vessels normal 0r

-Blood vessels clearly injected 1~~

-diffuse, carmine reddening, individual blood vessels not

distinguishable 

2

-diffuse, fleshy redness 

3
1STAGE 1 EXPERIMENTS IN INDUSTRJAL TOXICOOG

C!hrnosis aedemna of conjunctiva6

-no swelling 0

-sligyht swelling (any abnormal swelling,

includes'eyelid)

-clear swelling with partial turning out

of lid (ectopium)2

-swelling with half closed eyelids 3

-swelling With half to fully closed lids 4

A enix 2a: Evaluation criteria - eve

Evaluation based on following schema:

- SLIGHT IRRITATION eye irritation value

clouding of cornea 1.00 to 1.99

hyperaemia of iris, reaction to light more/equal to 0.5

erythema of conjunctiva 1.00 to 2.49

chemosis 1.00 to 1.99 -

Persistence longer than 24 hours

Reversible within 7 days

-MODERATE IRRITATION eye irritation value

clouding of cornea 2.00 to 2.99

hyperaemia of iris, reaction to light 1.00 to 1.50

-with use of 3 animals .1.00 to 1.99

erythema of conjunctiva more/equal to 2.5

chemosis more/equal to 2.0

Persistence longer than 24 hours

Reversible'within 7 days

-STRONG IRRITATION

see moderate irritation but

reversible within 21 days.

APy r
-CAUTERIZATION (DANGER OF SERIOUS EYE DAMAGE)

eye irritation value

Clouding of cornea more/equal to 3.0

Hyperaemia of iris, reaction to light greater than 1.5

-with use of 3 animals equal to 2.0

or other significant eye conditions which are not reversible within 21 days.

Calculation of the eve irritation value:

In calculating the values the DRAIZE scale was used approx. 24,48 and 72 hours after application. The

DRAIZE scale for cornea (degree of clouding), iris and redness and swelling (chemosis) of the-conj unctiva

are calculated separately. If more than 3 animals are used, the irritation values are calculated for all animals

and all the above-mentioned evaluation periods. If the number of animals used is 3, the irrtation value for

each animal is calculated individually. The evaluation is then carried out according to the individual

irritation values and the irritation values of the two most sensitive animals is recorded. If delayed reactions

occur or if no irritation values can be calculated (due to coloration of the test sample), then deviating

evaluation criteria are used.

Bibliographv

OECD Guidelines for testingy of chemicals no. 401. "Acute Oral Toxicity", adopted 12.5.1981

OECD Guidelines for testing of chemicals no. 402. "Acute Dermal Toxicity", adopted 12.5.1981

OECD Guidelines for testing of chemicals no. 403. "Acute Inhalation Toxicity", adopted 12.5.198 1

OECD Guidelines for testing of chemicals no. 404. "Acute Dermal Irritancy/Corrosivity", adopted

12.5.1981

OECD Guidelines for testing of chemicals no. 405. "Acute Eye Iffitancy/Corrosivity ",, adopted 12.5.198'1

2A~ UBI COP

itIa