# TS~CA NON-CONFIDENTIAL BUSINESS INFORMATION DOCUMENT DESCRIPTION DOCUMENT CONTROL NUMBER DATE RECEIVED p- - 

# Iv ". Sl0 

## COMMENTS: 

# DECLASS

DOIES NOT CONTAIN CII

### UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON D.C., 20460 OFFICE OF 

### ~ PROI~CHEMICAL SAFETY AND POLLUTION PREVENTIr" 

# Notice ofChange inCBI 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 forthe above referenced substance was dropped by the information submitter inthe 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-chlorophenyl)-4,4-dimethylCASRN: 1577-03-3

This notice as submitted to the NOIC files on 9/1/2011 Name ofstaffer: Jeffrey Santacroce Position: Asst. NOC Coordinator Branch: RDMB/IMD/OPPT/OCSPP 

# CONTAINS NO CBIForm 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 FORNEW CHEMICALSUBSTANCES
~~ ~DOCUMENT CONTROL OFFICER "- OFFICEOF POLLUTION PREVENTION 

## CompanySanitlzed AND TOXIC SUBSTANCES. 7407 U.S. E.P.A 401 M STREET, SW WASHINGTON, D.C. 20460j Enter the total number ofpages 90 ?5at E a bp in thePremanufacture Notice 70i; -" GENERAL INSTRUCTIONS 

### TS
- M 1C1 013 6 2 * You mustprovide all information requested in this form tothe extent that it is known toor reasonably ascertainable by you. Make reasonable estimates ifyou do

nothave actual data. * Before you complete this form, you should read the "Instructions Manual for Premanufacture Notification" (the Instructions Manualis available from the Toxic

Substances Control Act (TSCA) Information Service by calling 202-554-1404. or faxing 202-554-5603). * Ifa user fee has been remitted for this notice (40 CFR 700.45), indicate in the boxes above the TS-user fee identification number you have generated. Remember,

your user fee ID number must alsoappear on yourcorresponding fee remittance, which is sent toEPA, 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 tosubmit all test data in yourpossession or control and to provide chemical substance, even ifyou claim the identity as confidential You may authorize a description ofall other data known to or reasonably ascertainable by you, ifthese 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 willnot begin until EPA receives this information. A processing, distribution in commerce, use, or disposal ofthe new chemical letter in support ofyour subission should reference your TS user fee identification substance. Standard literature citations maybe submitted for data in the open number You must submit an original and two copies of this notice including alltest data If you claimed any information as confidential, a single sanitized copy must also be scientific literature. Complete testdata (writtenin English), not summanies ofdata, submitted must be submitted ifthey donot appear intheopen literature. You should clearly identify whether test data is on the substance or on an analog. Also, the chemical composition ofthe tested material should be characterized. Following are Part It- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE examples of testdata aisd other data. Data should be submitted according to the requirements of §720.50 of the Premanufacture Notification Rule (40 CFR Part If thereare several manufacture, processing, or use operations to be described in Part 11, 720). sections A and Bofthis notice, reproduce the sections asneeded Part III - LIST OF ATTACHMENTS Test Data (Check Below any included in this notice) Attach additional sheets ifthere 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, anytest data or other data and anyoptional 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 orcontrol ofthe submitter You may include any information that you want EPA io consider in evaluating the new *A physical and chiemical properties worksheetis located on the last page ofthis form substance On page II ofthis form, space has been provided for you to described pollution prevention and recycling information you may have regarding the new 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 bebound 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 finalproduct PMN) thatroutinely accompany the development ofconsent orders or Significant New Use Rules Except in the case ofexemption applications (suchas TMEA. LYE, LOREX) r where centain information provided in such notification is binding on the submitter when USNUN (Significant New UseNotice) the Agency approves the exemption application, checking abinding box in this notice -1 does not by itselfprohibit 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 claimany information in this notice as confidential, Toassert a claim on the LOREX (Law Release/Low Exposure Exemption) @4IFR 723,SO( (2) form, mark (X) the confidential box nextto 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. Ifyouclaim information inthenotices asconfidential, youmust also provide asanitized versionofthe notice, (includingattachments). For additional ISTHIS ACONSOLIDATED PMN? Yes instructions onclaiming information as confidential, read the Instructions Manual # ofchemicals

___________ Mark (x) ifany 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 ofEPA Form 77 10-25.Public reporting burden for this collection ofinformation isestimated to average 110 hours per response, including time forreviewing instructions, searching existing data sources, gathering and maintaining the dataneeded, and completing andreviewing thecollection of information. Send comments regarding theburden estimate or any other aspect ofthis collection ofinformation, 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 theOffice ofManagement andBudget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

### CERTIFICATION 

### Icertify that to the best ofmy knowledge and belief: I. The company named in Part 1,section A, subsection Ia ofthis notice formintends 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 ofthe date ofsubmission. 3. 1am submitting with this notice all test data in mypossession or control and a description ofall other dataknown to or reasonably ascertainable by me as required by §720.50 ofthe Premanufacture Notification Rule. Additional Certification Statements: Ifyou are submitting aPMN, Intermediate PMN, Consolidated PMN, or SNUN, checkthe following userfee certification statement that applies: Z The Company named in Part I,Section A hasremitted the fee of$2500 specified in 40 CFR700.45(b), or 

# Dj The Company named in Part 1,Section A hasremitted 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 PartI Section A is a smallbusiness concern under 40 CFR 700.43 andhas remitted a fee of$100

in accordance with 40 CFR 700.45(b). Ifyou are submitting alow volume exemption (LVE) application in accordance with 40CFR 723.50(c)(1) or a Low release andlow exposure exemption (LoRex) application in accordance with 40CFR 723.50(c)(2), check the following certification statements: 

# D The manufacturer submitting this notice intends to manufacture orimport the new chemical substance for commercial purposes, other than in small quantities solely for research and development, under the terms of40 CFR 723.50. 

# D-The manufacturer is familiar with the terms ofthis 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 ofthe exempted substance for commercial purposes within Iyear ofthe date ofthe fkVd
Treview

## period. 

# PUBLIC

Teaccuracy ofthe statements you make in this noticeshould reflect yourbest prediction ofthe anticipated facts regarding the chemical substance describedherein. Anyknowingand willfulmisinterpretation issubjecttocriminalpenalty pursuantto18usc 1001. Confidential Signature and title ofAuthorized Official (Original Signature Required) Date 

## x Signature ofagent - (ifapplicable) Date 

## x
EPA FORM 77 10-25 (Rev. 5-95) Page 2PartI -- GENERAL INFORMATION Section A -- SUBMMfER ]IDENTIFICATION ConfiMark ()the "Confidential" box next to any subsection you claim asconfidential -dential Ia. Person Name ofauthorized official Position Submitting 

### x Notice (in U.S.) Company

b. Agent (if Name ofauthorized official Psto applicable)

c. Ifyou are submitting this notice aspart ofajoint submission, mark (X) this box-- Joint Submitter (if Name ofauthorized official Psto applicable)

2. Technical Name ofauthorized official Position Contact (in U.S.)

_____________________

3 Ifyou have had a prenotice communication (PC) concerning this notice and EPA assigned a PC Numher to the notice, enter the numberMak()0 EX ifnone 4 If you previously submitted an exemption application forthe chemical substance covered by this notice, enter the exemption number assigned by Mark (X)

- EPA If youpreviously submitted a PMN for this substance enter the E PMN number assigned by EPA (i.e. withdrawn orincomplete

- ifNon 5 Ifyou have submitted anotice ofBona fide intent to manufaciure orimpori 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 ofNotice -Mark (X) Only Only3.Bt 

## D Binding Option 

## D Binding Option Mark (X) IMark (X) EPA FORM 7710-25 (Rev. 5-95) Page 3PartI -- GENERAL INFORMATION -- Continued Section B -- CHEMICAL ]IDENTITY INFORMATION: You must provide a currently correct Chemical Abstracts (CA) name ofthe substance based on the ninth Collective index (9C[) ofCA nomenclature rules and conventions. Mark (X) the "Confidential" box next to any itemyou claim asconfidential Complete either item I (Class I or2 substances) or 2(Polymers) as appropriate. Complete all other items. Ifanother person will submit chemical identity information for you (for either Item Ior 2), mark (X) the box at the right. ConfiIdentify the name, company, and address ofthat person in acontinuation sheet. 0 EJ -dential I.Class I or2 chemical substances (for definitions of class 1and class 2 substances, see the Instructions Manual)

a. Class ofsubstance -Mark (X) 1I E Class 1 or 2 E] Class 2 X b. Chemical name (Currently correct Chemical Abstracts (CA) Name that isconsistent with TSCA Inventory listings for similar substances. For Class 1substances a CA Index Name must beprovided. For Class 2substances 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 cf chemical structure diara.oFr rmcass2stncped in (1) is te : mm ediat e prcuso sutances with Intry Espet CSeistracoy Numbes ()destfcrtibeen atr of ethraton or rOces niaether Sore ran

eopotioandfrthe pCA compostony (wher eaproite). (4)t Prvdbeorcersnaierprilceiatutr darmscbmie as an hen oni oeicae raoa seie



# D dak.Xt boxua ifoulattcan ContRiuton shmeet. ube led xit o hesbtne EPA ORM77 1-25(Rev 5-5) PgeD.Poyesc(Frib th

denthods of plmeseenrthe astrciosorayusates: GPCOter (Secfy
a.Indica loest numberaverage ih ftels molecular weightcmoiino h oye yuitn omnfcue

ii) maximium weight %below 500 molecularweightiii) maximum weight %below 1000 molecular weight:

_________ 

## ED Mark (X) this box ifyou attach a continuation sheet. b. You must make separate confidentiality claims for monomer or otherreactant identity, composition information, and residual information. Mark (X) the "Confidential" box next to any item you claim asconfidential (1) - Provide the specific chemical name and CAS Registry Number (ifanumber exists) ofeach monomer orother reactant used in the manufacture of the polymer. (2) - Mark (X) this column ifentry in column (1)is confidential. (3) - Indicate the typical weight percent ofeach monomer orother reactant in the polymer. (4) - Mark (X) the identity column ifyou want a monomer or other reactant used at two weight percent or less to be listed as part ofthe polymer description on theTSCA Chemical Substance Inventory. (5) - Mark (X) this colun ifentries in columns (3) and (4) are confidential. (6) - Indicate the maximum weight percent ofeach monomer or otherreactant that may be present as aresidual in the polymer asmanufactured for commercial purposes. (7) - Mark (X) this column ifentry in column (6) is confidential. Monomer or otherreactant 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 boxifyou attach acontinuation sheet.

C. Please identify which method you used todevelop or obtain the specified chemical identity information reported in thisnotice (check one). 

### 0 Method I (CAS Inventory Expert Service - a copy ofthe 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, ifone can bereasonably ascertained,



# DMark (X) thisbox ifyou attach acontinutation sheet. EPA FORM 7710-25 (Rev. 5-95) Page 5EB 13 28003 14:24 FR BRYER CURPURI N 412 777 7484 TO 912025648955 P.03/03

Mark~ ar IX

(.i boxRA iNORATO

you aCchntoiinntonshet Sectyonym - EntCAr a]Dny r mINFOMAIO sy-n- orhenwCoia ntiedinsbeinIor2 (.Tad Identif c i urit yta a e r thc neabl santce idb pent in sube cheion l su-m or 2auatrdfr.omrilprpx.Poieh CAS a Rk gistr Nofyumaerach avconiua.Ion theeacuietfetmuiis ne Uietfe. (.benri cEsmate name maimyum weaigh %hec i pti y. s con eruidentiied yoimtp riie en tiaefoer yortaeighatreval thespcicch mic puienity ond CAe egistry Nubser Maxmu th axmm et osil.Ree t h 

### SenAtachdl



# El Mak

## (X) thisboxifyouatach 

### a continuation sheet. 

# I i 

# 1(' 

# C
7. Synrodncms - Eneri any bmca yonycs rhn rmtemnfcee rcsiguco ipslothe new chemical subsnfec rfled thcCASc licgisorry Cl) (2)dnial 

### SEe Aak( hed bxi o taha otnainse

Mark X) this box iyou attach acontinuation qheet, 6.A GeOrc 7710-25a (Re-I3) camcemclieniya cof t a gc yo 6utpoieagnrcnm o orsbtneta eel thespecific 

### ~ ~ ~ ~ ~ ~ ~ ~ ~ * chemica identity oftenw*cn*lusa otemaiu xetpsil.Rfrt h

### PartI -- 

### 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 maximumproduction volume during the first 12 months ofproduction. Also estimate the maximum production volume for any consecutive 12-month period during the first three years ofproduction. Estimates should beon 100%new chemical substance basis. For a Low Volume Exemption application, ifyou choose to have your notice reviewed at a lower production volume than 10,000 kglyr, specify the volume and mark (x) in the bindingbox. 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 ofthe category ofuse, the percent ofproduction 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 ofuse ofthe new chemical substance by function and application.. (2) -- Mark (X) this column ifentry column (1) is confidential business information (CBI). (3) -- Indicate your willingness to have the information provided in column (1) binding. (4) -- Estimate the percent of total production for the first three years devoted to each category ofuse. (5) -- Mark (X this column ifentry in column (4) is confidential business information (CBI). (6) -- Estimate the percent ofthe new substance as formulated in mixtures, suspensions, emulsions, solutions, or gels as manufactured for commercial purposes at sites under your control associated with each category ofuse. (7) -- Mark (X this column ifentry in colurm (6) is confidential business information (CBI). (8) -- Indicate %ofproduct volume expected for the listed "use" sectors. Mark more than one box ifappropriate. 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 ofuse (1) CBtI Binding Produc- CBI %in CBI %ofsubstance expected per use CBI Option tion % Form-

_________ (8) (by function and application i.e. adispersive dye Mark (x) ulation Site- Con-* Indus- Coin- Binding for finishingpo1yester 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 ofthe use(s) ofthischemical substance in consumer products.

In addition include estimates ofthe concentration ofthe new chemical substance as expected in consumer products anddescrnbe the chemical reactions by which this substance loses its identity inthe consumer product. 

# DMark (X) thisbox ifyou attach acontinuation sheet. b. Generic Ifyou claim any category ofuse description in subsection 2a as confidential, enter ageneric description ofthat category. Read the use Instructions Manual for examples ofgeneric use descriptions. description 

# DMark (X) thisbox ifyou attach a continuation sheet. 3. Hazard Information -- Include in the notice acopy ofreasonable facsimile of any hazard wamning statement, label, material safety data sheet, orother Binding

information which will beprovided to any person who is reasonably likely tobe exposed tothis substance regarding protective equipment or practices Mapki(x forthe safe handing, transport, use, or disposal ofthe new substance. List in part Ill hazard information you include. 

# ZMark (X) this box ifyou attach hazard information. EPA FORM 7710-25 (Rev. 5-95) Page 7

### Part11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE Section A -- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER Mark (X) the "Confidential" boxnext to any item you claim as confidential Complete section A for each type of manufacture, processing, oruse operation involving the new chemical substance at industrial sites you control. Importers do not have to complete this section foroperations outside the U.S.; however, you may still have reporting requirements if there are further industrial processing oruse operations after import. You must describe these operations. See instructions manual 1. Operation description Confia. Identity -- Enter the identity ofthe site at which the operation will occur. dential Name 

# x Site address (number and street) City. County, State, ZIPcode
Ifthe same operation will occur at more than one site, enter the number ofsites. Identify the additional sites on a continuation sheet, and ifany ofthe sites have significantly different production rates or operations, include all the information requested in this section forthose sites as attachments.



# D Mark (X) this box ifyou attach acontinuation sheet. b. Type-- 

## X Mark (X) ElManufacturing EProcessing D]Use



# _____ c. Amount and Duration -- Complete I or2 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)toindicate your willingness to have yourprocess description binding.X (I) Diagram the major unit operation steps and chemical conversions. Include interim storage and transport containers (specify- e.g. 5gallon pails, 55 gallon drum, rail car, tank truck, etc). (2) Provide the identity ,the approximate weight (bykg/day or kg/batch ona 100%new chemical substance basis), andentrypoint ofall starting materials and feedstocks (including reactants, solvents, catalysts, etc.), and ofall products, recycle streams, and wastes. Include cleaning chemicals (notefrequency if not used dailyor perbatch.). (3) Identify by number the pointsofrelease, including small or intermittent releases, to the environment ofthe new chemical substance,



# DMark (X) this box ifyou attach a continuation sheet. EPA FORM 7710-25 (Rev. 5-95) Page 8

### Part11-- 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 ofworker activity, physical form ofthe new chemical substance, number ofworks exposed, and duration of activity. Mark (X) the "Confidential" box next to any item you claimas confidential. (1)

-- Describe the activities (i.e. bagdumping, tote filling, unloading drums, sampling, cleaning, etc.) in which workers may be exposed to the substance. (2) -Mark (X) this column ifentry 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) ofthe new chemical substance (e.g., solid: crystal, granule, powder, or dust) and %new chemical substance (ifpart ofa mixture) at thetime ofexposure. (7)-Mark (X)this column ifentry in column (5)is confidential business information (C131). (8)

--Estimate the maximum number ofworkers involved in each activity for all sites combined. (9) --Mark (X) this column ifentry in column (8) isconfidential business information (CBI). (10) and (11) --Estimate the maximum duration ofthe activity for anyworker in hours perday and days per year. (12)

--Mark (X) this column ifentries 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 ifyou attach acontinuation sheet. 3. Environmental Release andDisposal -- You must make separate confidentiality claims for therelease number and the amount ofthe new chemical substance released and otherrelease anddisposal information. Mark (X) the "Confidential" box next to each item you claim as confidential. (1) -Enter thenumber ofeachrelease point identified in the process description, part 11, section A, subsection Id(3). (2)

-- Estimate the amount ofthe new substance released (a) directly to the environment or (b) into control technology (inkg/day orkg/batch). (3) -Mark (X this column ifentries 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 landor incineration. POTW, orother (specify)) to which the new substance will be released from that release point. (5)

-- a. Describe control technology, ifany, and control efficiency that will be used to limit the release ofthe new substance tothe environment. For releases disposed ofon land, characterize the disposal method and state whether it is approved for disposal ofRCRA hazardous waste. On a continuation sheet, for each site describe any additional disposal methods that will beused and whether the waste is subject to secondary or tertiary on-site treatment. b. Estimate the amount released to the environment after control technology (in kg/day). (6) -Mark (X) this column ifentries in columns (4)and (5) are confidential business information (C131). (7)

-- Identify the destination(s) ofreleases to water. Please supply NPDES (National Pollutant Discharge Elimination System) numbers for direct discharges or NPDES numbers ofthePOTW(ublicly Owned Treatment Works). Mark (X) ifthe POTW name or NPDES # is confidential business information (CBI) Release Amount ofnew 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 X aera releases to water. to Mark (X) thisbox ifyouattach acontinuation sheet. EPA FORM 7710-25 (Rev. 5-95) Page 9

### Part11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- 

### Continued Section B -- INDUSTRIAL SITES CONTROLLED BY OTHERS Complete section Bfortypical processing oruse operations involving the new chemical substance at sites you do notcontrol. Importers do not have tocomplete this section for operations outside theU.S.; however, you mustreport any processing or use activities after import. See the Instructions Manual. Complete aseparate sectionBfor each tpieof processing,oruseoperationinvolviing the new chemicalsubstance. Ifthe same operation isperformed at more than one site describe the typical operation common to these sites. Identify additional sites on acontinuation 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 stepsand chemical conversions, including interim storage and transport containers (specify - e.g. 5gallon 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, theapproximate weight (by kg/day orkg/batch, on an 100%new chemical substance basis), and entry point ofall feedstocks (including reactants, solvents and catalysts, etc) and all products, recycle streams, and wastes. Include cleaning chemicals (note frequency ifnot used daily orper batch). (3) -- Identify by number the points ofrelease, including small or intermittent releases, tothe environment of the new chemical substance. (4) Please enter the# ofsites (remember to identify the locations of these sites on a continuation sheet): 2 # ofsites The material made in will go to two (2)different "tollers" that we will use to evaluate capabilities and cost issues. Iam told this would be done via intermodal containers. We may choose to return the tolled product inthese same containers. This isnot 

### decided yet. Infuture years we have not determined how itwill be shipped and who will clean the container. We are considering returning the inthe same containers used to ship the .The toilers would probable 'flush" the railcars with the as theflush solvent. No other "firm" data at this time. Information on how the toiler will handle the material has not been determined yet. Itshould be readily available once we make decision onwho to send itto. The willthen be returned to forconversion to inthe


# D: Mark (X) thisbox ifyou attach acontinuation sheet. 2. WorkerExposure/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 ofworkers exposed for all sites combined. (4)

-- Estimate the typical duration ofexposureper worker in (a) hours per day and (b)days peryear. (6)

-- Describe physical form ofexposure and %new chemical substance (ifin mixture), and any protective equipment and engineering controls, ifany, used to protect workers. (7) -Estimate the percent ofthe new substance as formulated when packaged or used as a final product. (9)

-- From the process diagram above, enter the number ofeach release point. Complete 9-13 for eachrelease point identified. (10) -- Estimate the amount ofthe new substance released (a) directly to the environment or (b) into control technology tothe environment (in kg/day orkg/batch). (12) -- Describe media ofrelease i.e. stack air, fugitive air (optional-see Instructions Manual), surface water, on-site or off-site land orincineration, POTW. or other (specify) andcontrol technology, ifany, that will be used to limitthe release ofthe 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 ifany ofthe proceeding entries are confidential business information (CBI).

_______ Letter #of CBI Duration CBI Protective Equip./ %in CBI Release Amountof CBI MediaofRelease 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 ifyou attach acontinuation 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 claimas confidential. 

### In this section you may provide information not reported elsewhere in this form regarding your efforts toreduce or minimize potential risks associated with activities surrounding manufacturing, processing, use and disposal ofthe 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 thereduction in the amount or toxicity ofchemical wastes by technological modification, process andprocedure modification, product reformulation, raw materials substitution, and/or inventory control. Recycling refers to thereclamation ofuseful chemical components from wastes that would otherwise be treated orreleased as air emissions or water discharges, orland disposal. Descriptions ofpollution prevention, source reduction and recycling should emphasize potential riskreduction subsequent tocompliance 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 ofrisks to other environmental media ornon-environmental areas (e.g., occupational or consumer exposure). In addition, information on the relative cost or performance characteristics ofthe PMN substance topotential alternatives may beprovided. All information provided in this section will be takeninto consideration duringthe review ofthis substance. See Instructions Manual andPollution Prevention Guidance manual forguidance 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 ofwaste materials through recycling, source reduction or other means; (4) a reduction in potential toxicity or human exposure and/or environmental release; (5)an increase inproduct performance, a decrease in the cost ofproduction and/or improved operation efficiency ofthenew chemical substance in comparison to existing chemical substances used in similar application; or (6) the extent to which the new chemical substance may be asubstitute for an existing substance that poses agreater overall riskto human health orthe environment.



### Normally we would have sentthe for .This 

### material was then returned tothe
. 

### Our method will prevent the need to shipthese chemicals transatlantic with corresponding reduction inriskto the environment and humans. The currently used process in uses inthe of .Our process will not use asolvent. The current method recyclesthe solvent but there issome wastage involved. Also, we will have a any unreacted to be recycled and re-used inthe reaction step. The will most likely be shipped in .This 

### isa reduction in the shipments and containers being transported. The company will realize significant inthe compared with shipping to for conversion to


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## PartIII -- 

## LIST OFATTACHMENTS Attach continuation sheets for sections ofthe form and test data and otherdata (including physical/chemical properties and structure/activity information), and optional information after this page. Clearly identify the attachment and the section ofthe form to which it relates, if appropriate. Number consecutively the pages ofthe attachments. In the column below, enter the inclusive page numbers ofeach 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 ofthe notice form a sanitized version ofany 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



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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 484,



### This page is intentionally left blank for the purpose of sub-'- mitting administrative information that is required by regulations promulgated by various countries.

Z2

- -. A51. 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,

3oh

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 -10 391. 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 9Wmix 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 

# :~IoEprmnare fmledion Dtearhvsote 

# StudPULy COPYtin at

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

9molecular 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.

104.2 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 'ousrate 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 0C 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 370

### C, new samples of individual colonies from these plates were transferred to flasks containing approximately 20 ml normal nutrient broth. T his inoculum was incubated overnight at 370

### C, 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 0

### C 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 370

### C 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 370

### C. 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.

144.2.4.5 

### 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 ,sixseconds -(TA1535 and 'TA 1537), 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 40

### C 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 (4W), 0. 15 MKCl 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 40

### C 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 filled with ice to keep the S9 mix cold. seventy ml of cofactor solution are composed as follows: MgCl 2 

### x 6 H 2

### 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). bacterialcolonies

0.1ad compound 0.1ml bacteria . * 0.5 ml S-9 mix or * * 

### b~uffer 2.0 ml softagar00 45degrees C inwaterbath petri dishwith solidagar colonycountusing max. 30 sec. automatic counter mixing ircubationat 37degree C for48 hous 

### For the mutant count, one plate was used, both with and without S9 mix, for each strain and dose. One plate, filled 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 soft agar were added to the tubes, the content mixed and plated.

2ml

soft Do

bateralckm

01lt bactons 01I

ml

compound sdubat*

# - 0.5 i$9mix orbutffer pitri d~ In t

orseeraon adtioiof#WACdue

toegolnlt 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, 'isnoted 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 0

### C 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 370

### C. 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 sufficient effects, 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 tt o 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 1

### groups, 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 found 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.

237. 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
247. 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. Ames: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 .negatiiejandpositive controlso ex ri~entifedNrmfrom 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 dimethylether274- 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. 16 538 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


### ~UBUC 

# C VON*&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 COPYsummary 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 9 301 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


# 30PULCCPSummary 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 - EGDE2 + 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 208- 2) Ethylene glycol dimethylether 

# 31PULCC 

# y

43Summary of historical negative and positive cotop.2~, from usnmean vauespreset 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

32Summary 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

338.2 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

34Summary 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

35Summary 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

36Summary 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



### 38Table 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 

## / 0 lP 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

51Table 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 31P 31 

## / 46P 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

40Table :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 2P 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 3P 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

41Table :4 SuyMStudy Director Dose/Plate 1echDilu in r (j~g/P~ate) -9 M SD +S9 M Da1te 1+ 9 ,s

Do0e7Plat 170 17uto pe 95 950.l.

158 7 7 

## / 13 13 

## / 33 3.3** 0.5 0.7

5000 7P 7 

## / 14P 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

42Table :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 US l 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

43Table :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 9P 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 liP 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

44D~se3 le 70 siludyo pmer m (jig/Tube)~~~sud DirMector S 0. 1+ -9 5 106' 06 

### I 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. 50 not tete 86 8664 6. 07 . S9 t15t S90 Mix 949 wit S9 Mix08 .

500 78 7 61 61 01** 0.7 0.

158178 P78 5 P 5 P 07 0.

5000 86 P86 5 7 . . 

# NF431 3. 8 83579Table 8 Studyr Number

JIM Dlutio er (~Lg/ube) 59 M D +S M SD 10-6........+5 5 5 

### / 6 6 83 .3'~ 1z0~,-1. a'd

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 

# 4,7Table 9 study Numbier, DoeStudy DiS9ctor (ag/Tue) -S9 M SD S9 M S 10.6 0i
- UvTS w13 13 

## 1 .17 7 

## / 29 12.9 1.Date~

158b 15 15- 6 18+ 18q 

### / 5 ** 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

%nottested P: Precipitatioi M: Y9am SD: Standard-Deviatiori -S9: without S9 Mix +S9: with S9 Mix

47Table 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 73 73 23 2 27 275 0. 0.By

Unti tme as the results inthisreport are published, they mayonly be usedwiththepermission of Nocopying ofthisreport whether inits totality oras an extract orextracts, ispermitted.



# 705Table ofContents 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 Acuteoral toxicity.............................................. 4 3.3.3. Acutedermal toxicity.............................................4 3.3.4. Acute inhalation toxicity (powder).............................. 4 3.3.4.1.Analysis oftestatmosphere.................................... 5 3.3.4.2 Particle analysis intestatmosphere............................ 5 3.3.5. Examination ofirritation andcauterisation ofrabbit 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 forirritation and cauterisation ofthe 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.Acuteoral toxicity............................................. 8 4.2. Acute dermal toxicity.......................................... 8 4.3. Acute inhalation toxicity (powder)..............................9 4.4. Examination ofirritation and cauterisation ofrabbit skin........ 10 4.5. Examination ofirritation and cauterisation ofrabbiteye......... 10 5. EVALUATION................................................... 12 APPENDIX.......................................................... 13 Analysis ofparticle 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......................................................... 18STAGE IEXPERIMENTS ININDUSTRIAL TOXICOLOGY 1.SUMMARY

The methodology adopted was in accordance with therelevant OECD guidelines. LD50-ora Rat (fasted) :biggerthan 5000mg/kg bodyweight. LD50-derma Rat (exposure 24hr): biggerthan 5000mg/kgy bodyweight. LD5O-inhalation (powder) Rat (exposure 4hr)biggerthan 1267mg/rn air (maximum technicallyproducible concentration) Local irritation (rabbit) Skin: the experimental subject canbe considered not toexhibit irritation oftheskin. (exposure4 hr,flank). 

### ~the experimental subject canbe considered nottoe Evaluation: theexperiments indicate Itha xhibited no evidence oforal,dermal orinhaledtoxicity underte prevailing"experimental conitT nso significant toxicological potential to skin or eye couldbe established. The experiments indicate that properuse oftheproduct presents nopotential foracuterisk tothe health of human beings.

# - STAGE 

### IEXPERIMENTS IN INDUSTRIAL TOXICOLOGY 2.LIM TION onfidential


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

tested on rabbits. Theseexperiments werecarried outinorder toassess thepossibility ofanyrisk tohealth forpeopleusing the product forindustrial purposes. _________________________exhibits the following structural formula: [referto original]

Studies no.: Acute oraltoxicity: Acute dermal toxicity: Acuteinhalation(inpowderfo 

### Local Irritation (rabbits)-PAGE05/20 STAGE 1EXPERIME 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 abody weight ofapprox. 160- 200 gfrom the breeder WINKELMANN, Borchen inPaderborn. Theanimals were kept in aconventional manner inMakrolon cages, type 111, 5animals percage inan experimental animal laboratory: lab. Temperature 19-25* Celsius,relative humidity ofthe air40-60% light/darkrhythm 12 hours. As cagelitter woodgranules with alow dustcontent were used. The animals were fed on "Altromin 1324 - animal food forrats and mice" (producer Altromin GmnbH, Lage) and unlimited waterfrom thedomestic supply. Animals wereidentified by theircolourand cage. Animals were settled into theirenvironment foratleast 5days beforethe commencement ofthe experiment. All animals wererandomly allocated tothe experimental groups. Adult Albino rabbits, pedigree HC:NZW (breeder: Hacking &Churchill Ltd., Huntingdon, UK). They were feda standard diet("SsniffK4from Versuchstierdi~ten GmbH, Soest, Westfalen) and unlimited water. The rabbits were kept separately inindividual metal wire cages (type III,high). Forfurther livingconditions see rats. 3.3. Exnjerimental Methods The methodology adopted was inaccordance with the OECD "Guidelines forTestingofChemicals; section 4: Health Effects.STAGE 1EXPERIMENTS ININDUSTRIAL TOXICOLOGY Con 3.3.1.Observational period,conclusions andautopsy. Thebody weight oftherats was recorded before application/exposure and afterthatat weekly intervals, theirbehaviour and appearance was recorded several times on thedayofapplication/exposure and after thattwice daily. During the4-hour head/nose exposure noobservations wererecorded. Inthecase ofthe experiments foracute oral, dermal and inhalation toxicity theobservational period was 14 days. After that theanimals were putdown using ethyl ether (oral and dermal application) or with Evipan (inhalation) and were given apathological anatomical examination.

-- 4. Inthe tables the column 'Toxicological Results" the following abbreviations wereused: IS number = number ofanimals whichdied. 2nd number =number ofanimals with symptoms 3rdnumber =number ofanimalsused inthe experiment. 3.3.2.Acuteoral toxicity The testsample was dissolved indemineralised waterusingTylose (0.5%) and was administered at a volume ofl0mi per kgbodyweight for 16 hoursto fasted male and female animals (5rats perdose andper gender); thisprocedure was carried out onceusing agastric feedingtube. Two hours afterthis application the animals were offered food. 3.3.3.Acutedermaltoxicity The experiments werecarried outon male and femaleratsusingthe foil body-wrap method ofNOAKES and SANDERSON (Brit.J.Ind. Med. 26,59, 1969). Thedose was individually weighed out, dissolved inphysiological cooking salt, andapplied to theshaved skinofthe back ofthe rats (5animals ofeach gender ineach dose group). The application was covered withaluminium foil and fixed tothe skinusing bandages. Theexposure timewas 24hours. After the removal ofthe bandages theskin areas treated were cleaned with soap and water. 3.3.4.Acuteinhalationtoxicology(powder) Theexperimental material was administered inthe formofapowder inhalant kept indynamic condition in a20litre inhalation chamber. Theinhalation was carried outatintervals with adispersion pressure of approx. 50 kPa,the duration ofeach pulsebeing 0.5 seconds 

# PUBLIC copywitha pause of3seconds between each pulse. During theexposure the rats were kept stationary intubes (head/nose exposure) 5male and5female rats wereexposed tothe powdered inhalant at each session of exposure. 3.3.4.1.Analysis ofthetest atmospher The concentration ofthe inhalant wasgravimetrically determined on amembrane B-acetate filter.The sample extraction was carried outin thebreathing fieldofthe'experimental animals ata'suction velocity

... , which enables theanalysis oftheentire particle spect m (suction velo'city ap~rox125 cmp;'rsecod).' N .: _z__ The concentrations recorded referto mg of perm3ofair. 3.3.4.2.Particle analysis inthetestatmosphere The sampleextraction from the inhalation chamber was carried outunder conditions comparableto those in 3.3.4.1.Analysis oftest atmosphere maybe found below. Theparticle analysis wascarried outinrelation to its mass with the use ofan ANDERSEN CASCADE IMPACTOR, the specifications ofwhich maybe found inthe appendix, page 13. The particles impacted on the impactor gradations were measured on agravimetric basis. The calculation ofthe distribution-specific p-maraintewas carr~giwpawith.an anmnoriate computer programme on an HP3000computer oftiI Theparameters calculated were: theMMAD (Mass Median Aerodynamic Diameter, the NMAD (Nurber Median Aerodynamic Diameter) and thegeometric standard deviation. The alveolar mass was taken as the relative massproportion: thisshows aMMAD smaller than 5 3.3.5.Examination ofirritationandcauterisation ofrabbitskin 3.3.5.1.Procedureadopted On the daybefore application ofthe testsample the skin onthe flanks on both sides of3adultrabbits was shaved over an area of6x6 cmwith anelectric haircutter. Only animals withundamaged skin were used. 

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-74-STAGE IEXPERIMENTS ININDUSTRIAL TOXICOLO Y Confidential

- Each animal was administered 500 mg ofthetest sample made into apaste bythe addition ofwater and spread onto acellulose dressing (approx. 2.5 x2.5cm). Aseconddressing was dampened with water and placed on the oppositeflank ofthe animal.Both dressings were then fixed inplace with an elastic adhesive plaster. After 4 hoursthe dressings wereremoved andthe exposed areaofskinwas washedw wiA water. 3.3.5.2. Results Theresults ofthe tests at various times afterexposure aregiven intable4. The extent of erythema/encrustation formation, i.e.oedema formation was recorded inaccordance with DRAIZE (see appendix 1,*OECD Guidelines); all othernoticeable findings were also recorded. Thepartoftheskin nottreated served as acontrol. 3.3.5.3 Evaluation Criteria Assessment oferythema/encrustation formation onthe basisoftheDRAIZE scale ofmeasurement was madeby calculating the primary skin irritationvalues (see appendix Ila) at intervals of24, 48 and72 hours andbyaveraging overtherespective periods oftime.Further details ofthe assessment criteria aregiven in appendix Ia. 3.3.6.Testforirritation andcauterisation oftheeve 3.3.6.1.Procedure adopted 3adultrabbits ofeach gender were treatedwith 100 -- ofthe test sample inpowderform; this corresponds to aweight ofapprox. 60 mg,applied tothe conjunctiva tissueofthe eyelid. After application the eyelids wereheld together for approx. Isecond. The eye onthe opposite side was not treated and served as acontrol. A washofphysiological NaCI solutionwas applied tothe eyewhich was treated after aperiod of24hours. 3.3.6.2. Results Results ofthe application wererecorded atthe intervals indicated intable 5.Assessment ismade ofthe state ofthecornea (degree ofclouding and thearea affected), the iris (hyperaemia, reaction tolight) and conjunctiva (erythema and chemosis) in accordance with DRAIZE (see appendix 2,i.e. OECDGuidelines). 

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77STAGE IEXERIMENTS ININDUSTRIAL TOXICOLOGY The flow (outflow) oftearsis alsomeasured. All other findings notcovered by theDRAIZE scale were also recorded. Cornea and irisfindings are taken withthe use ofoptical instruments. Todetermine the area ofcornea affected after aperiod of24 hours Idropofa 1% fluorescein solution isappliedto the cornea oftheeye concerned: in thecase ofpositive results this isalso carried outfor anyfinding recorded after the24 hourperiod. After that awashofphysiological NaCI isapplied tothe eye. UVlightisused to determine the extentofcornea results.. 3.3.6.3. Evaluation Criteria In the assessment ofresults onlythose findings whichpersist for24 hours orlonger are used. The assessment criteria andthe mode ofcalculation foreye irritation values are given in appendix 2a. 3.3.7. LC50ILD5OCalculation Wherever acalculation ofaverage lethal dose/concentration (LD5OILC5O) ispossible, itiscarried outon the computer (HP3000) in accordance with A.P.ROSIELLO, J.M. ESSIGMANN and G.N. WOGAN (J. Tox.AndEnviron. Health3,797-809 (1977)). This procedure isbased on the Maximum Likelihood Method ofC.I. Bliss (QJ.Pharm. Pharmocol. 11, 192-2 16(1938). 3.3.8. Quality control (GLP) The experiments were carried outinaccordance with OECD Principles ofGood Laboratory Practice (GLP) (Bundesanzeiger 35 3-16 ( 2nd March 1983)). In accordance wijhiib~cenrinciples the experiments were recorded i nerimental planswhich are archived intq(Archive) together with all other papers relating to-theseexperim-ens.



# (pqY4. RESULTS 4.1.Acute Oral Toxicity Theresults oftheexperiments on fastedrats are listedinthe 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 5000mg/kg bodyweight. Symptoms: the treatment was tolerated without any symptoms. Autopsy results Animals killed atthe endofthe observation period: there was noevidence ofanysubstance-induced macroscopically recognisable organ damnage. 4.2.Acute dermaltoxicity Theresults oftheexperiments on rats arelisted inthefollowing table. Table2: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 LD50greater than 5000 mg/kg bodyweight. 

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# COPY'a '70W-TGE IEXPERIMENTS IN INDUSTRIAL TOXICOLOGY SYMptoms The treatment was tolerated without any clinical symptoms. Localised results onthe area oftreatment were notfound. Autopsyresults Animals killed attheend ofthe observation period: there was no evidence ofanysubstance-induced macroscopically recognisable damage to thevisceral organs. 4.3.Acute Inhalation Toxicity (powder) Theresults ofthetest foracute toxicity ofthe testsample powder withaperiod ofexposure of4 hours are summarised inthe following table: Table 3:AcuteInhalation Toxicity -powder (exposure 4 hours) Group Concentration Toxicol. Duration of Timeof Mortality (mg/kg result symptom death M% bodyweight) rat 1267 0/0/5

- 0 rat 1267 010/5

- -0 (maximum concentration technically possibleto manufacture) LD50 greater than 5000 mg/kg bodyweight. Theanalysis ofparticle distribution gave arelative alveolar mass of56%(MMAD 4.6- geometric standard deviation 1.8). The individual results ofthe particle analysis are given intheform ofgraph and tableinthe appendix (see page 13, 14). Symptoms:The exposure was tolerated withoutany symptoms. Autonsv - Animals killed attheend ofthe observation period: there was noevidence ofany substance-induced macroscopically recognisable damage to the organs.

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4.4. Examination ofirritation andcauterisation ofrabbit skin The individual skinreactions are summarized inthe following table 4. Table4: 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 0 000-
- 0 0 E99 (3.3k-.) 00 0 00 0 0000- 0 0 Evaluation see appendix I E--erythema 0 oedema

-=not tested The testsample istherefore considered "not irritating tothe skin"(Evaluation see Appendix Ila).



# PUBI ICloPYSTAGE 1EXPERIMENTS IN INDUSTRIAL TOXICOLOGY 4.5.Examination ofirritation andcauterisation ofrabbit eThe individual results atthe various times ofevaluation are summarized inthe table below. Table 5:Test forirritation oftheeye (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 ofhyperaemia oftheconjunctiva is seen as theresult ofmechanical irritation (caused bythe physical composition ofthe test material). Therefore the test material isconsidered "not irritant to theeye" (Evaluation see appendix 2a). 

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# )TAGE IEXPERIMENTS IN INDUSTRIAL TOXICOLOGY o tiail
xhbtdno acuteoral, dermal orinhalation toxicity on There was no significant potential fortoxicity inrespect oftheskin oreye. Given appropriate use ofthe product thereistherefore nopotentially acute riskto health forhuman beings.

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



# 7C pyAPPENDIX Analysis ofRarticle distribution N Impactor Cut Off Mass Relative Cumulative degree diameter (absolute) Mass M% Mass (%) 1 0.01-0.40.01 .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 ofuse 1200.00 seconds Alveolar proportion (small than5

- 1.relating to mass: 56% (measured) 2.relating to no.ofparticles: 82% (extrapolated) EFFECTIVE CUT-OFFDIAMETER (ECD): the cumulative distribution isrelated tothe Effective Cut-off Diameter. TheECDrepresents aconstant factor inthe useofthe impactor.TSI NUTRILTXCLG

MMAD= 4.62 Sigma--1.83
Norm.rel.Mass concentration

Aerodynamic diameter (mcm) 1.Mass- 2. surface- 3.particledistribution



# IL Ar
-4 

# Ilb%proportion smaller than ECD

Aerodynamic diameter (mcm)



# 177APPENDICES Appendix 1: Evaluation-table - skin Ervthema - reddening/encrustationl ofskin -noreddening 

## o0 -slight,hiardly perceptible reddening -easilyperceptible reddening 2 -moderate to strongreddening 3 deep reddening to slight encrustation (changes affecting deepinto the skin) 4 Oedema - swellin2 -no swelling 0 -very slight,hardlyperceptible swelling I -slight swelling, edges well defined 2 -moderate swelling (edges approx. 1mm raised) 3 marked swelling (edgesraised more than I1mm, all exposed area affected) 4 Appendix I a: Evaluation criteria - skin Evaluation Primary skinirritation value
-noprimary irritation 0 to 0.99 -slightprimary irritation 1.0 to 1.99 -moderate primary irritation 2.00 to2.99 -strong primary irritation 3.00 to4.00 (maximum value)

- cauterization: deep wounds suchulceration, necrosis, formation ofscars on removal ofthedressing or withinthe observation period. Calculation ofthe skin irritation values: Incalculating the valuesthe DRAIZE scale was usedapprox. 24, 48and 72 hours after application. The DRAIZE values oferythema! scarformation as well as thedegree ofswelling arecalculated separately. If more than 3animals areused, the irritation values arecalculated forall animals and allthe above-mentioned evaluationperiods. Ifthe number ofanimals used is3,the irritation value foreach animal iscalculated individually. The evaluation isthen carried out according tothe individual irritation values andthe irritation values ofthetwo most sensitive animals was recorded. Ifdelayed reactions occurorifno irritation values can becalculated (due tocoloration ofthe testsample), then deviating evaluation criteria areused.'SAG I$EXEIET -I NUTIA OIO

Appendix 2: Evaluation table
- ee A)ComeLa

-noclouding 0

- cloudiicg inindividual, scattered areas, fine structure ofthe iris clearly visible -

- easily observable, translucent areas of clouding, details ofiris slightly darkened 2 -motherofpearl typeclouding. No details ofirisrecognizable. Sizeof pupils hardly distinguishable. 3 -complete clouding ofcornea. Iris no longer visible. 4 In thecase ofpatchy cornea clouding thefollowing additional evaluation isused: Areaofcornea affected -44 

### orless butnot0(zero) I -more than 1/ 

### butlessthan V2 

### 2 -more than V2 

### butlessthan 34 

### 3 -from 34

### towhole area 4 B)Iris -normal 0 -clear, deep folds, hyperemia swelling, circumcorneal vascular dilation (one orall ofthese symptoms orcombinations). Iris stillreacts to, light(slow reaction to light istaken aspositive). 1 -no reaction tolight, bleeding, coarse destruction ofthe iris (one or allof these symptoms 2 C) Conjunctiva Reddening ofthe conjunctiva on the Lids and eyeball only on cornea and Iris -Bloodvessels normal 0r 

### -Bloodvessels clearly injected 1~~ -diffuse,carmine reddening, individual blood vesselsnot distinguishable 2 -diffuse, fleshy redness 31STAGE 1EXPERIMENTS ININDUSTRJAL TOXICOOG

C!hrnosis aedemna ofconjunctiva6 -no swelling 0 -sligyht swelling (any abnormal swelling, includes'eyelid) -clear swelling with partial turning out oflid (ectopium)2 -swelling with halfclosed eyelids 3 -swelling With halfto fullyclosed lids 4 A enix 2a: Evaluation criteria - eve Evaluation based on following schema:

- SLIGHTIRRITATION eyeirritation value clouding ofcornea 1.00to 1.99 hyperaemia ofiris,reaction to light more/equal to 0.5 erythema ofconjunctiva 1.00 to2.49 chemosis 1.00 to 1.99

- Persistencelonger than 24hours Reversible within 7days -MODERATE IRRITATION eye irritation value clouding ofcornea 2.00 to2.99 hyperaemia ofiris, reaction tolight 1.00 to 1.50 -with use of3animals .1.00 to 1.99 erythema ofconjunctiva more/equal to 2.5 chemosis more/equal to 2.0 Persistence longerthan 24hours Reversible'within 7days -STRONG IRRITATION see moderate irritation but reversible within21 days. 

# APy r-CAUTERIZATION (DANGER OFSERIOUS EYEDAMAGE) eye irritation value Clouding ofcornea more/equal to3.0 Hyperaemia ofiris,reaction to light greaterthan 1.5 -with use of3animals equal to2.0
orothersignificant eyeconditions which are notreversible within 21 days. Calculation ofthe eve irritation value: In calculating the values theDRAIZE scalewas used approx. 24,48 and72hours after application. The DRAIZE scalefor cornea (degree ofclouding), iris and rednessand swelling (chemosis) ofthe-conjunctiva arecalculated separately. Ifmore than3animals are used, theirritation values are calculated forallanimals andall the above-mentioned evaluation periods. Ifthe number ofanimals used is3,the irrtation value for each animal is calculated individually. The evaluation isthen carried out according tothe individual irritation values and the irritation values ofthe twomost sensitive animals isrecorded. Ifdelayed reactions occurorifno irritation values canbe calculated (due tocoloration ofthe test sample), then deviating evaluation criteria are used. Bibliographv OECD Guidelines fortestingy ofchemicals no. 401. "Acute Oral Toxicity", adopted 12.5.1981 OECD Guidelines for testing ofchemicals no. 402. "Acute Dermal Toxicity", adopted 12.5.1981 OECD Guidelines fortesting ofchemicals no.403. "Acute Inhalation Toxicity", adopted 12.5.1981 OECD Guidelines fortesting ofchemicals no.404. "AcuteDermal Irritancy/Corrosivity", adopted 12.5.1981 OECD Guidelines fortesting ofchemicals no. 405. "Acute Eye Iffitancy/Corrosivity ",, adopted 12.5.198'1 

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