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### date 01 submiAion. 

### 3. I am.**~brnitt1ng** with this 

### DCtice 

### aD test c!ala in 

### my poaasioft or

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# I if

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### c::erti6catkm. statement that

### applies:

. I 

### I I 

### [8lThe Company twned in Part I, Sedion 

### A has remitted. 

## the fee of

### S2500 spe::ified. in 40 

### CFR 700.45 (b), 

### or 

# I o The 

### Company named. in Part 

### I. Section A has remittaci the fee of51000 !or an 

### Inten:Md.i:W! PMN (clefi.ned • 40 CFil7CO.43) in 

### ac:cord.anc::e with40 CFR 7CO.4S(b), 

### or 

# I o The

### Company named in 

### Part I, Section A is a 

### smaIl bwsi.ness 

### coru:em under40 

### a:R. 700.43 and

### hal

### mnittecl a fee 

### of5100 in**a~c:e** With 40 

### CFR 700.45 (b). if

### you are submitting a low 

### volume exemption (L 

### VEl application in 

### aa::ordance with40 

### CFR i23.SO 

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### tow t mease 

### mc:lloW' exposure exemptlOD (LoREX) 

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### CfR

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# I i 

# o The llW\u!actu.rer submittingthis

### .notice intends to 

### manutaCture or import

### the DeW' c:hemial wbetance for 

## ! conune:rd.al purposes, other than in small 

### quantities solely for 

### research 

### and development..·under the terms of40 CFR 723..so. .• 

# I 1 

# o The IN.nuJac:t:ure: is familiar with the 01 this 

### Medon aDd will

### comply t:boIe terms; aDd. I 1 I I I 

# o The

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### lorwhich the not:ice 

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### USCIOOL



# .

Date 

# '.-2

## Part 1- CENERAL INfORMATION
Ccnfl**dtn~** 

### Marketing 

### &



### New 

### York. NY 10022 b. Agent(if Name 01 authorized offidal applicable}

MailIng address (number and street) 

### Motohiro "Mike" Sonoda Sales Specialty Materials



### Floor INC. Manaqer,

Telephone

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

## i Position I -;:;;;;iipji;Y

______________-'--

__________________

Telephone
Position 

### Consultant

Type of Notice

- Mark (X) 

# 1.0

FORM EPA 77l(}'2S (Rev. 5-95) , Telephone I 

### 703 5907337

c. If you are submitting thb notice as part of ajoint submission. mark 00 this box.**J~t N~e~mthmU~~oow**  SublJlitle:r (if applicabJe)

Mai.ling address 

### mumber and street) State, ZIP Code

2. "'ame Contact 

### Dr. William A. 

### Olson (in US.) Company 

### Center for Regulatory Services, Inc. MailinglcUUe. (number

### ilia street) 

### 5200 Wolf Run Shoals Road CIy, State. ZIP Code 

### Woodbridge, VA 22192 3. U you have had a prenotice communication (PC) concerning this notice and EPA assigned a PC Number to the notice, enter thenumbOilr. Mark (X) )to if none

## ~ 4. Uyou previously**subaUtt~** iIIt exemption application for the chemical substance covered by thb notice, enter the exemption number assigned Mark (X) by EPA. If}'l'upreviously submitted. a PMN for this substance enter the )toif none 

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# o Both 

# O BindingOption Mark (x) Page 3

### Part I - GENERAL lNPORMATION - 

## Ccmtfnued. Sec:t:lon B - CHEMICAl. IDEN'ITl'Y t:Nf()1lMAnONt

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- 

## HtJMAN EXPOSURE AND ENVIRONMENTAL R.£LEASE

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### SectioA B - INDUSTJUAL SIrES CON'TROLLED BY OTHERS 

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.. ".**caoee~** 11_ dI!II1C1l'l*' batc:b). (3) -1deD1lfy by D'I2:IZIber tbe paintI ofreleIte, bdwIDs II'Ddcar 1DIIa:mIltIiatreIeufII. to tIM taYk'OlDmtof tile DIW'dMm.IcalJahetiDc:a. £nta' • ol.ila 

# I Dia.ram for the .ajor unit operation Itepa(k./batch)

The new chemical substance 4
.. lIixln. 0) Bilphenol A type liquid epoxy 

# 18=t Coat tn. re.ln with pi

.. ent and diluent (2) Cured

# epoxy~co.tin. film**~** Che.ical conversion (Typical one)**~~HCH2-R-CH2~7H2** +**H2NH2C-~CH2NHCH2CH2-~** Epoxy re. i n New chemical .ubstance (Cur in. a.ent) 1

## -CH2d:CH2-R-CH2~:CH2~H2C-~CH2~CH2CH2-~ 

# o Cured epoxy re,in Mark 00 this box IE you att.ch • ClXltlnuatl.on sMet. R:Bllphenol A re.idue 2. Worker**~ta1** l1et.. 

### n> - 

### ;rem. th. dIapm Il:Icwe. p1'OYIde the lett.- fer -=!:i 'W'CIrSer dvlty. Ccap&at 1-1 fer

# -= W'ClCkIr IdlvSty de.c::lbecL (2)
- EstI:mate the DUZnlMroi wcr.bnapoeecl for 

### an ... c:a:nbIMd. (4)

­ &timalll the typICIl duraI:kla olllXpCllUl'e per WCII'ket 

### mCal hours per dayad (b)"daysper ywr. " (6)

- Dac:x:I.be phySc:al foa:n of UId. S DeWc:bemtc:al_JbItmc:e Ofm zn1laure), IDd. D1 pObidbeeqW.pme:at md. Clpeed:Igcaatrols. if any used. to proted workm. (7)
- Estimate the petCI!lt 01 the Dew' subIItmaI.rormulated. wbeD p!ICkIp:l 01' UMCl.a 1INIl product. 

## en ­ Pram the 

### dlaPm**a~** enlet the D\IIZIbcof

# -= releaepaIrIt. CampIIte,.13 fer

# -= rete.epaiDt 1

......t!6ed (10)

­ Estimate the 1m<Nllt 01 the newsubItanc:e :eleaedCal d.iredlylO tNt or (b) IDIO ccatrcl-=wosrlo tbe e:t:I.VIroI.uDIt an kg!day or

### ksfbatcb). • .". " (12.)

­ Dac:x:I.be media of:eJe.e (Jtac:X air, Iujftlw IIr**(~~** MamaD,1UIfiIce WIIIiII;. CIiIHIItIe or CilJ.dI1IDcl or IIIdDer:II:Ica. POTW, or other 6Jped.fy) aDd castrol tech&:Iclosy.llaDY, tbat 

## wm be UMIto IImI.t 1M of tba DI'WtI.1bItmc:e 10 Ibe cmtlDlDiillt.**(1~)** - IdeDIlfy b)'l'fCd'Qds wbk:h 21'1&yftlUlt &oat Ibecpaatk& .' " G)' (5), (8). Ol). (13) ad.AS)

- Mark OOtbll c:okI:ilm If .,. ollbepo _Dzl,fIIIlda a..c:calldedl1 !I:I:fcmuIIk:G (CBI). Latter 'ol CBI Ount:k::a ICBI**P.n.dl"'~** I 

# 1ft!.. CBZ 

### a.a- It.IDaaDtof CJI Caatrol 

## em

"01 

### worm. 01**~.Ctiatr.rbl** " " New 

### Ttdmdosr Act­ .ExpoIed 

### Expaan 

# . Pbjlkal

### rar. tIDd '" u1aIka**~** !.vUy DCWIQIwtMn . III IIBI (1) I 

### aOIl : 

### nOlI) (2) (2) (3) (4a) I "II) (5). (6) Q) (I) (9) 

### nt (13) I I 

## Burn 1 1 1.01 

## Rubber groves 

## <D 0.2/b1 I 

## Respirator I 

## , • I**~atety** 

## goggles I' 

## 2 1 " 1. 0 I 

## Rubber groves -(2)**0.02/~** 

## Burn I 

## Respir.ator I I 

## 0.011, 3 1 

## 1.0: 

## Respirator 1.0 @ 

## Not applicable I I I I I I**(1')-~** (15) 

# o wmoo**~_IIJ'Oflatt.ch.~** .... 

### hplO

### OPTIONAL POLLunON PREVENTION INFOR.M:ATION 

## I I i To c:laiJn information In this !IIlCtion u c:on.flde:ntLal d.rcle orbracket the spedftc 1nlormadon that you cla.Im u a>n.ftdentiaJ. 

### i
, 

### In this section you may provide information not reported elsewhere in this form regarding your efforts to reduce or mi.nimize potential risks associated with activities 

## SUlTOunding manufacturing, processing, use and disposal of

### the PMN 

### substance. Pleaseinclude new information pertinent 

### tQ pollution preventiqn, including source reduc:tion, recycling activities and 

### safer processes or products available due 

## to, the new 

### chemical 

### substance. Sourcereduc:C6n includeS the reduction in the amountortoxicity

## ofchemical wastes

### by technological modification, process and procedure modification, product**refonnu1atio~** 

### rawmaterials substitution, and/or inventory controL Recycling refers to the reclamation of useful 

### chemical components from wastes thatwould otherwise 

### be 

## treated orreleased as air 

## em.issions or :water 

### discharges, orland 

## disposal Desaiption's ofpollution**preventio~** 

### source reduction and recycling should**emp~** 

### potentialrisk reduction 

## subsequent tQ compliance with existing regulatory requirements and can

### beeither quantitative orqualitative. The 

### EP 

### is interested in this 

### information to assess overall 

## net reductions 

### in toxicity orenvironmentalreleases and exposures, not the shifting of risks 

### to other environmental media 

### ornon-environmental areas (e.g., occupational or consumer exposure). In addition, information on the 

### relative cost or performance characteristics of 

### the PMN' substance

### to potential altematiyes maybeprovided.­ All informationprovided 

### in 

### this section 

### willbe taken into consideration during the 

### review of this 

### substance. See InstructionsManual and Pollution Prevention Guidance 

### manual for guidance and examples.

_ De5cn"'be the expected net benefits, such u (1) an overall reduction inrisk tohuman healthorthe e.tMroru:ttent; (2) a reduction in the volume manufacturecl; (3) a reduction in the generatlon of waste rnaterials through rec:yding" sou.rce reducDcn orother means; (4) a reduction in pote:l'Itial toxicity orhuman 

### expowre and/or envIroruN!l:l\ta.l reJ.eue; (5) an inaeue In product perfcnnance,. a de:rease in the eM ofprocbx:don 

### wI or improved operation ef6denc:y ofthe new c:hemical subetance in comparison to existing chemical substances used in :si.m.Da.r applk:ations; or (6) the extent to which the N!Wc:hemical subttanc:e ma.y be a substItute (or an existing

## substan.ce that poses a 

### greater overall risk to hu:man health or the envirorune:nt. 

### 1. 

## New chemical substance can reduce release of 1,3-benzenediaethanaline into air when used as ­ an epoxy curing agent as compared with neat 1, enzenedlaetnanallne
2. 

## New chemical substance provides long pot life for an epoxy res In cmpound and gOOd propertI es for cured epoxy resin as an epoxy curing**agen~**



# o Mark (X) thia box " 

### you dIc:h • ccnt!nuclon IhMt. Paptl

### Pan 

# m- UST OFATIACHMENTS
AttKh CIOIItlnUab._ far IICtIcM cllbe farm

# _tilt data

.. otMr

_ 

### ODcluc:lbts phJlbllcl:Ma:llcal propenlelmc:1**·,tnlCt\lz'e/lldMty~, -~~dlrtlU,....** **a~** aItIdIIIMDt_ thlldclaol 1M fcrm to whIdlltrUe.,1f**~CCIDIICIltlWIl7"""** ctibi

........ _ .. I'll c::Dlu:IaIIl beIaw,.

__**tbe~** pep mamba 01 1Mb attIcbllMllt

. Mark 00 11M "Con8ded'" box oatto ID'f an.

## "'!.MDt
.... 7OUcIIbD. cr:cfIcIeDttll

.. 1IIt
.......cI:bIa

## MiIaaal_ 

# C OIl how to dU:D 

## -rid

.. ii·h I'll ••IhrbNDt.CI'ID8<IeotW Youat tnclDdI w1IIa
....... ".,.,capy oltbellOtlcl _lIIIIlIzed 'V'CIioIl**QI.,~** III

# \IIIhIdl,... cIIbD 1Mi'll

..... ClXlIldeIlIl 

# . I A,,*hIlNt AtlltWwtDllDJt 

### o.s**pqe~) ~**  

# . Material Safety Data SheetOOOS) 

# /:3- 

# /q Physical and chemical properties 

# 20 GC chr(lllatogram 

# 2J 1 spectnJII 

# 22­ 13C-NIR spectrum 

# 23 I R spectrum 

# 2JI CAS Inventory Expert Service Report 

## 2.5 Reverse Mutation Assay "Ames Test" 

# Z"-57

.



# -

' 

## .

..



# o 'Ida: 00 ddIt 'baz If 1O'111ti1dla CNlittII W!c.

......... alllldl-* 1IIIIDII1IId

..... 

# ,.1% ,

## MITSUBISHI GAS CHEMICAL AMERICA, INC. Page 1 of7 MATERIAL SAFETY DATA SHEET Product Name: GASKAMINE 240 Preparation date: 

### 817102

PRODUCT NAME: GASKAMINE 240 MSDS REVISION 

### #: New
SYNONYMS: None DISTRIBUTED BY: Mitsubishi Gas Chemical America, Inc. 520 Madison Avenue, 17 th Floor New York, NY 10022, USA PHONE NUMBERS: Inquiries
- (212) 752-4620 (business hours) CHEMTREC
- (800) 424-9300 (transportation emergencies)

Component CAS No Exposure I jmjts**~** 1,3-benzenedimethanamine, >99 404362-22-7 Not established reaction products with styrene 1,3-benzenedimethanamine <1 1477-55-0 ACGIH STEL 0.1 mg/M 3

­ (skin) 

## I;·'.••••••••..{···••••·••••···.·.;••· •• ;··. ·.·.·.·.·.;···.···••••••••••·.···;•••••; •••

# "S~TIQI$··l· 

# •••**H~RP$** 

# ••**~O~N.t~~jW~** 

# •• ; •••••••.·

••••••••.•·••••· ••••·• ••r

••••••••• i·.·.•..•.· .......;.· ..•...) 

### *********************************** EMERGENCY OVERVIEW 

### ********************************** Product is a pale-yellow. clear liquid. Causes severe skin and eye irritation. even bums. Vapors. especially upon heating, may cause severe irritation. Can cause allergiC reaction. Toxic to fish. Avoid contact with skin. eyes and clothing. Avoid inhalation of vapors. Wear protective goggles and gloves and impervious clothing when handling this product Wash thoroughly after handling. ********************************************************************************************************
Section 3 continued on next page 

# 13>

## MITSUBISHI GAS CHEMICAL AMERICA, INC. Page 20f7 MATERIAL SAFETY DATA SHEET Product Name: 

### GASKAMINE 240 Preparation date: 8/7/02

POTENTIAL HEALTH EFFECTS EYES: Vapor, especially when heated, can cause irritation, redness, tearing, blurred vision; liquid can cause severe or permanent eye damage. SKIN: Can cause severe irritation, even burns, on prolonged contact. Can cause allergic skin reaction. INGESTION (swallowing): Results in severe damage to mucous membranes. INHALATION (breathing): Vapor, especially upon heating, may cause irritation of the respiratory tract. CHRONIC EFFECTS/CARCINOGENICITY: This product (or component) is not listed in IARC Monographs, the NTP Ninth Annual Report or the ACGIH TLVs as a carcinogen or potential carcinogen. OSHA does not regulate it as a carcinogen.

EYE CONTACT: Immediately flush with large amounts of water for at least 15 minutes, lifting upper and lower lids occasionally. Get medical attention immediately. If physician is not immediately available, continue flushing with water. 00 not use chemical antidote. SKIN CONTACT: Immediately wash exposed area with soap and water for at least 15 minutes, then flush with water. If reddening persists, or if open sores or blisters develop, see a physician. Remove contaminated clothing and launder before re-use, using hot water and heavy-duty detergent.
Section 4 continued on next page 

# If

## MITSUBISHI GAS CHEMICAL AMERICA, INC. Page 30f7 MATERIAL SAFETY DATA SHEET Product Name: GASKAMINE 240 Preparation date: 

### ant02

INGESTION (swallowing): If conscious, immediately give two large glasses of water. Never give anything by mouth to an unconscious person. Call a physician. INHALATION (breathing): If affected, move to fresh air. If a burning sensation develops in throat or chest, or shortness of breath occurs, get immediate medical attention.

FLASH POINT: 401° F (205° C) FLASH POINT METHOD: CDC UPPER EXPLOSION LIMIT: Unavailable LOWER EXPLOSION LIMIT: Unavailable AUTOIGNITION TEMPERATURE: >752° F (400° C) EXTINGUISHING MEDIA: Dry chemical, water fog, and regular foam FIRE AND EXPLOSION HAZARDS: None known FIRE-FIGHTING EQUIPMENT: Keep personnel removed from and upwind. Wear full protective clothing and selfcontained breathing apparatus with full face-piece. Cool containers with water. Water or foam may cause frothing which can be violent, especially if sprayed into containers of hot, burning liquid. Combustion/decomposition products may include amines and other nitrogen compounds as well as carbon monoxide and carbon dioxide. SECTION 6·

......

## .6.~CI~~TAt.R~tEASEMeA$URES/! 

# •••••••· ••••••• (·······
Persons not wearing protective equipment should be excluded from the area of the spill until clean up has been completed. Dike area of spill to prevent spreading and pump liquid to salvage tank. Absorb remaining liquid on vermiculite, floor absorbent or other absorbent material and shovel into containers. This product is toxic to fish; prevent run-off to sewers, streams, lakes and other bodies of water. 

# /5

## MITSUBISHI GAS CHEMICAL AMERICA, INC. Page 4 of7 MATERIAL SAFETY DATA SHEET Product Name: GASKAMINE 240 Preparation date: 

### ant02

HANDLING: Avoid contact with skin, eyes and clothing. Avoid inhalation of vapors. Wash thoroughly after handling. STORAGE: Keep in closed or covered containers when not in use. Store in cool dry place with adequate ventilation. Do not store near heat or open flames.

RESPIRATORY PROTECTION: A NIOSHtMSHA approved respirator with organic vapor cartridge is recommended, especially if product is heated or where there is insufficient ventilation. SKIN PROTECTION: Wear protective gloves such as Neoprene or BUNA-N. EYE PROTECTION: Chemical splash goggles in compliance with OSHA regulations and full face-shield are advised. OTHER PROTECTION: Impervious clothing and boots are recommended. Leather shoes and boots cannot be decontaminated if soaked with liquid material. ENGINEERING CONTROLS: Provide sufficient ventilation to maintain exposure below level of overexposure and established exposure limits.



# lip

### MITSUBISHI GAS CHEMICAL AMERICA, INC. Page 50f7 MATERIAL SAFETY DATA SHEET Product Name: 

### GASKAMINE 240 Preparation date: 

### 817/02

APPEARANCE: Pale yellow, clear liquid @ 100 0 F (380 C) ODOR THRESHOLD: Unavailable SPECIFIC GRAVITY: 1.05 @ 770 F (250 C) VAPOR PRESSURE (mm Hg): 1.3 x 10.5 Pa @ 77° F (25° C) VAPOR DENSITY (Air 

# =1): Heavier than air INITIAL BOILING POINT: Unavailable FREEZE POINT: Below 

### -40 F (-20° C) EVAPORATION RATE: <1 (Ethyl Ether 

# =1) SOLUBILITY in WATER: 2.075 gm/L SURFACE TENSION: 49.9 mN/m @ 69° F (20.5° C) for a 90% saturated solution VOLATILE 

### %: <1 pH: Unavailable pH METHOD: Not applicable

# ~................................................... ••···••·•··..·.• •••.• ;

.••••.•$Eci"ION••• 1ij••••••

# ~· ••••

# $TiB'~;~[JR~ij.tti.r.· 

# ••· ••· .....·••••· .••··••.·••.•·••••.·••••••; ...;·••••••••••••·.i·
...·.·.·.··1 STABILITY (conditions to avoid): Stable under normal conditions. INCOMPATIBILITIES (materials to avoid): Avoid contact with strong oxidizing agents and strong mineral acids. DECOMPOSITION: Not available HAZARDOUS POLYMERIZATION: Not known to occur

Salmonella/microsome mutagenicity (Ames)
- no evidence of mutagenicity 

# 17

### MITSUBISHI GAS CHEMICAL AMERICA, 

### INC. Page 60f7 MATERIAL SAFETY DATA SHEET Product Name: GASKAMINE 240 Preparation date: 

### 817102

No data available. Amines, in general, are considered toxic to fish and other aquatic organisms.

Incineration is the recommended disposal method for all chemical wastes. Material collected on absorbent material may be deposited in a landfill in accordance with all applicable local, state and federal regulations. This product. if disposed of. may be considered a corrosive waste (0002) under current RCRA regulations due to the pH of the material. 

### 1 •••• •••·.....····< ••••••.••••..•••••••..•...........••..••••••.•••••.•.••.•••• •••••··•···•·....

# ·$~QtfdN 

# ••••··i4••••jj;•••

# ~~~$~~>lfj~~~~6tl·; 

# •. i ••;.···.·•••••·.·.·.I
...·.·.. 

### ···;····;················\;);1 Not regulated under current DOT, IMO, or ICAO regulations. 

# I>n•..•• ····>}···.··.<··•••> •.· .•

# ·SeCTIO"15.;REdQt.AT()~l'IN~PRMAf~)· 

# ····•••**·~I** TSCA INFORMATION: This product is, to the best of our knowledge, not listed on the TSCA Inventory. Use of this product is restricted to technically qualified individuals for research and development purposes only. SARA 313 INFORMATION: SARA requires submission of annual reports of release of toxic chemicals that appear in 40 CFR 372. This information must be included in all MSOS that are copied and distributed for this material. Components present in this product at a level that could require reporting under the statute are: None

# J~

## MITSUBISHI GAS CHEMICAL AMERICA, INC. Page 7 of 7 MATERIAL SAFETY DATA SHEET Product Name: GASKAMINE 240 Preparation date: 

### 8/7102

HAZARD RATING: HEALTH 

### 3 0- LEAST FIRE 1 1 - SLIGHT REACTIVITY 

## o 2-MODERATE OTHER 3- HIGH 4-EXTREME HAZARD RATING METHOD: HMIS REASON FOR REVISION: New MSDS
The product information contained herein is beHeved to be acrurate as of the date of the Material Safety Data Sheet, and is provided without warranty, expressed or implied, as to the results of use of this information or the product to which it relates. Recipient assumes all responsibility for the use of this information and the use (alone or in combination with any other product), storage or disposal of the product, induding any resultant personal injury or property damage.  END OF REPORT** *·

### PHYSICAL AND CHEMICAL PROPERTIES WOKKSHEBT To UIbt FJWI m1ew01

# pitT: aftCl cbtmIc:Il prOpIrtltIdata.**p-~** 

### ..**~~** foe 

### c:t.ta)'all 

### pn:mcs. Illd Udude It

### mthe ftdk& cIcIIly tbIpcOf*t1l1llU

..«I,.the 

### PIP01 theDOtka

.. wtddl plCIpCtJ'

...... iheVaNe 01**the~the** \DIlt:I 

### mwIdda**tlIiII~** ..**-*c.~_** ,......oraot
.. 

### P %:2!lIIc1aialect. coaJJdcItI.aI. 

# :=... ofthe

.. IUbiItDcIlbauidlie "l1MM**~~** lie

... the I*t Clc.lCl'5 pun) c:ltcNc:al PropiIt118 dud 

# "III'M" tar IIII:IctuN ortara&1atIoaI 

# _10 aOIIId

### <" PWN 

### m_).You Itt

# not:= to IUbaIIt tbiIi WlXbbeIc;lao

....., SPA

# = It( Qalllllllld. tMtJ'CIGdo 10,. It

# will...,.., lWfIew IZId. CIII.n**that~** lIIhmetSoft**:=~** YeN 1uIIadt

....woGabiiIt.al\lPf

..... 10 JWitaJhmilalloa 01
.. c!atl. ThIll

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....dIlL . " ". PropIJty WakOO.1I 

# :r. VIlDI w-.t (".Qd.

### ~ 4iIIIIW (I) ptcw.IcIai (b) (d

### ~OO 

# ".It Physlc:a1 ale of\.'leal IUbItmm X X

_ C:I)_ CD

_ (a:) Varel.::ur. 

## ac x 1. 

## 733x10-3 Tan' 

## x 1.05 OensltyIreiatlYw demtty 

### glcm3 SolubWty 

## 25 

## ac

• TCI.\pcaNre 

## Ethanol, Aceton, Xylene x 1000<

SolYmt**~** Solubility In water • Tea:rpe:rature 

## 20 ac 

## x 2.075gJL "-c 

## Freezing temperatur.e x <-20 -c MellinrtemJ:)cature Bci1inc/subUmatica teD:u:>eratln • tca'prean 

## x 

## lH-NIR, 13C-NIR, IR SptCtra DissodaI:ion const;mt

, Putide sf.:e dJstribW::ilxa O::tmcl/waterpc1ili«lcoelSdeat 

## x LogPow>6.20

Harr'1 LawCOlUlblllt Vollld.zatlcrl from walls' Vol1taB.utlCll from sell

. pH • t::'OIICIIIl1b'tkm FIammabiW:y 

## Flash point x 177±2CC**~** 

## x Negat i

## ve <fred i 

## cated)**~/~** 

## x LogKoc>4.42 0tMr-Sf*Ur

## Attached sheet 1 1.3-Benzenedimethanamine. N-(2-phenylethyl) derivs. 

# GC chromatogram
Detector : FID Shiaazu 6C-14A Col

.... 

### : Ultra Alloy EX UA-l(Frontier lab.) O. SMtb

### xts. df 1.

# 5". TellPerature : CoIUln initial**250~(31in** 

### hold),final**300~(18.** 1ain 

### hold), Inj. 

## & Det.**300~** 

### : 0.5" I (2wtl 

### lethano I solvent)

1.0

v Q I t 

## • 0.5

0.0 \ 

# \**~NIIC¥Ha'O** 1.0 

### I 

# ,tHpAl**IYIH~H2** 'cHzCMt'O 

## 2**V"""~2~Hl)** V 

### fe·S a I 

## 3 

# ,0 t s 

# u**HzCHpN~zC"2** 'cHP 

# llD . 4

IN 

# '"

# 'J.~ 

# :**~:** [,-,,0 

## :1·

0 5 10**MIn~** HI 20 25



### Peak1 2 3 4 Time



# ---------­ 2.33 6.40 7.14 14 .18 Area



# ---------­ 5327530

455067 3536434

387794 Area %



## ---------­ 54.9

4.7 36.4

4.0 Totals 9706825 100.0

### Attached sheet 2 1. 3-Benzened 

### i methanam 

### i ne, N- (2-pheny I ethy I) der

### i vs. 

# lH-NMR spectrum ,-----.--. -.----...-----..---. - .......-- ..----. .-. ---------- ----- -- DATIM
..**----~** l:::r.

- ":'1 

### oc :::;.::.c - :',1:""; '-C or. ::n 

## =- or.**~** x x Ir.::.c -, x m Ir.:") Ir. Nlt-::n '7;" x N t-**~:..o ~.-**  m '"'1" t-::n Nt-
....... :..o:::>**~:::>** 

### MENUF 1(; or. or.**~:-: ~** **~"; ~** **~::::** x 1·- '-C or. -r':") N:::: cr::::o:..o::n:..o Ir....,.. r-- t- <..0:..0 Ir. Ir..."...**~..",..** N::n x t- t-:..o 1r.10 "'" "'" 10:::> 1 

### t-r--: "': r--: r--: I

### ':"~**~ ~ .:;-.~** **':'~ r-~** **~ ~ ':'~**  -: '"': -: -: -: -: -: '"': 

# c:: c::**~lQ** 

### ex: 

## 0:: ex: ex: ex:**~ ~** 

### r--: r--: r--: r--: r--: r--: r--: r--: r--: ':

# c: OONUC it-I'-I"'-:r1r l

- t-I- t- t-t- t-t-t-t- t- t- t-t- t- t-t-**t-C":)~C":)~** NN N N N**N~~NNNNJ:rvuuJ(** 

### OFR

## l~~#ddJ JpJ,J 

## J J I 

## I I

## ,~)~~ddd.~ 

# . - OOSET**~~** 

### PlY 1 

### DEADT PREDL 

## IWT 

## POINT SPO TIMES DUMMY 

### FREQU FLT DELAY ACQ'IlI 

### PD 1...\ .....:J 

### ADOIT RGAIN OF T1 

### T2 T3 T4 EXMOD 

# (! EXPCM IRNUC 

## IFR IRSET IRFIN : 

### , I**~**



## 1 

## '---­ 

# L I**~** 

### IRRPlY

# Jir~ LKSET LKFIN LKLEV I I 

### n-MI LGAIN I I 

### LKPHS H 

### 7 

## o rn 'I :l :l () 

### LKSIG

. __________....... .1 CSPED 

### Mon Feb 18 09:12:45 2002 NON IH 399.65 MHz 124.00 KHz 10500.0 Hz 6.2 us 11. 9 

### us 0.2000 ms 1. 

### 0 sec 32168 32168 8 1 

### 8000.0 Hz 4000 Hz 50.0 us 4.096 sec 2.904 sec

0.00 6 

### O. 

### 12 Hz 0.00 0.00 90.00 100.00 

## NON NON:Single.coupled:PWl_ACQTM_PD lH 

### 399.65 MHz 124.00 KHz 10500.0 Hz 55 

### us 511**¥VMIYcYM~¥8¥PMS-02AINON_EI_FT.** 

### 02010901 61. 60 KHz

19.0 liz



### 180 23 100 

### 196

II Hz

## Attached sheet 3 l,3-Benzenedimethanamine, N-(2-phenylethyl) derivs. 

# 13C-NMR spectrum

----.--..------- --.-.----- DATIM Mon Feb 18 16: 16:54 2002 c C X :t;**~** - iC":.'

-- ":'f:'**~** r- C - -::""" It:' ...- t- 0 t'- r- t.C :.c M :.c C'J m to r- " oc**~ ~** c.o 00 0 lr:; -t to 0') <.0
-- N c.o 

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## Attached sheet 4 l,3-Benzenedimethanamine, N-(2-phenylethyl) derivs. 

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## 4000 3600 3200 2800 2400 2000 1600 1200 800 WAVENUMBERS(cm-l) RESOL : 4 cm-l 

### 3297.72 96.45 1452.15 67.25 SCANS 

### : 20 3058.59 93.58 1336.44 86.48 AMPGAIN P. tNT 

### : 1(4 : 2 cm-l 

### 3023.87 2919.13 89.32 88.58 1153.24 1112.74 82.39 74.03 BEAM : single S. 

### SPEED : lOS S.NUMBER: 2643 2815.60 1943.92 1870.64 88.33 97.33 97.38 1081.88 1029.81 1002.81 81.82 82.66 86.41 M.DAlE 

## F.NAME : 2/18/2 

## : MS0218-2.WSF 1805.01 1602.58 97.46 83.26 919.67 889.03 86.71 79.38 UQA 1;11 77 '1 A

# M6C.ieCc 

# ern Inventory 

# Expert Service

A division of the American Chemical Society Phone: 800-631·1884. 614-447.3870 Fax: 614-447·3747 E-mail: answers@cas.org Internee hcrp:llwww.cas.orglSupportldient.html

INVENTORY EXPERT SERVICE REPORT

IES-ORDER**NU~ER:** 66641
REGISTRY NUMBER: 404362-22-7

CA INDEX NA.'iE 1,3-Benzenedimethanamine, N-(2-phenylethy1) derivs.

Please print the above CA Index Name on the appropriate page of your PMN. 

# o If this box is checked, CAS has made correction(s) marked in red to your IES order. Please make the same correction(s) to your PMN before submitting it to the EPA.

2540 Olent.lngy River R03d .: PO Box 3343 Columbus. Ohio 432 10·0334PAGE 1 OF 31 PAGES



# SafePharm Laboratories

MXDAlSM Adduct: REVERSE MUTATIPN ASSAY"AMES TEST" USING 

### SALMONELLA TYPHIMURIUM AND 

### ESCHERICHIA COLI SPL PROJECT NUMBER: 930/074



### STUDY SPONSOR: Mitsubishi Gas Chemical Company, Inc. Mitsubishi Building 

### 5-2, Manmouchi 

### 2 chome Cbiyoda-ku TOKYO 100-8324 JAPAN

930-074.docIIDS 

### AUTHOR: PWThompson



### TEST FACILITY: Safepharm Laboratories Limited P.O. Box No. 45 

## DERBY DE12BT U.K. Telephone: (01332) 792896 Facsimile: (01332) 799018SPL PROJECT NUMBER: 9301074 PAGE 2


### QUALITY ASSURANCE REPORT This study type is classed as short-tenn. The standard test method for this study 

### type ("General Study Plan" 

### in OECD terminology) was reviewed for compliance once only on initial production

. 

### • Inspection of the routine and repetitive procedures that constitute the study is carried out as a continuous process designed to encompass the major phases at or about the time this study was 

### in progress. This report 

### has been audited by Safepharm Quality Assurance Unit, and is considered to be an accurate account ofthe data generated and ofthe procedures followed.
In 

### each case, the outcome of QA evaluation is reported to the Study Director and Management on the day of evaluation. Audits of study documentation, and process inspections appropriate to the type 

### and schedule ofthis study were as follows:



### § § 15 August 2001 Standard Test Method Compliance Audit 27 May 2002 Test Material Preparation 29 May 2002 Test System Preparation 30 May 2002 Exposure 30 May 2002 Assessment ofResponse 10 June 2002 Draft Report Audit Date ofQA Signature Final Report Audit 

### § Evaluation specific to this study

# ~~ 

# ...**~~.0\~** 

# ............... DATE:
.......... ..

## ?)M~3~~? 

# ............. For Safephann Quality Assurance Unit*

"Authorised QA Signatures: Head ofDepartment: JR Pateman CBiol MIBiol DipRQA FRQA Deputy Head ofDepartment: JM Crowther MISeT MRQA Senior Audit Staff: N Johnson BSe MRQA; G Wren ONC MRQA; R Hurst MRQA 

# 27SPL PROJEer NUMBER: 9301074 PAGE 3


## GLPCOMPLlANCESTATEMENT The work described was performed 

### in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106)). These Regulations are 

### in accordance 

### with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENVIMC/CHEM(98)17); and are 

### in accordance with, and implement, the requirements of Directives 87118IEEC (as amended by Directive 1999/111EC) and 88/3201EEC (as amended by Directive 19991121EC).
These international standards are acceptable to the Regulatory agencies of the following countries: Australia, Austria, Belgium, Canada, the Czech Republic, Denmark, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Israel, Italy, Japan, Republic of Korea, Luxembourg, Mexico, The Netherlands, New Zealand, Norway, Poland, Portugal, Slovenia, Spain, Sweden, Switzerland, Turkey, the United Kingdom, and the United States ofAmerica.
This report fully and accurately reflects the procedures used and data generated.

:-:::::'-"1"c.-.



### .................................. . 

## 2 2 j 

## U L 2002

_...................................... DATE
.................................................. .. Study DirectorSPL PROJECf NUMBER: 930/074 PAGE 4



### CONTENTS QUALITY ASSURANCE REPORT 2 

### GLP COMPLLANCE STATEMENT 3 

### CONTENTS 4 

### SUMMARY S 

### 1. INTRODUCTION 7 

### 2. TEST MATERIAL AND EXPERIMENTAL PREPARATION 8 

### 3. METHODS 8 3.1 Tester Strains 

### 8 3.2 Preparation ofTest and Control Materials 

### 9 3.3 Microsomal Enzyme Fraction 9 3.4 S9-Mix and Agar 9 3.5 Test Procedure 

### 10 3.6 Acceptance Criteria 

### 11 3.7 Evaluation Criteria 12 

### 4. ARClDVES 

### 12 S. RESULTS 

### 13 5.1 Preliminary Toxicity Study 13 5.2 Mutation Study 

### 13 

### 6. CONCLUSION 

### 14 

### 7. REFERENCES 

### 14 Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls) 15 

### 16 17 18 19 20 21 22 23 

### 24 Table 2 Test Results: Range-Finding Study

- Without Metabolic Activation Table 3 Test Results: Range-Finding Study

- With Metabolic Activation Table 4 Test Results: Main Study Without Metabolic Activation Table 5 Test Results: Main Study

- With Metabolic Activation Figure 1 Dose Response Curve: Range-Finding Study

- Without Metabolic Activation Figure 2 Dose Response Curve: Range-Finding Study

- With Metabolic Activation Figure 3 Dose Response Curve: Main Study

- Without Metabolic Activation Figure 4 Dose Response Curve: Main Study

- With Metabolic Activation Appendix 1 Report ofResults in Mutagenicity Test using Micro-organisms Appendix 2 History Profile ofVehic1e and Positive Control Values 29 30 31 Appendix 3 Chemical Structure Appendix 4 Statement ofGLP Compliance in Accordance with Directive 88/3201EECSPL PROJECf NUMBER: 9301074 PAGES



### MXDAlSM Adduct: 

### REVERSE MUTATION ASSAY "AMES TEST"



### USING SALMONELLA TYPHIMURlUM 

### AND ESCHERICHIA COLI
SUMMARY Introduction. The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MET!, MIlLW and MAFF. It also meets the requirements ofthe OECD Guidelines for Testing ofChemicals No. 471 "Bacterial Reverse Mutation Test", Method B13114 ofCommission Directive 

### 2000/321EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Methods. Salmonella typhimurium strains TA1535, TA1537, TA98 and TAlOO and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding study was determined in a preliminary toxicity assay and ranged between 0.5 and 1500 Ilglplate depending on presence or absence of S9-mix. The experiment was repeated on a separate day using a similar dose range to the range-finding study, fresh cultures ofthe bacterial strains and fresh test material formulations. Additional dose levels were included in both experiments to allow for the toxicity of the test material, ensuring there were a minimum of four non-toxic doses.
Results. The vehicle (dimethyl sulphoxide) control plates gave counts ofrevertant colonies within the normal range. All ofthe positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity ofthe assay and the efficacy ofthe S9-mix were validated.
The test material caused a visible reduction in the growth ofthe bacterial background lawn to all ofthe bacterial tester strains, initially at 150 and 500 Ilglplate without and with S9 respectively. The test material was, therefore, tested up to the toxic limit. No test material precipitate was observed on the plates at any ofthe doses tested in either the presence or absence ofS9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose ofthe test material, either with or without metabolic activation. 

# 3DSPL PROJECI' NUMBER: 9301074 PAGE 6 

### Conclusion. The test material was considered to 

### be non-mutagenic under the conditions of this test.



# J/SPL PROJECT NUMBER: 9301074 PAGE 7



### MXDAlSM Adduct: REVERSE MUTATION ASSAY "AMES TEST"

USING 

### SALMONELLA TYPHIMURIUMAND 

### ESCHERICHIA COLI
1. INTRODUCTION This study was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system. The study was based on the in vitro 

### technique described by Ames and his co-workers (1, 

### 2,3) and Gamer et al 

### (4) in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium 

### and tryptophan auxotrophs of Escherichia coli 

### to various concentrations of the test material. The method confonns to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MET!, MHLW and MAFF. This method also confonns with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471, Method B13/14 in EC Commission Directive 2000/32IEC and the USA, EPA (TSCA) OPPTS harmonised guidelines. A copy ofthe Certificate of Compliance with GLP, issued by the UK Department of Health, is included as Appendix 4.
The mutant strains of Salmonella 

### are incapable of synthesising histidine and are, therefore, dependent for growth on an external source of this particular amino acid. When exposed to a mutagenic agent these bacteria may undergo a reverse mutation to histidine independent forms which are detected by their ability to grow on a histidine deficient medium. Using various strains of this organism, revertants produced after exposure to a chemical mutagen may arise as a result of base-pair substitution in the genetic material (miscoding) or frame-shift mutation in which genetic material is either added or deleted. 

### In order to make the bacteria more sensitive 

### to mutation by chemical and physical agents, several additional traits have been introduced. These include a deletion through the excision repair gene (uvrB- Salmonella strains) 

### which renders the organism incapable of DNA excision repair and deep rough mutation (rfa) which increases the permeability of the cell walL A mutant strain of Escherichia coli 

### (WP2uvrA), which requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence, was used to complement the Salmonella 

### strains. This strain also has a deletion in an excision repair gene (uvrA-).
Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell, the test material and the bacteria are also 

# 32

### 3.1 SPL PROJECT NUMBER: 9301074 PAGES


### incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pretreated with a compound known to induce an elevated level ofthese enzymes. The experimental phase ofthis study was performed between 16 April 2002 and 05 May2002.


### 2. TEST MATERIAL AND EXPERIMENTAL PREPARATION Sponsor's identification MXDAlSM Adduct Description pale yellow viscous liquid Batch number PMS-02A Date received 09 April 2002 Storage conditions room temperature, in the dark until 12 April 2002, thereafter room temperature, in the dark under nitrogen Data relating to the identity, purity and stability of the test material are the responsibility of the Sponsor.


### 3. 

### METHODS Tester Strains Salmonella typhimurium 

### TA1535, TA1537, TA98 and TAI00 Escherichia coli 

### WP2uvrAThe Salmonella typhimurium 

### strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia coli 

### strain WP2uvrA- was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -196°C in a Stateboume liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to 

### confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.SPL PROJECI" NUMBER: 9301074 PAGE 9 

### 3.2 Preparation ofTest and Control Materials The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 

### 2 mm pellets with a nominal pore diameter of 4 x 10-4 microns.
Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control and the positive control materials were as follows: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 

### 2 IJglplate for WP2uvrA-, 3 IJglplate for TAtOO and 5 Ilglplate for TA1535 9-Aminoacridine (9AA): 80 IJglplate for TA1537 4-Nitroquinoline-l-oxide (4NQO): 0.2 Ilglplate for TA98 

### In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the absence of metabolising enzymes, were used in the series of plates with S9-mix at the following concentrations:
2AA at 1 Ilglplate for TAl00 2AA at 2 Ilglplate for TA1535 and TA1537 2AA at 10 Ilglplate for WP2uvrABP at 5 Ilglplate for TA98 

### 3.3 Microsomal Enzyme Fraction S9 was prepared in-house on 03 March 2002 from the livers of male Sprague-Dawley rats weighing

- 250g. These had each orally received three consecutive daily doses of**phenobarbitone/~-naphthoflavone** 

### (80/100 

### mg per kg per day) prior to S9 preparation. Before use, each batch of S9 was assayed for its ability to metabolize the indirect mutagens 2-Aminoanthracene and Benzo(a)pyrene. The S9 was stored at -196°C. 

### 3.4 S9-Mix 

### and Agar The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration ofthe test.SPL PROJECT NUMBER: 930/074 PAGE 10


### S9 5.0ml 1.65 M KCl/0.4 M MgCh 1.0ml 0.1 M Glucose-6-phosphate 2.5 ml 0.1 MNADPH 2.0ml 0.1 MNADH 2.0ml 0.2 M Sodium phosphate buffer (PH 7.4) 25.0 ml Sterile distilled water 12.5 ml
A 0.5 ml aliquot of S9-mix and 2 ml of molten., trace histidine or tryptophan supplemented, top agar was overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day ofeach experiment. Top agar was prepared using 0.6% Difco Bacto agar and 0.5% sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 ml of top agar. Vogel-Bonner Minimal agar plates were purchased from International Laboratory Services Ltd. 

### 3.5 Test Procedure 3.5.1 Preliminary Toxicity Study In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity ofthe test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 J.1g1plate. The study was performed by mixing 0.1 

### m1 of bacterial culture (TAtOO or WP2uvrA"), 0.1 ml oftest material formulation, 0.5 ml ofS9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition., 0.1 ml of the maximum concentration ofthe test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth ofthe bacterial background lawn.


### 3.5.2 Mutation Study

- Experiment 1 (Range-finding Study) Seven concentrations ofthe test material were assayed in triplicate against each tester strain, using the direct plate incorporation method. Doses were allocated as follows:SPL PROJECT NUMBER: 9301074 PAGE II


### All tester strains without S9: 0.5, 1.5,5, 15,50, 150,500 Ilg/plate
All tester strains with S9: 1.5,5, 15,50, 150,500, 1500 Ilg/plate
Additional dose levels were included in both experiments to allow for the toxicity of the test material, ensuring there were a minimum offour non-toxic doses.
Measured aliquots (0.1 ml) 

### of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml ofmolten, trace histidine or tryptophan supplemented, top agar, 0.1 

### ml ofthe test material formulation, vehicle or positive control and either 0.5 ml 

### of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial 

### strain and for each concentration of test material both with and without S9-mix.
All ofthe plates were incubated at 

### 37°e for approximately 48 hours and the frequency ofrevertant colonies assessed using a Domino colony counter. 

### 3.5.3 Mutation Study

- 

### Experiment 2 (Main Study) The second experiment was performed using methodology as described for the range-fmding study, using fresh bacterial cultures, test material and control solutions. The test material dose range was amended slightly (based on observations made from the range-finding study) and were as follows:
All tester strains without S9: 1.5 to 500 Ilg/plate
All tester strains with S9: 5 to 1500 Ilg/plate


### 3.6 Acceptance Criteria The reverse mutation assay may be considered valid ifthe following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2000 and 200 I presented in Appendix 2.

### 3.7 SPL PROJECI' NUMBER: 930/074 PAGE 12


### The appropriate characteristics for each tester strain have been confinned, eg rfa cell-wall mutation and pk:MIOl plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 

### bacteria per mI.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure 

### and the integrity ofthe S9-mix. The positive control historical ranges for 2000 and 2001 are presented in Appendix 2.
There should be a minimum offour non-toxic test material dose levels.
There should not be an excessive loss ofplates due to contamination. 

### Evaluation Criteria The test material may be considered positive in this test system ifthe following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression(5)) significant increase in the revertant count in at least one strain of bacteria.


### 4. ARCHIVES Unless instructed otherwise by the Sponsor, all original data and the fmal report will 

### be retained in the Safepharm archives for five years, after which instructions will be sought as to further retention or disposal.

# 51

5.2 SPL PROJECf NUMBER: 9301074 PAGE \3



### 5. RESULTS Preliminary Toxicity Study The test material was toxic initially at and above 150 and 500 f..I.g/plate without and with S9 respectively to the strains ofbacteria used (TAlOO and WP2uvrA). The test material fonnulation and S9-mix used in this experiment were both shown to 

### be effectively sterile.
The number ofrevertant colonies for the toxicity assay were:

With(+)or Dose (J.l.glpIate) without (-) Strain

S9-mix 0 

### O.1S O.S I.S S IS SO ISO SOO ISOO SOOO



# - TAIOO 109 131 

### lOS 106 

### lIS 103 95 53· 

### O· O· O· + 

### TAtOO lIS 108 

### 116 101 109 129 C 

### 124 90· 

## o· O·



## - WP2uvrA 24 24 26 25 23 24 33 22 

### O· O· O· + WP2uvrA 21 41 29 

### 3S 46 34 26 39 34 

### O· O· C 

### = contaminated • ;; partial absence ofbacterial background lawn 

### Mutation Study Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9-mix used in both experiments ofthe main study was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates perfonned on the same day as the Mutation Study.
The individual plate counts, the mean number ofrevertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 with the results also expressed graphically in Figure 1 to Figure 4.
Infonnation regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry ofHealth, Labour and Welfare are presented in Appendix 1.
A history profile ofvehicle and positive control values is presented in Appendix 2.SPL PROJECT NUMBER: 9301074 PAGE 14


### The test material caused a visible reduction in the growth ofthe bacterial background lawn to all of the bacterial tester strains, initially at 150 and 500 Ilg/plate without and with S9 respectively. The test material was, therefore, tested up to the toxic limit. No test material precipitate was observed on the plates at any ofthe doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose ofthe test material, either with or without metabolic activation.
All ofthe positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confinning the activity of the S9-mix and the sensitivity of the bacterial strains. 

### 6. CONCLUSION The test material was considered to be non-mutagenic under the conditions ofthis test. 

### 7. REFERENCES 1. Ames B N, Durston W E, Yamasaki E and Lee F D (1973b) 

### Proc. Natl. Acad. Sci. (USA), 70,2281-2285.
2. Ames B N, McCann J 

### and Yamasaki E (1975b) 

### Mutation Research, 31, 347- 364.
3. Maron DM and Ames BN (1983) 

### Mutation Research, 113,173-215.
4. Gamer R C, Miller E C, and Miller J A (1972) 

### Cancer Res., 32,2058-2066.
5. Kirkland D J (Ed) 

### (1989) Statistical Evaluation ofMutagenicity Test Data. 

### UKEMS Sub., committee on Guidelines for Mutagenicity Testing, Report

- Part III, Cambridge University Press.SPL PROJECT NUMBER: 9301074 PAGE IS MXDA/SM Adduct: REVERSE MUTAtION ASSAY" AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COU 

### Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls) Range-finding Study Number ofrevenants (mean number ofcolonies per plate) Base-pair substitution type Frameshift type TAloo TAI535 WP2uvrK TA98 TAI537 67 75 (68) 62 16 25 (18) 14 24 24 (25) 26 12 22 (18) 20 7 11 (9) 8

Main Study Number ofrevenants (mean number ofcolonies per plate) Base-pair substitution type Frameshift type TAtoO TAI535 WP2uvrA" TA98 TAI537 67 74 (73) 78 22 28 (22) 15 25 25 (23) 20 26 28 (24) 19 14 11 (l3) 15SPL PROJECf NUMBER: 9301074 PAGE 16 MXDAlSM Adduct: REVERSE MUTATION ASSAY "AMES TEST'" USING SALMONELLA TYPHlMURIUM AND ESCHERICHIA. COU 

### Table 2 Test Results: Range-Finding Study

- Without Metabotic Activation
TEST PERIOD FROM: 23 APRll.. 2002 TO: 26 APRll.. 2002 Test Nwnber ofrevertants (mean nwnber ofcolonies per plate) With or without substance Base-pair substitutiO/l type Frameshift type S9-Mix



# -

·



## -

·

-

-



## -

- Positive COIltrols S9-Mix

· concentratiO/l (Ilwplate) TAIOO TAI535 88 (89) 28 (22) 0 86 20 94 4.2# 17 5.7 85 (85) IS (l4) 0.5 87 13 82 2.5 14 l.0 81

# ~.~ 

# I 22 (19) 1.5 73 14 76 21 4.4 79 (78) 14 (14) 5 63 14 92 14.5 15 0.6 88 (88) 13 (16) 15 87 17 88 0.6 18 2.6 62 (78) \3 (19) 50 67 21 106 24.1 24 5.7 57 • (53) 16 • (16) ISO 49 • 18 • 52 • 4.0 14 • 2.0 9" (14) O· (0) 500 26 • 

### o· 8 • 10.1 O· 0.0 Name ENNG ENNG Concentration

(J,IgIplate) 3 274 267 No. colonies 292 (305) 197

per plate 348 38.6 262 5 (242) 39.1 WP2uvrA­ TA98 TAI537 25 (23) 25 (21) 12 (2) 19 20 9 25 3.5 17 4.0 14 2.5 18 (19) 22 (22) 5 (9) 18 22 14 20 1.2 21 0.6 8 4.6 29 (26) 24 (23) 16 (15) 31 23 IS 19 6.4 22 1.0 15 0.6 25 (22) 16 (19) 8 (II) 18 21 13 24 3.8 21 2.9 13 2.9 29 (25) 21 (22) 8 (9) 25 22 11 21 4.0 22 0.6 7 2.1 20 (21) 28 (24) 12 ('12) 19 28 12 24 2.6 17 6.4 13 0.6 18 (21) 25 (22) IS (16) 18 20 16 26 4.6 20 2.9 16 0.6 O· (0) 12 • (12) O· (0) O· 12 • O· O· 0.0 12 • 0.0 

### o· 0.0

ENNG 4NQO 9AA

2 0.2 80 369 \02 1381 342 (355) 84 (llO) 1899 (1424) 353 13.6 144 30.8 992 455.0

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine 4NQO 4-Nitroquinoline-I-oxide 9AA 9-Aminoacridine 

### * Partial absence ofbacterial background lawn # Standard deviationSPL PROJECT NUMBER: 9301074 PAGE 17 MXDAlSM Adduct: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHlMURIUM AND ESCHERICHIA COM 

### Table 3 Test Results: Range-Finding Study

- With Metabolic Activation TEST PERIOD FROM: 23 APRIL 2002 TO: 26 APRIL 2002 With or without S9-Mix

+

+

+

+

+

+

+

+ Positive controls 59-Mix
+ Test Number ofrevertants (mean Dumber ofcolonies Del' Dille) substance Base-pair substitution type ooncentration TAIOO

(u2lDIate) 103 0 96 79 73 J.S 76 77 110 5 116 88 87 15 93 96 97 50 105 106 108 150 103 108 72· 500 68 • 66 • 

### o· 1500 

### o· 

## o· Name Concentration

(jJ.gIplate) 1661 No. colonies 1556

per plate 1364 (93) 12.3# (75) 2.1 (105) 14.7 (92) 4.6 (103) 4.9 (106) 2.9 (69) 3.1 (0) 0.0 2M

1(1527)

150.6 TAt535 17 (17) 21 14 3.5 8 (II) 14 12 3.1 11 (11) 11 11 0.0 11 (14) 17 13 3.1 16 (15) 17 11 3.2 8 (17) II 31 12.5 12 • (10) 9· 8· 2.1 

### o· (0) 

### o· 

## o· 0.0 2M

2 335 (334) 337 3.6 330 WP2uvrA39 (32) 29 29 5.8 38 (29) 24 2S 7.8 36 (27) 23 23 7.5 16 (27) 24 40 12.2 26 (31 ) 26 41 8.7 35 (28) 24 25 6.1 31 (29) 26 31 2.9 

## o· (0) 

## o· o· 0.0 2M

10 709 (762) 700 100.3 878 Frameshift type TA98 TAt537 31 (44) 25 (20) 45 17 55 12.1 17 4.6 44 (39) 15 (21) 36 26 36 4.6 21 5.5 35 (37) 11 (16) 34 12 43 4.9 25 7.8 34 (41) 18 (18) 34 18 55 12.1 18 0.0 50 (44) 21 (18) 52 20 31 11.6 13 4.4 C (42) 16 (16) 44 17 39 3.5 15 1.0 55 (45) 12 (18) 38 29 43 8.7 13 9.5 

### o· (0) 

## o· o· o· (0) 

### o· 0.0 

### o· 0.0 BP 2M 5 2 196 360 (193) 187 338 (350) 5.2 196 353 11.2

BP Benzo(a)pyrene 2AA 2-Aminoanthracene Contaminated 

# • Partial absence ofbacterial background lawn # Standard deviation CSPL PROJECT NUMBER: 9301074 PAGE 18 MXDA/SM Adduct: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUMAND ESCHERICHIA COll 

### Table 4 Test Results: Main Study

- Witbout Metabolie Activation TEST PERIOD FROM: 01 MAY 2002 TO: 04 MAY 2002 With or without S9-Mix Test substance concentration

h,g/plate) Number ofrevertams (mean number ofcolonies ocr olate) Basc-pair substitution type TAloo TAIS3S WPZuvrA· Frameshift type TA98 TAIS37



# - 0 63 (64) 61 67 3.1# 11 (11) 8 13 2.5 IS (21) 29 18 7.4 19 (18) 9 25 8.1 11 (12) 14 10 2.1



## - 1.5 S9 (59) 58 60 1.0 17 (IS) IS 13 2.0 19 (20) 22 19 1.7 23 (20) IS 22 4.4 4 (10) 12 13 4.9



## - S 64 (68) S9 81 11.5 7 (15) 19 18 6.7 23 (20) 19 18 2.6 23 (23) 28 19 4.5 19 (17) 18 14 2.6



# · IS 56 (60) 70 S4 8.7 9 (12) 8 18 S.S 24 (21) 21 17 3.5 2S (21) 20 18 3.6 11 (13) 17 12 3.2



## · SQ 61 (69) 68 79 9.1 11 (16) 22 16 5.5 19 (21) 19 24 2.9 31 (27) 21 29 5.3 21 (16) II 16 5.0



## · 150 2S • (28) 25 • 33 • 4.6 14 • (IS) 19 • 11· 4.0 22 (20) 19 19 1.7 14 (18) 23 17 4.6 13 (IS) 16 17 2.1



## · SOO 

## o· (0) 

### o· 

## o· 0.0 

### o· (0) 

### o· (0) 

### o· o· o· 0.0 

### o· 0.0 

### o· (0) 

## o· o· 0.0 

### o· (0) 

### o· o· 0.0 Positive controls S9·Mix


# · Name Concentration

(1I8IpIate) No. colonies

per plate ENNG ENNG ENNG 4NQO 9AA 3 S 2 0.2 80 SOO S46 (510) 484 32.2 418 432 465 706 (438) 741 24.1 549 (665) 102.3 ll3 124 (26) 140 13.6 1300 1138 (1246) 1299 93.2

ENNG N-ethyl·N'-nitro-N-nitrosoguanidine 4NQO 4-Nitroquinoline-I-oxide 9AA 9-Aminoacridine

... Partial or complete absence ofbacterial background lawn # Standard deviationSPL PROJECT NUMBER: 9301074 PAGE 19 MXDAlSM Adduct: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHlMURlUMAND ESCHERlCHL4. COU 

### Table 5 Test Results: Main Study

- With Metabolic Activation TEST PERIOD FROM: 01 MAY 2002 TO: 04 MAY 2002 Wither without S9-Mix Test substance concentration

(J1g/plate) Number ofrevertants (mean number ofcolonies per plate Base-pair substitution type TAIOO TAIS3S WP2uvrAFrameshift type TA98 TAIS37

+ 0 76 (79) 78 83 3.6# 12 (12) 9 14 2.5 26 (32) 36 35 5.5 3S (37) 38 37 1.5 22 (23) 18 28 5.0

+ 5 75 (80) 90 76 8.4 11 (14) 12 18 3.8 29 (27) 24 27 2.S 23 (29) 27 36 6.7 28 (23) 25 15 6.8

+ 15 94 (83) 81 74 10.1 9 (10) 6 14 4.0 33 (26) 14 31 10.4 48 (39) 38 32 8.1 29 (22) IS 21 7.0

+ SO 89 (93) 103 86 9.1 16 (13) 14 8 4.2 2S (26) 32 21 5.6 S3 (SO) 41 55 7.6 27 (20) 16 17 6.1

+ ISO 85 (88) 103 76 13.7 14 (13) II C 2.1 31 (24) 18 23 6.6 43 (36) 35 31 6.1 18 (18) 20 16 2.0

+ 500 49 • (61 ) 60 • 73 • 12.0 15 • (9) 4· 9· 5.5 31 (26) 26 20 5.5 41 (43) 33 55 11.1 IS (IS) 14 17 1.5

+ 1500 

### o· (0) O· O· 0.0 

### o· (0) O· O· 0.0 

### o· (0) O· O· 0.0 0* (0) O· O· 0.0 

### o • (0) O· O· 0.0 Positive controls S9-Mix

+ Name Concentration

(J1gIplate) No. colonies

per plate 2AA 2AA 2AA BP 2AA 1 2 10 5 2 2673 2653 (2666) 2673 11.5 154 119 (128) 110 23.2 1235 1216 (1197) 1140 50.3 109 139 (127) 134 16.1 441 428 (413) 369 38.4

BP Benzo(a)pyrene 2AA 2-Aminoanthracene Contaminated 

## • Partial or complete absence ofbacterial background lawn # Standard deviation CMXDAlSM Adduct: REVERSE MUTATION ASSAY"AMES TEST" USING SALMONELLA TYPHIMURlUM AND ESCHERICHIA COLI


### Figure 1 Dose Response Curve: Range-Finding Study

- Without Metabolic Activation
1"---------------------

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

### ---------71**L~!~~QQ_~!~'!.5}~ ~P2_~v~J\~** **_~_!A~~ _~_I~~_~~~J**  

### 100

90 80 70

- Q) 

# -**~** as 

### 60 

# .e­**~** 

### c 

### 50**~** Q) > 

### 40 Q) 

### a::: 30 20 10

-

0 1·----­**----.-.~-** .. --.-----. --­ r­ _.­ -- 'r-'­ T 

### 0 0.5 1.5 5 

### 15 50 150 500 j..Ig/plate

SPL PROJECT NUMBER: 930/074 PAGE 20

### Figure 2



### S co 

## c.. tJ)

# ~ 

# - 1: co 1::

### ~ CD 0::: MXDAlSM Adduct: REVERSE MUTATION ASSAY"AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI



### Dose Response Curve: Range-Finding Study

- With Metabolic Activation

-_._- ------------------.--­ 

### -+-TA 100 -TA1535 .....-WP2uvrA- --*-TA98 --TA1**~--** _.- - - ---_.-. -- - - -- _.- -**-~-.--** --­ 

### 120
100

80
60

­ 40 20
.. )1(

__

-- 1­ 

## o +----------­ 

# o 1.5 5 

### 15 50 150 JAg/plate 

### 500 1500

SPL PROJECT NUMBER: 9301074 PAGE 21MXDAlSM Adduct: REVERSE MUTATION ASSAY" AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI


### Figure 3 Dose Response Curve: Main Study

- Witbout Metabolic Activation

## TA10~**~TA1535** -a-WP2uvrA-

## ~TA9~~_~_-~T,~~537J 80

0 1.5 5 15 50 150 500 JJg/plate 

### 70 60 .; 50

# ~ C. (j; 

### 1: 40 m t= Q) 

# ii 30 0::: 

### 20 10



## o i--­ T

SPL PROJECT NUMBER: 9301074 PAGE 22MXDAlSM Adduct: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURlUM AND ESCHERICHIA COLI


### Figure 4 Dose Response Curve: Main Study

- With Metabolic Activation 

# r=-- 

# --------------------J**----~-----~--** 

### l=!:" TA 100 - TA 1535**~"'!f'~I},!r'2-** 

# __**~_I~~~~!.~ 15~?_**  

### 100

90 80 70 Q) 

# - as 

### 60

­ fI) c:

# ~ .e-


# - 50**~** Q) > 

### 40 Q) IX: 

### 30 20 10

0 0 5 

### 15 50 150 500 1500 -_ .. ----- 

### .---- -----**~g/plate**

SPL PROJECT NUMBER: 930/074 PAGE 23SPL PROJECT NUMBER: 9301074 PAGE 24 MXDAlSM Adduct: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COU 

### Appendix 1 Report ofResults in Mutagenicity Test using Micro-organisms
1. GENERAL ITEMS (to be completed by the sponsor)

Name ofthe new chemical substance 

## - (JUPAC nomenclature) Other name MXDAlSM Adduct Structural formula or rational formula (or outline ofmanufacturing See Appendix 3 method, in case both are unknown) Purity ofthe new Lot No. ofthe new chemical substance 99.73% chemical substance PMS-02A tested tested Name ofimpurities and MXDA: 0.2 (wt %) SM: <0.0 I (wt %) concentration Water: 0.06 (wt %) CAS No. 

## - Vapour pressure 

# - Molecular weight See structure Partition coefficient 

## - Melting point (OC) 

## - Appearance at ordinary Boiling point (OC) temperature Pale yellow viscous liquid



## - Stability Stable under nonnal temperatures and pressure
Solvent Degree of solubility Degree ofsolubility in solvent Watertt Immiscible at 50 mglml Acetone 

### t Miscible Stability Solvent in solvent



## -

# ~t

- Tetrahydrofuran 

## t Degree of Stability solubility in solvent Miscible at 

### '" 50mglml Miscible 

## -

t Solubility data provided by the Sponsor 

### tt Solubility data generated by the testing facility 

### '" No visible change in test material fonnulations throughout each experiment, therefore, assumed stableSPL PROJECT NUMBER: 9301074 PAGE2S MXDAlSM Adduct: REVERSE MUTATION ASSAY"AMES TEST" USING SALMONELLA TYPHlMURlUM AND ESCHERICHIA COU 

### Appendix 1 (continued) 

### 2. TESTER STRAINS Tester strain Obtained from Date obtained TAIOO UCB· 4 August 1995 TAI535 UCB· 4 August 1995 WP2uvrA' BmRA" 17 August 1987

TA98 UCB· 4 August 1995 TAI537 UCB· 4 August 1995


### 3. S-9 MIX (1) Source ofS9 (encircle the applicable number and fill 

### in the relevant entries) Made in-house or purchase 

## t) Made in-house 12. Purchase (Supplier) Prepared on 03 March 2002 Lot No. (in case ofpurchase) 

# - Storage temperature -1 96 D C 

### (2) 

### Preparation of S9 Animal used Inducing substance Species strain Rat Sprague-Dawley Name Phenobarbitone/p-naphthoflavone Administration method Male Oral 

# I Sex

Age (in weeks) 7 weeks 3 days Administration period and amount (mglkg bodyweight/day) Weight -250g 8011 00 mg per kg per day (3) 

### Composition of S9-Mix Constituents Amount in 1 ml S9-Mix Constituents Amount in I ml S9-Mix 4.0 ""mol S9 0.1 ml NADPH MgCIz 8.0 ""mol NADH 4.0 lJInol KCl 33.0 ""mol Na-phosphate buffer 100.0 ""mol Glucose-6-phosphate 5.0 ""mol Others ( ) Glucose-6-phosphate dehydrogenase

·UCB: University ofCalifornia at Berkeley "BIBRA: British Industrial Biological Research AssociationSPL PROJECT NUMBER: 9301074 PAGE 26 MXDAlSM Adduct: REVERSE MUTATION ASSAY"AMES TEST" USING SALMONELLA TYPHlMURlUM AND ESCHERICHIA COli 

### Appendix 1 (continued) 

### 4. PREPARATION OF THE TEST MATERIAL IN SOLUTION (encircle the applicable response) Name Supplier Lot No. Grade Purity (%) Solvent used Dimethyl sulphoxide Sigma Aldrich 31K.3641 ACS reagent 99.9 Reason for selection ofsolvent Miscible at 50 mglmJ Appearance ofthe test material 

## <: preparation Solution 

## .:::> Suspension Others ( ) Suspension and other methods when test substance difficult to dissolve Solution achieved by action on an autovortex mixer Storage time and temperature of solution from preparation until use 25 minutes room temperature (range-finding study) 50 minutes room temperature (main study) Conversion ofpurity Yes 

## CE:)
5. CONDITIONS OF**PRE~CULTURE** 

### ETC. (1) Conditions
Nutrient Broth Name Manufacturer Lot No. Oxoid Oxoid Ltd. 250177 05106 Period ofpre-culture 10 hr Culture flask (form, size) Costar 75 cm2 tissue culture flasks Amount ofculture medium 5 ml Amount ofstrain inoculated 2Oll1 

### (2) Cell viability at the end of**pr~culture** Base-pair substitution type TAl 00 TAI535 WP2uvrA" Cell number Range-finding study 2.2 2.1 3.3 (x 1091ml) Main study 1.2 1.6 2.7 Count method 

# o Conversion from 0.0. value (encircle the applicable number) 2. Stepwise dilution method 3. Others ( Frameshift type TA98 TAI537 2.5 

# 1.6= 1.0 1.6
) I

# ~/SPL PROJEer NUMBER: 930/074 PAGE 27 MXDAlSM Adduct: REVERSE MUTATION ASSAY"AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COU 

### Appendix 1 (continued) 6. MINIMUM GLUCOSE AGAR PLATE MEDIUM (encircle the applicable number and fill in 

### the relevant entries) Made in-house or purchase l. Made in-house 

# o Purchase (International Laboratory Services Ltd) Prepared on 12 April 2002 (range-finding study) 19 April 2002 (main study) Lot No. in case ofpurchase 81371S-2 10/0S Name ofsupplier and lot no. ofthe used agar 

# -

7. TEST METHOD (encircle the applicable number and fill in 

### the relevant entries) (1) Test Method and the reason why it was selected
Test method used l. 3. Pre-incubation method Others ( ) 

# CD Plate incorporation method

Reason in case ofothers 

## . (2) Test Conditions

i Pre-incubation Plate Method Bacterial suspension ml 0.1 ml Test substance solution ml 0.1 ml Na-phosphate buffer ml O.Sml Composition S9-mix (in case ofmetabolic activation method) ml O.S ml Top agar solution ml 2.0 ml Others ( ) ml 

# - Temperature °C 

## - Pre-incubation Time min 

# - Temperature °C 37°C Incubation Time hours 48 hours



# 52SPL PROJECT NUMBER: 930/074 PAGE2S MXDAlSM Adduct: REVERSE MUTATION ASSAY"AMES TEST' USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COU 

### Appendix 1 (continued) 8. COUNT METHOD OF COLONIES (Encircle 1 and 2 if

### both methods used)
Count method 1. By hand 

# WColony counter Conduction ofcorrection 1. No 

# o Yes (Correction method: Area and miscount correction) 

### 9. TEST RESULTS (1) The results should be reported on the attached form (2) Judgement of the results Judgement (encircle one) 

# I Positive Qegative=::> Reason for judgement: No significant increase in the frequency ofrevertant colonies was recorded for any bacterial strain used with any dose ofthe test material in two separate experiments either with or without metabolic activation.. (whenjudged as positive, attach a table for relative activity) 

### (3) Referential matters Dimethyl sulphoxide was selected as the vehicle for this study because the test material was soluble at 50 mglml (the most concentrated stock dilution) and was non-soluble at the same concentration in sterile distilled water in solubility checks performed in-house. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of4 x 10-4 microns. For a substance to be considered positive in this test system, it should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression(5» significant increase in the revertant count in at least one strain ofbacteria. Phenobarbitone/p-naphthoflavone was used to induce higher enzyme levels in the rat liver S9. The S9-mix used in each experiment of this study was shown to be sterile. The test material formulations showed no evidence of contamination when tested for sterility during the preliminary toxicity test. The use ofapproximate half-log dilutions instead oftwofold dilutions was agreed with the JMOL in January 1995. [REMARKS] "Referential matters"

- (fill in the view etc ofthe Study Director on the test results) 

### 10. OTHERS
Name Safepharm Laboratories Ltd. Tel No: 0044 1332 792896 Address P.O. Box 45, Derby, United Kingdom Fax No: 0044 1332799018 Title Senior Genetic Toxicologist Study Director Years ofexperience: 14 930/074 10 April 2002 

## 2 2 JUt 

## 2002 Protocol authorised b DirectorSPL PROJECT NUMBER: 930/074 PAGE 29 MXDAlSM Adduct: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COU 

### Appendix 2 History Prome ofVehicle 

### and Positive Control Values COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2000
5train TAIOO TAIS35 WP2uvrA" TAI02 TA98 TAIS37 TAIS38 WP2uvrA" pkmlOI TA97a 59-Mix -59 +59 -59 +59 -59 +59 -59 +59 -59 +59 -59 +59 -59 +59 -59 +59 -59 +59 Mean 115 118 22 16 22 24 284 312 23 32 12 16 14 31 

### m 198 102 125 50 22 22 5 4 5 6 41 35 6 6 4 4 6 3 99 42 14 4 Min 63 63 8 9 8 10 193 224 II 13 4 8 8 29 113 168 82 122 Max 198 181 40 31 45 47 381 377 55 63 25 27 21 33 377 228 123 127 Values 801 621 761 588 706 S40 149 81 764 594 749 575 6 2 5 2 6 2 POSITIVE CONTROL VALUES 2000
5train TAIOO I TAI535 WP2uvrA" TAI02 TA98 TAIS37 TAIS38 WP2uvrA" pkmlOI TA97a 59-Mix -59 +59 -59 +59 -59 +59 -59 +59 -59 +59 -59 +59 -59 +59 -59 +59 -59 +59 Mean 488 1620 382 278 663 664 877 794 138 2S9 1071 466 225 607 2089 1389 261 68S 50 137 490 26I.J 89 243 212 137 123 34 70 327 121 28 40 175 967 I 141 Min 234 443 125 128 203 185 522 388 89 129 290 191 193 568 1965 705 260 585 Max 1190 3079 1843 630 1520 1534 1161 1090 262 506 2160 768 245 648 2213 2073 261 785 Values 172 172 172 170 166 166 69 70 173 172 171 170 3 3 2 2 2 2 COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2001 WP2uvrA" WP2uvrA" TAI02 TAl537 TAlOO TAI53S TA98 TAl 538 TA97a TAl04 5train pkmlOI 59-Mix -59 +59 -59 +59 -59 +59 -59 +59 -59 +59 "59 +59 -59 +59 -59 +59 

## - Mean 106 116 22 26 278 305 23 32 II 13 16 IS 14 

# .. 144 147 116 131 512 

### :g 5 6 45 39 50 23 23 4 4 7 8 4 4 5 61 14 21 24 93 

## j ;;**~** Min 58 62 II 13 195 201 8 7 10 !3 2 4 74 131 84 114 389 ii 6 

# .. " > 

# .. ii ii Max 178 178 37 37 41 52 405 421 23 29 56 58 20 "Q 292 164 134 134 616 "'Q 0 0 c:: ValUe1l 817 658 790 621 244 157 793 630 656 501 814 662 5 10 4 5 2 5 c POSITIVE CONTROL VALUES 1001 5train 59-Mix TAlOO -59 +59 TAI535 -59 +59 WP2uvrA" ·59 +59 TAI02 -59 +59 TA98 -59 +59 TAI537 -59 +59 TAl 538 -59 +59 WP2uvrA" pkmlOI -59 +59 TA97a -59 +59 TAI04 -59 +59 Mean 50S 1582 431 336 685 756 849 830 140 303 1415 428 153 501 1946 2102 618 750 82S 1445 SO 190 S41 412 110 323 243 179 159 43 96 713 146 60 246 794 844 62 142 209 202 Min 259 4S4 98 113 248 242 461 485 72 136 197 139 113 322 672 956 547 649 644 1224 Max 1829 2738 2039 990 1960 1340 1541 1303 362 679 3616 792 133 413 2790 3100 655 850 1125 1621 Values 165 163 163 161 156 155 92 93 166 164 162 160 4 4 7 7 3 2 4 3

SD 

### = Standard deviation Min 

### =Minimum value Max Maximum value54.9 SPL PROJECT NUMBER: 9301074 PAGE 30


### Appendix 3 

### Chemical Structure

STRUCTURAL FORMULA MOLECULAR MOLECULAR COMPOSITION FORMULA WElGlIT (wt%)

CloH:toN:z 240.34

# ~ 

# I**I~**

# --U~

CHzCHl

# n**~** /**~4Hz6Nl** 344.49 4.7

# ~~%~v

344.49 36.4

448.64 4.0SPL PROJECT NUMBER: 9301074 PAGE 31


### Appendix 4 Statement of GLP Compliance in Accordance with Directive 881320lEEC

TIlE DEPARTMENT OFHEALTH OFTIlE GOVERNMENT OFTHE UNITED KINGDOM GOOD LABORATORY PRACl'lCE STATEMENT OF COMPLIANCE IN ACCORDANCE 

### wnu DIItECI'JVE III3ZO EEC LABORATORY TESTTYP£ Sd'eI'IIaJw LabontorieilAd AuIyticaI 

### 0emiIt:r7 SIwdIow BaiDea Pull EaviroIlmeaUll'ate I.AHadoD IIaad J:avinJuaeatlll

### TaidtJ SIumIIcnr MDtqellidty 

### DemJdaire PlayllClaem Tillis DE722GD 

### ToskoIoaJ

DATE OF INSPECTION 21 FebnutrJ 2080
A BeDeral inspection f'or complia.nce with the Principles of' Cioocl Laboratory Pr.Iaicc

....as carried out at the above laboralof)' as pan of UK OLP Compliauce Programme. At the time of the inspection no deviations were found of' sufficient magnitude 10 affect the validity of non-elinical studies performed al these fadlilies.

# ~{. ­**~--** 

# 2J./'I( 60---'; Dr. Ropr 0. AIa.mder Had, UK OLP MoniloriDa AIlIbariIy**SAFEP~LABORATORIESLDVUTED** 

### MXDAlSM Adduct: REVERSE MUTA nON ASSAY"AMES TEST" USING 

### SALMONELLA TYPHIMURIUM AND 

### ESCHERICHIA COLI SPL PROJECT NUMBER: 930/074
I verify that this 

### is an exact copy ofthe original 

### report which is located. in 

### the Archives ofSafePharm Laboratories Limited, Derby, 

### UK.

P W 

### Thompson HNC Study Director
DATE 

# ..................................**?~** 

# ..**J~J:.~Qn~** 

# .................... .



# 51