--------- ~r 

'd ~111··'< . fff{ 

I.r ~. 

:Wll 

~!IJr ~ 

~U~ 

~8S~ 

2:5°8 

~~~~ 

~~~~ 

~81~ ~~ 

-...--.--... --..... --'".-~ ......-.-.. ------..•., 

~ . 

> 8 "10< 9 f 9-;: "'ill'i1~ fA-fO< [F! >- ~ ~= i ElliJ 'i1iio< i 0 

qf tdlU If" ~ t.U ~ ~ 

hrU'[ Ii 

ill! 

i ~j fit .nffiurf in ij~ 

h 

If 

a ~n 1!r 

~ UjHjU 

I 1ft Ilfll!~u tf~ hi iii UfU~!1 

q. a

~I b. w wl·.,lll IJ • i ~I! f rr~~ Sa' . °

:z: 

~ .. 00000 D~ ~ ~~ ~ r f r'~ mrurmm! I~! ~~ ~I----

l

r PI iJ~ 1II U'i } tiiI UUln!H!U I ilP! q1i ~ I i 

tl 

I 

~ r, f L 11 1 t j ! mhih 

If

i 

1~I!tUfni 

LI [!'H mL _ ~ :3' 

2 

0 It I ''f ~.1 lis g 

l /t) ,- 

1 If I'

t ~i . \ '

'I ~ II r/?r; i - f .1') r, .f I 0 '(I H I v' ,j 

~ II c~'l~'"'' fill J . iii t q ~ H . l0 

Ii .,. J to"> 
Pub}1c: rt:pCll1:lflg burcIcft far thia coI1ec:I:ica oIlNom1atlallil -ama.t Ie...... 11Q bola per ' .... DI.... 1DdudI:D11lIM IcIr NVIIwiDI ~ 

MN'Chlns cQtI.rIs c:lata pthctns ad. ZMI:tWIINDI the data aDd. CllXDpLIb, ad. lW'oillWfDllM =lid,.ofIzt.fcIaIIIIIk& s..t 

COWMl\tI reprdinlltut burdc ..c:lmatll g/f -1 otbc upect of tbIa of1Dlccm••lDduc:llas IU6I haa Car Ndu.c:Ias dda ~ 

to Ct.1ef. h!lcrm.tk:ID PoUcy 8n.Dch. PM-22l. Us. EnvlraN:nlatll ProtIIdSoa Aa-c'I.4O'I M. St:, S.W ... W .....D~ o.c. _Ie" 0&Ice of 

JduIa.-ment aM. BuQet. R.lud:I.oc Act C2CJ1O.OO1.2), W1lhiDtdCa. nc. 2DS03. 

IS- M6c... '1 Oh 	 CERTIFICATION 

I c:ezti£y that to the best of my bowled~ beIW: 

1. 	 The company named in Part t IedIon A, su.bte:::tkm ta of tJda DOtb torm lzodadalO manutac:tare or fmpoItfar. 

couunerdal pwpote, other than In smaD. qwmtltieIlOIeIyfiIr NIeIl'Ch ancl d~tlwsWMt,.",..1denti&d Ir& 

Put [, Sec:tkm B. 

2. 	 All iNon:n.adon providec:1in this notice is complete an4 t:ruthfa1 as ofthe date 01 submiAion. 

3. 	 I am. ~brnitt1ng with this DCtice aD test c!ala in mypoaasioft or con.trol mel a d.eIcription of aD other data JCDcrwn to 

or reasonably a.scertalnable by me as requi:ecl by sno.so ~tbe Notific:atb:l Rule. 

Additional Certification Statements: i 


I ifyou are submittinga PMN, IntermedJate PMN, Consolidated PMN, orSNUN, cbec:k the !olJcwing 1IMrfee c::erti6catkm. 


statement that applies: . I 

I 

I [8lThe Company twned in Part I, Sedion A has remitted. the fee ofS2500 spe::ified. in 40 CFR 700.45 (b), or 

I o The Company named. in Part I. Section A has remittaci the fee of 51000 !or an Inten:Md.i:W! PMN (clefi.ned • 40 CFil7CO.43) in 

ac:cord.anc::e with 40 CFR 7CO.4S(b), or 

I 

o The Company named in Part I, Section A is a smaIl bwsi.ness coru:em under 40 a:R. 700.43 and hal mnittecl 


a fee of 5100 in a~c:e With 40 CFR 700.45 (b). 


ifyou are submitting a low volume exemption (LVEl application in aa::ordance with 40 CFR i23.SO ec) ru or a tow t 

mease mc:lloW' exposure exemptlOD (LoREX) appUcatibn in ac:crd.a.ru:e with 40 CfR 723.SO (e) (2), c:her:k the foDowing 

c::ertification statements: I 

i o The llW\u!actu.rer submitting this.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 

of 40 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 new chc:alc:al substance lor which the not:ice 1I.mbmitted meeIs all appBcable ex.aptian coaditionL ! . . . 	 . I 

, o ifthis appUca.tion i.s £Or an L VE In at:alrd.a:ac:e with 40 CPR m.50 (c:)(l), the manuf..u:tmer !I:IieM.I to CDINZIIfIDCIt 

manufac.ture of tlle exempted subIt:m:e m com.me.rc::ial purpoIIS within 1 yeez of the cSate of the expirat!Da. of the 

I 30 day review period.. 

! 

I 

The aa:uni::t of !he"a"""" JOG mab&. lhillIJOI:iaIlhcaIc:l Je6ect.,.beIt~~!be IIItIo~" r.d.lep&i"ciabl r-------i 

clfIIC.riW benia. J.zrtkwwilig" wm£a1 ~leldljld~cdmfIIlpatallrf'D''''lOli USCIOOL . 

Date 

'.-2 
Part 1-CENERAL INfORMATION 

Ccnfldtn~ 

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 

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

iPosition 

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 a joint 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. 

Mailing lcUUe. (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. 	 U you previously subaUtt~ iIIt exemption application for the chemical 

substance covered by thb notice, enter the exemption number assigned Mark (X) by EPA. If}'l'u previously submitted. a PMN for this substance enter the )toPMN numbef: _gned by EPA a.e. withdrlwn or incomplete). if none C&1

---J.... 

If you have submitted a notice Bona tkle intent to manufacture Of Mark (X) )l­ iDlpan for the chemical substance covered by thb notice, enter the ii none notice num ber assigned by EPA. 	 .,. 

Manufacture ~. I'Vl Import 

Only Only 3. o Both O BindingOption 

Mark (x) 

Page 3 
Part I - GENERAL lNPORMATION - Ccmtfnued. 

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

1. ea,,1 w2 cIIemkal .......(for def!nltkw 01c:S.-la4dMl21U",."., _dieIat.nutIIau MaaDaD 

.. o-oIlIIIMUzIcI-MckOO 1 Da..l·or 2 c..i . 

1. 3-Benzenedi!llethanallline. N-(2-phenylethyl) derhs. 

Molecular Formula: Variable 

404362-22-7 

1. Precursor 

<D 

Chemical Name: 

CAS No.: 

1,3-Benzene dimethanamlne 

1477-55-0 

2. Reaction Scheme (fypical one) 

tylIftCUC~N~ + ~H=CH2 

~ Michael addition 

<D 1,3-Benzenedimethanamine 

® Styrene 

mol S 

42 

58 

(4) 4.0wt% 

wt% 

49 

51 

® 

Styrene 

100-42-5 
P ". P" O 

I po 

~D 

~ f l r 0. 0 . ~ .~~ ~~~7fi f . 

:t S' i 1 

UI . f !llfDn!U~1 i i

l1 h

5

~ f K Ilo~i ~ I If rlflrrlftr~ r 

~ i 1 fa i t t~ hfitfh· i It 

~ I p 

• 

g 

J 

I 

! f! It! l . f Ililnlllf 

i J Jll' . 'ttJtt tft 

\II 

.• ! If . . I~'II i~1 

t I ,.il ' 1I1~ U J J r 

B , " £ . ! it tID 

f ~ 01·. . ell if·' 1 

i

B I ( -~ ----1- ! I II 

f 

Nt -~f I.. ~ 

,I .... i!)~" i i If 

r " far 8 It II' "1 

i II· iU f i I I 

~ ,. ~l f i I: if 

,I I 1 rr , ~l 0 f I f ! ~ ~ ~ ~ ~ ~ '" Po f PI 

, ~ , 

I , CID 9D~ 
,.I Part I - Gg.,rr' AT. INFORMATION L- .'" 

~ Sed:lOJl B - CHEMICAL IDEN'I'ITY INPOl.NAnON - Ccmtimaed 

:I. Impullill 	 . • c,u-W...."..a.p.dIflWaqbot ........,.aiIcI........................--._.•' .... r.•••..w,.,.... 
 " ,........CAS I.IaI*1NulMIf aniJIIIIe, If..._ ..waH'" .........._ 
 ...."'0...... . 1)'-r.tIIIItie".........spt .. fIf..:ll~. If"'__'. ......,...,.................... 


Impadtfad.CASJttalttrTNumMr Caa.fI. 

fit) dmtkl 

(1 " 

(I))' 

l,3-8enzenedimethanamine 	 1417-55-0 

... 

1-' 

I­

... 

... 

,. 

o Mark 00 thII box If WJtl atti.dt a continuation meet:: '- S)'lIOIIl'IU- EDter Illy c:hemk:a1 JYDClIIya» fer the new chemical sul:Jetuca Idmtlfled: Ja 1 or 2. :, 

o 00 tbIa box Uyou. atIac:b a caatmUIf.iaD sheet. 

5. Trade - LIst trade fer the ."'... !deatlWIn I'Illwc::tlm 1 or 2. 

GASKAIII HE 240 

o Marlc 00 tbls box U,YOIlattach a caatlmlatl.an sheet. . 6. 	C=cic chem1calDame - JE you cbrim c.ile:ml.calldfilltlty • caafldeatial, 1WIIl1Utpr.ovtde a pnerJc D&IIIIe lor your IlUbllUce 

tMt the spec:l!II: c.il1Ddc:al1dedt,r of tile ne,r c:beiDIcIl sa1BtImce to the maJllia:l;ml8ldlslt poBble. 

ReI..to the TSCA <:MmJcal Su1IIaDcI mftllllar7. U&S EdI.t5ca. AppedIx Bfer pdaIlce CD .~ • 

grae:ric l\CMS. . 

D. MIlk00 this 'bole U yea iIltada a CIXI.I:lrmatIcD .... 

1. s,proc:trada - ... I114Y byproductII ~S&om .._afactm~ prOQilllllns, ,.., ar C&pc:.l 01 !be DeW c:lMmIcIIlIUhNDce Provide \be CAS 

~~.-- .. ~ 

Omfi.. 

{l) 

Byproduct dmtIal 

the \lill ualnaL~h:fo!1 new lea ~~b~ta~ (r~~t WI ~~~~~~10XI LO • ~f)e 

CAS Rest*7NlIIIlt. 

(2) 

o MIlk00 1tdI'beat I J"DVo aIta:b • CiCllltlmaatica ...... 
-- ~ I.~ i 0

~l td 


:1 ilf i 


& If!: 


I 


[If I


~ffll 

11~l 

i 
I f ~ 

Irt


ill


-il


!b


( 1 


II


ali 


f l


PI 

,.. 0 n.;:• 


11 rf q Ri
 iSHIi:


i all 


g ~ e:; B. ~ 

· h 

. . it ,HI 

[1'III' 


j Iff 

1& r···' {I ~il' ~ ~. ~ I 1.1 r· f f(l 

t 8 ii .' 


1 I51


Ip i Ifl. 


. 

g.~,. ~G' i9 89 89899iljH t-'. 

. 

. it f~~fi fl frf'H=!f 

m III s.. i st sl stili

! iU fr~filflflfh!~h C .... f a. 1 ~ 8 


!§ ~Ir Ir 1
 Ir 

. . ~ i HlUUrin h 


9 
 Q il~UUi II jl 

. 8 is f f L~ I 


. ~ f 'Sla ~ Sa f 

~ .H ~I!l 8 U" giII. 1.9
 \' 9 t!1 W .fa. 

I': 1j~ . d 

I~ t f 't U· (pa . r-
 I~ t xf 


U I f 

~ 1

§!t 

HI

g.lI 

Ii h

if §

,- ..

-~ U

i

g

I! H

_It i

§ 1. Jt

It I

(~ 1

-'4 5

u1

III t

:9 ~ .•
p. 

~ ,0 


~ f 


!j 8· 

:J , 

~ ! . 
 . 1 


~ I 


• 


§ 

I 

t 


I

.. 

9 e ';l

Jtln~ if I p" Illi 'ltt,jll [ If f f! .. (I ;.~f.a 1 l 8 til K =1' ..... :-' iii 8 0 P rfb" .'Y I 0 ~ f ~ f '1·'gl. § Ifi r i r l~il~l a ~ '" f 

S 

a. l is -f 

IIG' IN ' ~ f 

flJ, 4'~ f fi I hi fl" ~i , l II" jj 0 § IIli ~ 

.t1ji (t I it~ (I l'iI ~!l1 g ~ ! I • f . i

l 

i., (I i tff J I t It s-IrI 

J Ii. 

~j J 1 r' g 0 I.J 

r 1111 r i i [. IU IUl I · r.r 

f nlli . . I~ I r 

ItlJ tfl t tr: o} IJ I r I , r I 

t: 

I 

I 

[I

"R
Zl . 	 i" D' r ~§989 SS

~ O i 9 ail ge ; 9 ~geeiC' i ~ ~l~f' fir!

II: .• I I I I I I I ~ It .. 11-= Ii- .. Ii"

/I. 	 ~3'. 0 Ii' o. 18 • I - Ii-	 

t: I 	 . t a 

flfq "~~Jir! " h! e S

~ 1 . J~frb~ii!' II. i 	 ilUt!ih

9 Q f ~ ~ ~It 1 	~ li ..d9sg.r

ILl. . ~ .. hrifUi~HJt ~Q. !hhLI 

. , -a 	 . ~'" .IILI t. .. 

o i! Il'll rtJ~lh 1 f 	 ~f181D 19 

i 	 'ldllli l Hiir~ .. ~!Ufi U 

• 	

1 ~·~iDf-it 1t1'~lt 19 Q~ i 

! i4 Uh rjll'ri !flL~ l 

1---.---lIJ.. - -I-- - ... - _ ... ­

I.{I t - l 	 I If 

I 

II i 1-1 Jf i [ ~f 9 . Q [

l-+-+--t-r--r-.., I ~ VI f Sl II §' Ifl 	 

IltPI ilh Jf If 	 §J·1 

I ~ tl.- 11111 ~ II 	 g{ g i 

• ! if 

\1-

lSI ~I!: t 'ott J!: 

l.-.J~+-+--t--r-t-;-i)"'OlQ . r J Q' 1 9 Q. 
Part II - HtJMAN EXPOSURE AND ENVIRONMENTAL R.£LEASE - Continued 

~ SectioA B - INDUSTJUAL SIrES CON'TROLLED BY OTHERS i 

Compla MCtlCllIlIor ~~~'"or,. optradoU 1DYClviDl tM DIW' -CIIat ...:J'O'& cia IIICIt ~ lleX' typk:Il " 

manulac:tu.tt:D1 or prQCilellzl' operatioN volYUII tM MW dMaNcaI. tubttmcI ....0GtIkIe tIMI U.s. tl'IIIt ,au do ..caslzal.tiilll ~ I 

MaUll. c:amplN. Mpanta Mdlca Bfar.=ofpoc..tnlo 01''' =-=aa:,tMlWIW'''''''''Ical Utbe_~II I pcfccmed. ~t~~OM'" lb. :u_~tI.aa COIIIZI:IOIl to.... addItIoIIIIl .....CID .............11......optkIa.lfar I 

site outslde· sm.&See 

L 0paI_ 'De1Cript!aa - To claim 1rIfcrma_1Il thIa NdScG • cas8dmMeL dzdItor bncIr:It the I!pICI6:lDImD"'aD dud,. c:WIIIl. cas8deHal 

(1) - 0Iapm the majcr UIIiloperlllkla IDd. dMmIcal ~==""'acllnDlpcxteaa....=:pIJDa pII1I,SS pIloD dnaM, nilen, taDk I:nIdar.. etc). the ktaIUyby 1ItW and. " deIcr:ll:Ie.m ___ac::IttIlJ'. a.- IbI • 

the approxll:na MIIip.t (by~daJiorksJbatcb,~"'dMIDlcIl~ lDd.e:trrpolDtofII. feIoMl 1:. GDda'!:l~ dveDti mel catotl."... etc) of productt,ric:yda1tNmDl, CICl .....&Iclac:s. dAabIa..".caoee~11_ dI!II1C1l'l*' 

batc:b). (3) -1deD1lfy by D'I2:IZIber tbe paintI of releIte, bdwIDs II'Ddcar 1DIIa:mIltIiat reIeufII. to tIM taYk'OlDmt of tile DIW' dMm.Ical JahetiDc: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 

re.ln with pi ..ent and diluent Coattn. (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 ~ta1l1et.. 

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.bn apoeeclfor an ... c:a:nbIMd. 

(4) ­ &timalll the typICIl duraI:kla olllXpCllUl'e per WCII'ket mCal hours per day ad (b)"days per ywr. " 

(6) - Dac:x:I.be phySc:al foa:n of UId. S DeWc:bemtc:al_JbItmc:e Of m zn1laure), IDd. D1pObidbe eqW.pme:at md. Clpeed:Ig caatrols. ifany 

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\IIZIbc of -=releae paIrIt. CampIIte,.13 fer -=rete.e paiDt 1......t!6ed 

(10) ­ Estimate the 1m<Nllt 01 the new subItanc:e :eleaed Cal 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,1UIfiIceWIIIiII;. 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'W tI.1bItmc:e 10 IbecmtlDlDiillt. (1~) - IdeDIlfy b)'l'fCd'Qds wbk:h 21'1&yftlUlt &oat Ibe cpaatk& .' " 

G)' (5), (8). Ol). (13) ad. AS) - Mark OOtbll c:okI:ilm If.,.ollbepo _Dzl,fIIIldaa..c:calldedl1 !I:I:fcmuIIk:G (CBI). 

Latter 'ol CBI Ount:k::a ICBI P.n.dl"'~ I 1ft!.. CBZ a.a- It.IDaaDt of CJI Caatrol em 

"01 worm. 01 ~.Ctiatr.rbl " " New Ttdmdosr 

Act­ .ExpoIed Expaan . Pbjlkal rar.tIDd '" !.vUy DCWIQIwtMn 

u1aIka . III 

~ 

IIBI 

(1) 

I 

(2) (3) (4a) aOIl : nOlI) (2) I "II) (5). (6) Q) (I) (9) nt (13) 

I I 

1 1 1.01 Rubber groves <D 0.2/b1 Burn

I Respirator I , • 

I ~atety goggles I' 

2 1 " 1. 0 I Rubber groves -(2) 0.02/~ Burn 

I Respir.ator I 

I 

3 1 1.0: Respirator 1.0 @ 0.011, 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 or bracket 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. Please include new 

information pertinent tQ pollution preventiqn, including source reduc:tion, recycling activities 

and safer processes or products available due to,the new chemical substance. Source reduc:C6n 

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

modification, process and procedure modification, product refonnu1atio~ raw materials 

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

components from wastes that would otherwise be treated or released as air em.issions or:water 

discharges, or land disposal Desaiption's of pollution preventio~ source reduction and 

recycling should emp~ potential risk reduction subsequent tQ compliance with existing 

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

this information to assess overall net reductions in toxicity or environmental releases and 

exposures, not the shifting of risks to other environmental media or non-environmental areas 

(e.g., occupational or consumer exposure). In addition, information on the relative cost or 

performance characteristics of the PMN' substance to potential altematiyes may be provided.­

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

of this substance. See Instructions Manual and Pollution Prevention Guidance manual for 

guidance and examples. 

_De5cn"'be the expected net benefits, such u (1) an overall reduction in risk to human health or the 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 or other 

means; (4) a reduction in pote:l'Itial toxicity or human expowre and/or envIroruN!l:l\ta.l reJ.eue; (5) an inaeue In product 

perfcnnance,. a de:rease in the eM of procbx:don wIor 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!W c: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 propertIes 

for cured epoxy resin as an epoxy curing agen~ 

o Mark (X) thia box " you dIc:h • ccnt!nuclon IhMt. 

Paptl 
Pan m- UST OF ATIACHMENTS 

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 oat to ID'fan. "'!.MDt....7OUcIIbD.cr:cfIcIeDttll ..1IIt .......cI:bIaMiIaaal_
 C OIl how to dU:D -rid.. ii·hI'll ••IhrbNDt. CI'ID8<IeotW YouattnclDdI w1IIa .......".,., capy oltbellOtlcl 


_lIIIIlIzed 'V'CIioIl QI.,~III \IIIhIdl,... cIIbD 1Mi'll.....ClXlIldeIlIl . I AtlltWwtDllDJt A,,*hIlNt o.sMaterial 

pqe~) ~ 

. Safety Data Sheet OOOS) /: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 If1O'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, 17th 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/M3 ­

(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 @ 1000 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 m1ew 01 pitT: aftCl cbtmIc:Il prOpIrtltIdata. p-~..~~foe c:t.ta)'all pn:mcs. Illd Udude It mthe ftdk& cIcIIly tbI pcOf*t1l1llU .. «I,. the PIP 01 theDOtka ..wtddl plCIpCtJ' ......ihe VaNe 

01 the~the \DIlt:I mwIdda tlIiII~ ..-*c.~_,......oraot..P %:2!lIIc1aialect. 

coaJJdcItI.aI. :=...of the..IUbiItDcIlbauid lie "l1MM ~~ lie ...the I*tClc.lCl'5 

pun) c:ltcNc:al PropiIt118 dud "III'M"tar IIII:IctuN or tara&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 ..rxblWltllllOt a of .... dIlL . " ". 

PropIJty WakOO.1I :r. VIlDI w-.t (".Qd. 

~ 4iIIIIW 

(I) ptcw.IcIai (b) (d ~OO ".It 

Physlc:a1 aleof \.'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 

MellinrtemJ:)cature -c

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«l coelSdeat 

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 icated) 

~/~ 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 

Peak 

1 

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 imethanam ine, 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

I':"~ ~ ~ .:;-.~ ':'~ r-~ ~ ~ ':'~ -: '"': -: -: -: -: -: '"': c:: c:: ~lQ t-r--: "': r--: r--: 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 M - 1- ;:--11- ::lC .:r. 1- c; - IC '='I - en ':'l10 10 r- -' 00..,- .--; IC 1- .e c c:c r- .-. en r- to en t.C r- 0 10'='1 cc r- 0 r- 00 en 10 10 MENUF BC a - - :n Ir., 0':: ~ ="I - 0 :n en oc' r- r- - ....., 0 :.c 10 -<:;' ~ ~ .-. a t.C "'1' '::'? "'l" 0 r- .-. "'" -:'0 1:"';111': Ie C"O N N 00 ...... M M I:D OBNUC 13C ............... , lit ..... ( ....... ,. .... , .... 11 ............................ , • II ......... . 


<":) 0 0 en en co X> 00 00 00 00 r- r- r- r- r- r- I:D t.C !.O !.O 10 10 Ie 10 LO 10 10 "'l" '7' "'l" r- r- !.O 00 In 10 10 In M M 0 I:D I:D 0 I:D M c") N -,. "7' ~ ~ N N N N N N '='I '='I N N N N N N '='I N N N '='I N N N N'='I N N r- r- r- '0 In 10 10 Ie-; In 10 10 "<I' '<I' '<I' M c") M M OFR 100.40 MHz 

n HI 125.00 KHz l.rQtL4JJ@JJJ1TnJn.LTili J d J J J J J ~rr; 10500.0 Hz +l 1r--- .. rr~ Pll 5.0 us , DEADT 19.5 us 

PREDL 0.2000 ms 

lIT 1. 0 sec 

POINT 32168 

SPO 32168 

TIMES 8000 

DUMMY 1 

FREQU 27322.4 Hz 

FLT 13650 Hz 

DELAY 14. 1 us 

ACQTM 1. 199 sec 

PD 1. 191 sec 

ADBIT 0.00 

RGAIN 24 )..} 

~ BF 1. 20 Hz 

Tl 0.00 

T2 0.00 I T3 90.00 

T4 100.00 

EOOD ~ 

II 

~I I, EXPCM Bilevel.complete.decoup1ing:Set IRNUC IH I IFR 399.65 MHz 

IRSET 124.00 KHz ~ IRFIN 10500.0 Hz 

, ~ 55 us f::~~ 511. 

DFILE ¥¥Ml¥c¥M~¥B¥PMS-02A1BCM_E1_FT. 

I ~ I~ ~l 1 02010901 

61. 60 KHz .-_., __ ,, ___ ...._L1J '------------ lJ l] t~~ 19.0 Hz 

LKLEV 180 

I I In! LGAIN 25 r-r-I I I I I I I I LKPHS 100 


170 Ii)O 12:') 100 15 50_._~~~ 318
 _______ ___ ~~~~g 11 liz 
Attached sheet 4 


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


IR spectrum 


... '... _. J' 

\ 

96.00 i 

88.00 

! 


i 


w 

(.) .... ··l·····.. Ii ~ ~ ".~'~'--~. 80.00'" 

;......... L .........:•.
 -t..­ .rJ5 .. ···.. :::::::i:~.: .. :~ .. .. ~ .., ··..F· ....j.. .. I I : .. .,-.._----:..__. ,- ~-- --,~,~",,-- , .. .:'g." 72.00 -'-i---"'--:-- : . 

.......;....... 
 .­ •... , ...... ,.. ! ,~: 

~:.. , "­ ~.-...... .~~~ ...~.-

-.. , .-:t:.:~ ... :.. : ... ' ~ .-.~-.~-~ ...... 64.00 

·1 .. ·.... , I 

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 '1A 
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 10·0334 
PAGE 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) 799018 
SPL 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 of QA Signature Final Report Audit 

§ Evaluation specific to this study 

~~...~~.0\~............... DATE: .......... ..?)M~3~~? ............. 
 For Safephann Quality Assurance Unit* 

"Authorised QA Signatures: 

Head of Department: JR Pateman CBiol MIBiol DipRQA FRQA 

Deputy Head of Department: JM Crowther MISeT MRQA 

Senior Audit Staff: N Johnson BSe MRQA; G Wren ONC MRQA; R Hurst MRQA 

27 

SPL 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 of America. 

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

:-:::::'-"1"c.-. .................................. . 2 2 j UL 2002 _...................................... DATE .................................................. .. 


Study Director 
SPL 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 of Test 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 of Results 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 88/3201EEC 
SPL 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!, MIlL W and 

MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 

"Bacterial Reverse Mutation Test", Method B13114 of Commission 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 of the 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 of revertant colonies within 

the normal range. All of the 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 of the assay and the efficacy of the S9-mix were validated. 

The test material caused a visible reduction in the growth of the bacterial background lawn to all 

of the 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 of the 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. 

3D 

SPL 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 TYPHIMURIUM AND 


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!, MHL W 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 of the 

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 of these enzymes. 

The experimental phase of this 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 

spectrophotometric ally for turbidity with titres determined by viable count analysis on nutrient 

agar plates. 
SPL PROJECI" NUMBER: 9301074 PAGE 9 

3.2 Preparation of Test 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 TAl 00 

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 of the 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 of each 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 of the 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 of the 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 of the bacterial background lawn. 

3.5.2 Mutation Study - Experiment 1 (Range-finding Study) 

Seven concentrations of the 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 of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the 

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 of revertant 

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 of four 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 of bacteria used (TAlOO and WP2uvrA). The test material fonnulation 

and S9-mix used in this experiment were both shown to be effectively sterile. 

The number of revertant 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 of the 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 of revertant 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 of Health, Labour and 

Welfare are presented in Appendix 1. 

A history profile of vehicle 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 of the 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 of the 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 of this 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 of Mutagenicity 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) 

15 
SPL 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 of colonies 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 deviation 
SPL 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 of colonies 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 

C 
SPL PROJECT NUMBER: 9301074 PAGE 18 

MXDA/SM Adduct: REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND 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 of bacterial background lawn 

# Standard deviation 
SPL PROJECT NUMBER: 9301074 PAGE 19 

MXDAlSM Adduct: REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHlMURlUM AND 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 of revertants (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 of bacterial background lawn 

# Standard deviation 

C 
MXDAlSM 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 

-_._- ------------------.--­

-+-TA100 -TA1535 .....-WP2uvrA- --*-TA98 --TA1 _.- - - ---_.-. -- - - -- _.- - -~-.-- ~-- --­

120 

100 

80 

60 ­

40 

20 .. )1( __ 

o +----------­ -- 1­

o 	 1.5 5 15 50 150 

JAg/plate 

500 1500 


SPL PROJECT NUMBER: 9301074 	 PAGE 21 
MXDAlSM 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 22 

MXDAlSM 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=!:" TA100 - TA1535 ~"'!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 23 

SPL PROJECT NUMBER: 9301074 PAGE 24 

MXDAlSM Adduct: REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COU 

Appendix 1 Report of Results 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 of impurities 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 stable 
SPL 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 of purchase) - Storage temperature -1 96D

C 

(2) Preparation of S9 

Animal used Inducing substance 

Species strain Rat Sprague-Dawley Name Phenobarbitone/p-naphthoflavone 

I Sex Male Administration method Oral

Age (in weeks) 7 weeks Administration period and 3 days

Weight amount (mglkg bodyweight/day) -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 

S9 0.1 ml NADPH 4.0 ""mol

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 of California at Berkeley 

"BIBRA: British Industrial Biological Research Association 
SPL PROJECT NUMBER: 9301074 	 PAGE 26 

MXDAlSM Adduct: REVERSE MUT ATION 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 of solvent Miscible at 50 mglmJ 

Appearance of the 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 of purity Yes CE:) 

5. CONDITIONS OF PRE~CULTURE ETC. 

(1) Conditions 

Nutrient Broth 

Name Manufacturer Lot No. 

Oxoid Oxoid Ltd. 250177 05106 

Period of pre-culture 10 hr 

Culture flask (form, size) Costar 75 cm2 tissue culture flasks 

Amount of culture medium 5 ml Amount of strain 

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 oConversion 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 of supplier 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 of others . 

(2) Test Conditions 

Pre-incubation i 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 of metabolic 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 

52 

SPL 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 of correction 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) IPositive Qegative=::> 

Reason for judgement: No significant increase in the frequency of revertant colonies was recorded for any bacterial 

strain used with any dose ofthe test material in two separate experiments either with or without metabolic activation.. 

(when judged 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 of 4 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 

of bacteria. 

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 of approximate half-log dilutions 

instead of twofold 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 of experience: 14 

930/074 

10 April 2002 2 2 JUt 2002 

Protocol authorised b Director 
SPL PROJECT NUMBER: 930/074 PAGE 29 

MXDAlSM Adduct: REVERSE MUTATION ASSAY "AMES TEST" 

USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COU 

Appendix 2 History Prome of Vehicle 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"

5train TAlOO TAI53S WP2uvrA" TAI02 TA98 TAl 537 TAl 538 TA97a TAl 04 pkmlOI 59-Mix - -59 +59 -59 +59 -59 +59 -59 +59 "59 +59 -59 +59 -59 +59 -59 +59

Mean 106 116 16 IS 22 26 278 305 23 32 II 13 14 .. 144 147 116 131 512 

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

Min 58 62 8 7 II 13 195 201 10 !3 2 4 74 131 84 114 389 6 ii .." 

> 

.. Max ii ii 178 178 37 37 41 52 405 421 56 58 23 29 20 "Q 292 164 134 134 616 "'Q 

0 0 c:: ValUe1l 817 658 790 621 656 501 244 157 814 662 793 630 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 value 
54.9 

SPL PROJECT NUMBER: 9301074 PAGE 30 

Appendix 3 Chemical Structure 

STRUCTURAL FORMULA MOLECULAR MOLECULAR COMPOSITION 

FORMULA WElGlIT (wt%) 

CloH:toN:z 240.34

--U~ ~ I I~

/ 

CHzCHln~ 

~4Hz6Nl 344.49 4.7 

~~%~v 

344.49 36.4 

448.64 4.0 
SPL PROJECT NUMBER: 9301074 PAGE 31 

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

TIlE DEPARTMENT OF HEALTH OF TIlE GOVERNMENT 


OF THE UNITED KINGDOM 


GOOD LABORATORY PRACl'lCE 

STATEMENT OF COMPLIANCE 

IN ACCORDANCE wnu DIItECI'JVEIII3ZO 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 MUTAnON 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 of SafePharm 

Laboratories Limited, Derby, UK. 

P W Thompson HNC 

Study Director 

DATE ..................................?~..J~J:.~Qn~ ..................... 


51