Company Sanitized 

REGULATORY ASSISTANCE CORPORATION 

17 Clearview Cirele 

Hopewell Junction, New York 12533 

PLEASE NOTE 845-896-4340 

NEW AREA CODE 845--896-4341 (FAX #) 

July 9, 2003 

Document Control Officer Company Sanitized 

Office ofPollution Prevention &Toxic Substances, 7407 

US EPA 

1200 Pennsylvania Avenue NW 

Washington, D.C. 20460 

RE: Letter of Support for PMN Identified ./u TS-SPR WAY 

Gentlemen: 

On behalf ofour client [ ], we are hereby submitting this Letter of 

Support for the above referenced PMN. The Letter ofSupport submitter is: 

We are enclosing a copy ofpage 2 ofthe PMN fonn signed by [ ] as confirmation 

ofthis Letter of Support. 

For the agency's convenience, we have provided copies ofthe PMN form with the 

information supplied by [ ]. 

We would like to once again remind the agency that in addition to the confidential data 

on this and the other submission being treated as CBI and, therefore, not disclosable, the 

CBI data in this submissioJl (Letter of Support) should also not be disclosed to the P:MN 

submitter. 
-2­

We would also like to inform the agency that Regulatory Assistance Corporation is the 

technica1 contact for this submission. Please contact either myself (203-262-1316) or 

Mrs. D. Kuter (845-896-4340) ifyou have any questions on this submission. 

Ifyou have any questions, please do not hesitate to contact me. 

Very truly yours, 

- George S. Dominguez 

GSD/dk 
U. S. ENVIRONMENTAL PROTECTION AGENCr 

A EAIA PREMANUFACTURE, o c ~ r\ N01ICE 

AGENCY USE:ONLY 

O&teof~pt 

FOR,NE\V CHEMICAL SUBSTANCES 

When. ' DOCUMENT CONTROL OFFICER.., Company Sanitized 

I, I compl('tl'l 

"end thl" 

f(~rnl to 

OFFICE OF POLLlJTION P~""" .. ~ .. AND TOXIC SUBSTANCES, 1401 ,:, " , .... ;.- . 

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

WASHINGTON D.C. 20460 ' 

Enter the total nUll'iq,e.t of P#lge. 

in the Prem.anufa.c~e Notice 

Pad 1-~LINPOllMATlON 

Yo••ut~UM cmredO....cd AllIIbIIdII (CA) N_.onll.' 

a_ cIlemicIIl.1lINItD.o.t, _IfJH cIaIa tile Ict_tIIr • cUJldeaHaL , Yn riui; 

aat1lod.&e lIIOIher pIaIoa to ......c:Ia..aI kIeatIIT IaIoIllUldoa -,011,. bat 

youaaballlato. will _ be atiIipWI ..d tIle..n-wIIl_.,......tIl'IPA 

,lWCIi_thlilalo.....AIetIU....ppoatoi7OV .........aIlftld -' 

,ouTS _r..WnIffIcIIIoa""'" YOll .....INItaJI ........ , ..d two cop..oftldllllltb bIahad... aU WIt ...... If,..daJaM _, , 

uu-.tIaa • cu8d1llllJW, .......taaiIb:N ~.utaIIo be~, ' 

'Pad:U;" HuMANEXPOStJIUI AND ENWlONMlNl'AL ~ 

If 111_an--.._ufldui,proceItIiIIl;or-~ to be cl~ Ia 

Pad ll, MCIIou A u.d .. ofthJiI reproqllCl UMsadJD_ ........ , ' 

Part m - USTOPA'ITAat'MENTs ' 

Atu.cJa addllloUi ...... Iftha.1a aot _ap..-to __aqa.dt~fiilly. I.abel-=llatatiautfoa alufait wJtIa tIlliHlD-.-ua& MCdoa ~Mdlq. IaPI!d 

m;, Uat lbM. Mtri.......1....d_or oduIrd_..cl ..yopttoaaf 

iIlfaDllJlllGa IllCb.HelIa tM aatlIa • 

OPnONALINPOlMA1lON 

,Y01l1llllf bldllde !IIIY~tI!at 7011 __VAto ClIIIIIIHrill-.i1&lllla&' thit-.~O' ....UofW. .......-_............ _J'O.jt , 

dMczIJtepollaQoa~UIII""'."""""J'Oo ..,...,..... , the............. ' '.' " ' 

CONPlDEN'nAurYCLAIMS 

Yoli IIUIJ' cIaI:ci _, lafomuIda iaW. iIotica. ClIafWeatW. TO"';'. cWIit Oil the &iDll, lIWk OOu..-""-tfaIboxHIt 110.....,...,........70- cWa • c:oafld..tIaL To .......c:Iablfa _ ........cI:fItI ort.1iIII:bttM . 

iDlllrmaHoa '"dala • cm.8datIIIL Up defa"fmmet!im I. tltc.,...t1m.. CODff4mMai. Y9P mgt aIM JPtRdd••M.JttW""'sa gflit. PAlka. OWpdP' 

,"",hmcplai. POr: addltlaullMtntdiou CIa daIabi'iafOno...... mafld.uw. hIoIiII theIlllllndiou MIa.... . 

ra Ma1t 00 If lay ba.thia a~CII'lidAlmad c:oafldeaIbL . 

• ~'ri__fat .....,' 

, • Hea&tIl cilia 1lIIII:, , eta.... 

• ~hl....cIaII. 

·PIl~,..,.....·OY-

~nlailouh1,. 

r......·iia ..po..... wllDldlQl rri ..........., . 

:0 PMN ~NoIW 1" 

w o IHIDMEDIA'IlJMN ..................._wIIII fIaa1ptDli1l4 ::2::' ~ (PMN) . ,,··,'·::-2 C:>· 

n· ;mm(Slplfbid NeW Va Notfca), , ::; 

. 0 'll\GlA. (1"..¥edrado.BU."" A~ (..) 

IXl LVlo:..wV~a-pcioaJ •• (ft72S.!O(d(I)' U1 

o LOlIIXa- I!xpoIaa 'llUiaptlal). 7'2UO(C)W , , '..' 

o LVI r.f,odlflatlot 0 LODX MadUlaliIlll ... :...... 

. mnDSACoNSOIJDATlDPMN1 .0 y.' 

• oId"bla . ~-- IPreDDIlca c-.1aakatIoa , mqaiDlcl,1Dtar. oa ,....·3) 

P9RM mo.2S, (ReY. 5-95) Replaces previoUs ed1tkms Of BPAForm 7710-25. 

compan~anitiZed :;2{; c? J ,-:; {­
Public reporting burden for thiscoilecti.on of lniormation is estimated to average 110 hours per responll!!, including time for reviewing Instru,ctions, 

sea.rchinS--.rosting data sources, gathering and maintaining the data needed.. and completing and reviewing the collection of Iniormation. Send. 

comments regarding the burden estimate or any other aspect of this collection of lniormation, including suggesti01\ll for redudng this burd~ 

, to Qliei, Information Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M. St:, S.W." Washington, D.C. 20460; and to the Office of 

,Manajil;E!ment VldBudget,. PapenY'ork.-Redudion Act (2070-0012), Washington, D.c. 20503. ~ ~. -,'.._.­ ~ 

CERTIFICATION 

I certify that to the best of my know~edge and belief: 

1. The complnynamed in p~ I, section A, subsection lit of this notice form intends to manufacture or iu\port fo~ a 

commercial purpose, other than in small quantities solely ror research. and development, the substance identified in 

Part I, Section B. 

2. All infurmatfonprOVided in this notice is cOmplete and truthful as of the dat,e of submission. 

3. I am submitting With this notice all test data in my possession or control and. a desaiption of allotherdataknown to 

or reasonably ascertainable by me as required by §720.50 of the Premanufa.cture Notification Rule. ,', ' 

Additional Certification Stat~ments: 

If you are submitting a PMN, .Intermediate PMN, Consolidated PMN, or sNtJN, check the follOwing UJet fee certification 

statement that applies: 

o 'The Company named in Part I, &lction A has remitted. the fee !>f $25QO sped.tiedin 40CFR 7Oci.45 (b), or ' 

d '. The Company named. in Part I, Section A has remitted the fee: oi'$l000 foranlntermedlate PMN (de&ed • 40CFR 700.43)·in 

accordance with 4()CFR700~45(b)~or " '. ",,', I ' 

D'TheCompa.ny named irrPart1, sectionAis a small biainess mncem: 1,Wier40CFR. 700.4.3 and has remitted 

a fee cif $iOOin acconiananvith 40 CFR700..cS (b). , , 

Ifyou are su~g'a: lowv~bD1M .ption(tV19 applicationb\acCQrda1u:e.witb.40~123.50(-cnllora'Low ,. 

releaSe and'lowexpoeure exemption (LoREX) applicatibn in accordance with 40CFR 723..50 (c) (2); ,check the following , 

certifk:ation statements: THIS INFORMATION HAS. BEEN PROVIPEDBY mE SUBMITTER. 

, 0 ;rt'temanulacturer submittinS this, notice intend. to manufacture or il'tlportthe new cheinicalsubstance for 

cotri.m~ purposes, other than in small quantities solely for reIJea!Ch and,development. under the teims 

of 40 CPR 723.50. «' ." 

·0 The manufacturer is fa.miU.ar With the terms of this sec:t:ion and will comply withthoae terma; and 

..0 The tiew chemical substance:£or which the notice is lubinitted meet:s all applicable ~nmnditions. 

o U this application is for an'L VB in accordance with 40 CFR 123.50 (c)(l), the ma:n~ Jnteridl to mmm.enc:e 

, .manufacture of the exempted for mmmercial purpoees within l.year ofthe date oftheexplration of the 

30 day revie~ period. ' 

The 8.Cl:U1'icy of the statements you make in this noticeshould reftect your best pndic:tioo of the mtldpailld f.da repdiDg the chemical 

sUbstance desCribed hereiD.. Any knowing and willful misinlerpfelatioaiuubject to crimiDaI peaa1tJ punwmt to18 USC1001. r-eo-nl-ld-en-tlal--I 

Date 

Signature ~f alent. (lhppllcable) Date

. 

FORM EPA m~2S (Rev. 5-95) Pagel' any Sanlt\zeO OOmp~1' _ ' 
"'ec:OIJln B' ..... 

Part I - GENERAL INFORMATION - Continued 

C"U"t:"llITCAL IDENTITY INFORMATION You mgt provlde a currmtly comtCtOllmlicaJ. Abltracta (CAl IIIUne ofthe subet:ance - H.QJ.YU 	 . . : bued. on the ninth Collective Index (9(l) of CA nomenclature rules and COt\ventloN. 

Mark 	 item you. claim as 

","'_.n""., or 2 (Polymen) 1.lpproprLate. Complete III othu'iteml. 

Hanother person will submit c:he.mlcaJ. identity information for you (foIo either item 1 or 2), mark 00 the box It the risl!t. 

the and address of that In a continuation sheet. . .... 

1. Cla.. I or 2chemicallllbatancea (for defl,.nitions of class 1 and clasIl2 substances, see the In.truction. Manual) 

a. aass of substance':" Mark 00 1 0 Class l' 01' ' ,2 0 Case 2 ' 

b, QemicaJ. name (Cummtly correct OI.emiall Abstracts (CA) Name that Is cims.lste.nt with 1'SCA Inventory listings for similar substanc:es., 


. For Case 1 substances a CA Index Name must be provided. For Class 2 substances eitha' a CA Index Name or CA Preferred Name. 


be prov1~ whichever aappropr:tate bued on CA 9CI nomenclature rules and ~ventlons.). 
 . ,,; 

c. Please identify wh1cb method you used to deVelOp or obtain the specUIed c:hemicaJ.lndent1ty information reported in thiJI notice: 


, (c:heckone~.. . 
 Method 1 (CAS lnventoryExpert Servic:e • a copy of the Identification OMethod 2 (Other Source) 


'. 	 report obtained'from the CAS Inventory Expert SeMce D\WIt be 


attachment to thiJI notice) , 
 , 


d•. Molecular formula and CAS Reptry Number Ofa number alread.y ~tI for the subetance) 


e. Fora'cla.. 1 substance,. provide a complete ~d oorrect.d.\emk:al strUcture d:iagrlim. For. due 2subltaDce - (1) lJIttheimmectiate 

precursor with their respective CAS RegiJay Numben,. (2) DeIc:rihe the JiatuN of the r.c:tlcia orpiocea (3) Indkate the , range of compoiitlon and the typical compoaltlon (where app.ropdatel. (4) ~ oorrect ~latlye ot" partial d,\emic:,aI 

.strUctUre diagram, as complete as can be known..If one can be reuon.ably·asC:aitaIned. .' . . .'. 	 . 

FORM EPA 1710-25 , (Rev. S-95) 	 Plge 4. 
, 

Part I - GENERAL INFORM~TION- Con:.l!J:1t1~d. 

~.' Section B - CHEMICAL IDENTITY INFORMATION - Continued 

3. Impurltlell 

(a) ...., Identify e.acllimpurlty that may be reuoubly Ultldpated 10 be preM1lt ill the c:hem1a1lubllbnc:e • _u&d:Iued for Q)mmadal P1U:pOlllll. 


provide the CAS Reg:i.Itry Numb« If available. U there an1lJlJdmti8ed lmplUltlel, ft.r "uJdmtifled.• : ;- ~ .. 


(b) -Estlm" the maximum weight'" of -=II ilDplll'lty. U there an1lJlJdmtl.fied lmplU'iti.., I!IIf:Imale their total weJiht "'. 

Impurity and CAS Registry Number Maximum Confi­ percent (a) dential (b) 

" X 

~ 

" - " , % 

% 

'Jr. . 

" 

.~ 

o Mark 00 this box. 1.£ you attaCh aamttnuatlon sheet. 

4. Synonymt - Enter any .clIem1calsynOllyms for the new dtem1calsubstanc:e _.__in . 101'2. -­ . :. 

5. Tnde -LIst IrIIdenameetor the new 1 or 2. 

HSY-2.701 : ., 

o Mark 00 this box ifyou attadt a amtinuatiOll sheet. 

6•. Ceneric chemical name - IfYOU.claIm dtemical Jd.endty ..confldeatial. you mult provide apneric dtem1cal name fOI' youuubetance 

that revea!I the spedfk dtemk:al1dendty 01 the new chemlcalllUbltance to the maximum extent ~le. 

. . Refer to the TSCA Otemk:al ~cemwnay. 1985 Bdltlcxl, Appmdbt B fOI'guldance aD developing . pneriCnam.. . • . 

-: 

Pyrazolone derivative 

D· Mark 00 this box if you attach. amtlnuatioli Iheet. 

CAS RegIatry' Number 

(2) 

::J Mark 00 this box if you attach a c»nI:lnuatkla sheet. 

FORM EPA mo..2S (Rev. 5-95) Pap 6 

s 

x 
Part III - LIST OF ATIACHMENTS 

, 

Attach continuation sheel3 for sections of the form and test data and other data {including physica1/chemiCal properties and 

structu:rl1/ activity information}, and optional Information after this page. Oearly identify the attachment and the section of the form to 

, which ittelate!!. if apPropriate. Number consecutively the pages of the attachmentIJ. Tn the column below,~ter the incl~ve page 

numbers of each attachment. .' .- . ' . ' " 

Mark 00 the "Confidential" box next to any attachment name you claim as confidential. Read the In.tractioa. Manual for 

guidance on how to daimany Information In an attachment as confidential You must include with the unitized copy of the notice 

form a sanitized version of any attachment in Which you claim information as confidential. 

Attachment" Conti· . Attachment name ........ -_I " ~,., ... ___ '" '" 
 .;page number(s) A dl!ntlal 

Material Safety Data Sheet (MSDS) 14-15 lX 

Spectral Data ­ - Hi - 17 [Z(.

Reverse Mutation Assay 1A _ L..n 

- .X 

Phl.\SlCD..! ~ ~MI'~ I ((".,~y-t,\~ c. -r~ ... ~ 

. 

. -

. 

. . 

. 

-

. . 

., 

. 

0 Mark (X) this box if you attach a continuation sheet..Cnter the attac:hment name and number. 

FORM EPA 7110-25 (Rev.S-9S) Page12 " 

\4-IS 


Fina) report 

Title 

Reverse mutation assay...• ' ..........
 • ,.1in bacterial systenlS 

1_-­

KOEI Techno Service Co., Ltd. 
CERTIFICATION 


Title: Reverse mutation assay of•••I.'•••••••__•••;.Ul........' ­ __in bacterial sYStems 

£ 

Type ofDocument: "Final report" 

The attached English d.ocuirient is the transJation of1apanese oris;inal. 

Trnnslatedby: 

Date: ,Jj~ b l %002 

MiekoKanao 

KOEI Teclmo Service Co... Ltd. . . .. . . 

Approved by ; 

Date: 

Toshio Kawaguchi, 

KOEI Techno Service Co., Ltd. 
QUALITY ASSURANCE STATEMENT 

Title : Reverse mutation assay of. • d , I 7A? "Je •••••••in bacterial systems 

• 

St1.¢y No. 

The periodic inspections and audit for tills study were conducted in accordance with thtJ 

procedures described in the standard opcratine; procedures of the QuaI.ity Assurance Unit. The 

dates of inspections and audit n:t.ade and the dates of findings reported to facility manager and 

study director are as follows. 

[tem Dan, ofins,pect:ion I audit OAtfi of ~!)rtina 

Facility manager and 

QAU Study director 

Master schedule sheet January 11, 2002 

. Assay protocol January 22, 2002 

Reference or inspection schedule January 22. 2002 January 22, 2002 

Oose~flndins assay (termination) . January 25, 2002 Jan\W')' 25, 2002 

Main assay (start) January 30, 2002 January 30. 2002 

Rmew ofdraft report Fcbmary 8, 2002 Febnwy S, 2002 

Audit ol.final report February 21,2002 February 21,2002 

This report accurately describes the methods and procedures used in the study and the reported 

results accomte1y reflect the raw data ofthc study. 

Quality .Assurance Manager: Date: 

. Ryotaro Kimura February 21,2002 

KOEI Techno Service Co;, Ltd. 
Study No •• 

-~~itle: Reverse mutation- assay of __ 

..in bacterial systems 

. ,'­

Compliance with .GLP.(Good Laboratory Practice) standard , 

This study was performed in compliance with the following Good Laboratory Practice' 

Standards: 

." Japanese Ministry of. Labor, Notification No.76 (1938), and Revised Notification No.13 

(2000), 

Ja.paneso Ministry of Health and Welfare, Seieinatsu Notification No.268, Environmental 

Health Bureau (March 1, 2000), and Revised Japanese Ministry of Health, Labour and 

Welfare, Iyakuhatu Notification No.43, Phannaecuticai and Medica.l Safety Buteau 

(January 24, 2001). 

Deviations from the protocol 

'I'hcre were no deviations from the assay protocol. .­

Date of report preparation 

Study Director: Date: 

Miek:o Kau.ao February 21,2002 

KOEI Techno Service Co., Ltd. 

Date of approval 

Facility Manager: Date: 

Toshio Kawaguchi February 21,2002 

KOEI Techno Service Co., Ltd. 

1 

" 
Study No. '0. 

'>fist dates 

Commencement of the study January 22, 2002 


Start of experiment January 23, 2002 


Finish of experiment February 1, 2002 


, Study completion February 21, 2002 


Sponsot 

Name 

Address • a n SUt 

Test faciUty 

Name KOEI Techno Service Co., Ltd. 

Address 2-12-13. Hanaten-nishi, Jato-ku, Osaka 536.0011. Japan 

Tel. +81-6-:-6961-0254 

Fax. +81-6-6961-0124 

Responsible personnel 

Facility Manager: Towo Kawaguchi 

Archive Manager: Mitsu.h.iro Takeda 

Business Division, KOEl Techno Service Co., Ltd. 

Study Director Mieko Ka.nao 

Safety Evaluation Group. KOEI Techno Service Co., Ltd. 

Staff Scientist Miyuki Tsuji 

Saf'otyEvaluation Group, KOEI Techno Service Co., Ltd. 

Staff Scientist Mai Morita 

Safety Evaluation GrouP. KOEI Techno Service Co., Ltd.. 

Staff Scientist Ayako Hamada 

Safety Evaluation Group, KOEI Techno Service Co., Ltd. 

2 

Study No.: F 

'tJmma.-y••a.1It was evaluated for its mutagenic potential by a reverse mutation assa.y with four 

strains of Salmonella typhimurium (TAlOO, TA98, TA1535 and TA1537) and one strain of 

Escheriohia oolJ 0NP2 uvrA) . 

. In Dose-finding assay. the highest dose: of the test chemical was set at 5000J.lglplatc as the 

maximum dose recommended in the guidelines, and the assay was condueted with 7 doses ,at 

a dilution faotor of 4 for each strain. 

Ih Mainassay, the highest dose was set at 313J.lg/plate. and the assay was conducted with 5 

doses at a dilution factor of2 for each strain. 

Throughout Dose-finding assay and Main assay. the test chemical did not show any 

dose-dependent increase in the number of teVertan.t colonies to at least twice as many as that 

afthe vehiole control with or without S9 Mix jn any of the strains. 

On the other hand, positive control chemicals induced more than twice as many revertant 

colonies as that of the vehicle: control in each' strain. which indicated the assays had been 

performed properly: 

Based on these results. it is concluded 'that a • is not mutagenic· under the test

conditions. 

In Dose-finding assay and Main assay, the growth inhibition of bacteria induced by the test 

Clhcmi.eal was not observed either with Ot without 89 Mix. ' 

Red precipitate of the test chemical was observed at and above 1 S6jlg/platc with 89 Mix. 

78jls!plate without 59 Mix. 

3 

Study No. ; • 

,~ *' ' -",. J~~oducdolt <'. • •

The mutagenic potential of~was txnmined by a bacterial reverse mutation assay. 

Test chemiealand vehicle 

1. Test chemical 

Name: • II E • a 2 


(wpAC nomenclature) .. 


Other name: . - • . • 


Lot No:: -'.0 I E 


Purity: 


Physical description: 


Structure formula: 


. \ 

Supplier. . 

2. Vehicle 

As a vehiole for tho test cl\emica1, dimethyl sulfoxide (DMSO) was lIelected. A suspension of .. 

the test c.heniicalwas ptepatCd just before usc, 

Tho reasotl for selection of this vchiQle is that the teat. chemical is not.solUble in water, and is 

solublo under 1% in DMSO, and is bomogenu8 suspenaion and stable in DMSO at a concentration 

of50/0. ________________M ___.._______• ___________ 

~---...---­ Name: DMSO 

~ufacturer: Waleo Pllre Cbemical Industries, Ltd. (OsalaL. Japan) 

LotNQ.: DWLS2S2 

Grade: .. Biochemical grade 

Purity: 99.9%

-_.._--­

4 

StudyNa. • 

~~ * 

Control chemicals 

1. Vehicle control chemical 

DMSO; the same lot that was used for the preparation ofthe test chemical suspension 

2. Positive control chemicals' 

-..,-_.._.-------------......--..._-..-----_._-_.---_..------_..._....._---_... _-­

Bacterial strain Without S9 Mix With S9 Mix 

(J~ g/p1ate) (p. glplate) .. 
 --------_......-_.._--------------....-----..-----_... __.------_..._-_._--

TAlOO AF-2 ( 0.01 ) 2AA ( 1.0 ) 

TAl'3S NaNa ( 0.5 ) 2AA ( 2.0 ) 

WP2uvrA AF~2 ( 0.01 ) 2M ( lO.O ) 

TA98 AF-2 ( 0.1 ) 2AA ( 0.5 ) 

TA1537 ICR·191 ( 1.0 ) 2AA ( 2.0 ) 

-----.---_..._-_.._----......__....._--------------..._-----------_....­

* AF-2: 2-(2-Fury1)-3-(5-nitro-2-furyl)acrylamide 

(Lot No. CKQ1402, purity 99.0%, Wako Pure Chemical Industries. Ltd.) 

* NaN3: Sodium azide 

(Lot No. ACQ2S42, purity 99.4%, Wako Pure Chemical Industries, Ltd.)

*ICR-191: 6-Chloro-9-[3-(2..chloroethylantino)-propylatniDO}-2-methoxyacridine 

dihydroch1oride (Lot No. 412795, Polyscience, Inc., USA) 

* 2M: 2-Aminoanthracene . 

(Lot No. DSR3206, purity 96.5%, Wako Pure Chemical Industries, Ltd.) 

. Positive control ohemicals except NaNl were dissolved in dimethylsulfoxide (DMSO: 

biochemioal grade, purityiii;99.5o/o, Wako Pure Chemical Industries, Ltd.) and aliquots were 

stored at -20t. NaN! was dissolved in distilled water (fixed 'impurities :,i 2ppm. Wako 

PllfC Chemical Industries. Ltd.) and aliquots were stored at -20t. These stock solutions 

of each positive (;()J1trol chcnrica1 were thawed just befure usc. 

Test bacterial &trains 

1. Bactorial strains. origin and the date of reception. .~_________• ____••_________• ___•••_______• _______• ________________________• ______~w_~_____~~____ 

Salmonella typhfmuTium TAlOO, TAlS3S, TA98 and TAl537 

Obtained from: Japan Bioassay Research Ccmter (KanagaWfI., Japan) 

Date November 28. 1996' . 

F..schsTichta colt WP2uvrA 

Obtained from: Japan Bioassay Research Center 


Date: November 28, 1996 
 -........ ....·-------_........----.__• ______________.w__________..___......_ ...... 


2.Reason for the sele<::tion aftho test bacterial strains 

The selected strains of S. typhtmurtum and E. coIl are widely used for the reVerse mutation 

assay because oftheir high sensitivity to detect mutagens. 
Study No. 2 • 

~.it,Storage ofbaoterial strains 

, 	 The baoterial suspension was mixed with DMSO (bacterial suspension; DMSO'" 0.8 : 0.01) 

and aliquots were stored at -80"C. The stock suspension of each bacterial strain were 

checked for the amino acid requirement, UV sensitivity. and presence of rfa and R-factor. 

The results· confirmed that all strains rctain~ their own phenotypic ·characferiStlcs.'·' ... ., '.. 

Experimental procedure 

1. Testing'guidelines 

This study was conducted in compliance with the following testing auidelines . 

• 	Japanese Ministry of Labor, Notifica.tion No.n (1988) (Revised Notification No. 61, 

(1997» .. 

• 	 Japanese Ministry of Health and Welfare, Eisei Notification No. 127• Environmental 

Health Bureau (October 31, 1997), and Revised J~paneseMinistryofHea1th, Labour and 

Welfare. lyakuhatu Notification No.43, llha.nna.ceutical and Medical Safety Bureau 

(January 24,2001). 

2. Method (Preincu.bation method) 

Each bacterial frozen stock culture was thawed, and 2~L of bacterial suspension waS' added 

to 10mL of Nutrient Broth medium No.2 (Oxoid, England). Each bacterial Su~penSion was 

kcpcat 2°C until cultivation (maximum 5 hours and 35 minutes}, and then cultured at 37°C 

for IO hours with sbaking (75 revs/min with 2S~ width) by aCODstant-tClbtperature water 

bath-type shakcr(Prothcnno Shaker NTS-2100. Tokyo Rika, Japan). After tho cultivation, 

each bacterial suspension was kept at room temperature until use (maximum 2 h01l1'$'and IS 

minutes). 

The ten snspellSion «(UmL) and O.5mL of O.IM Na-phosphate bu.ffer .sblution(pH 7.4) (or 

O.SmL ofS9 Mix in the presence of metabolic activation) were mixed, and then O.1mL of 

each bacterial susPension was added, and incubated at 37t for 20 'minutes with shaking 

(Preincubation method). After addition of 2.0mL of top agar solution, the mixture was 

poured onto a minimal glucose agar plate, and allowed to spread evenly. An agar solu~on 

(O.6o/oAgar:BACTO-AGAR. DIFCO, USA, o.So/aNael) containing amino acid solutions of 

O.SmM L-histidino and O.SmM D-biotin for S. typhimurjum or O.SmM L-tryptophan for 

E. coli in 10:1 by the volume was used as top agar solution. 


Tho assays were carried out in duplicate plating for each dose level exCept for the vehicle 


control. The vehicle control groups were treated in triplicate. 


Aftt')r 48 bour-inoubation at 37"C, the srowth inhibition of bacteria was IOOI.mined under a ' 


stereoscopic mioroscope, the number of revertant colonies on a plate was counted. 


6 

Study No• • ,. 

bbsenation and measurement 

1. Colony count 

. The number of revertant colonies within about 81mm diameter of a 90mm diameter plate (inner 

diameter 86mm) was counted with an Ilutomatic colony counter (Automatic' Colony Analyzer 

CA-U;Systcm science Co., Tokyo, Japan) .. The counted values were adju~(rto'~orrecrt the-' 

area and the number of miscounted colonies by using a personal computer. When the number 

of revertant colonies on a plate was over 1500, it was counted manually at S different places in 

the plate using a stereoscopic microscope and the average of count values was acljusted to tako 

:rea into account, since-the accuracy of the automatic colony oounler was lowering for plates 

with over l~OO colonies. The numbers of revertant colonies were counted manually at and 

. above 1250Ilg/plate with and without S9 Mix, because an automatio colony counter was not 

available due to precipitation of the test chemical. 

For ~ch dose, the avera.ge number of revertant colonies on two or more plates were 

calculated and rounded offto an intcaraJ number. 

2. Growth inhibition and precipitate 

For all plates, the growth inhibition of bacteria by the test chemical waS cxainined using a 

stereoscopic microscope (magnifying rate of 40) and tb.e precipitate of the test;'chemical was 

observed by unaided eyes. 

3. Bacterial conoentration 

Bacterial . concentration in lO:'fold saline diluted preculture was measured using 

spectrophotom.ete,r (660 run, lcm cell). 

4. Sterility test 

89 Mix (O.SmL)or the test chemical suspension (O.1mL) was poured ontO two minimal 

glucose agar plates I'('j$pectively, and allowed to $pread c,venly with 2.0niL of top agar 

solution. After incubated at31"C for 48 hours. sterility was examined by uD.aided·CYcs. 

:Evaluation criteria 

When tho following oonditions were all sa.tisfied. the test chemical was judged to have 

mutagenic potential. 

1) A dose--dependcmtincrcase in the average numbe~ of revertant colonies is observed. 

2) The average number ot)evertant colonies induced by the test chemical at a certain dose 

is at least twice that ofthe vehicle control. 

3) The reproducibility ofthe results is confinned. 

Otherwise, it was judged to havc no mutagenic potetttial. No statistical analysis was 

performed. 

7 

Study No.8 • 

Protocol, protocol amendment, raw data, documentation records. final report, quality 

assurance report, quality assurance statement, a sampJe of test chemical, record for test 

chemical and all data. collected dwing the study' are retained in the archive ofthe test facility 

in accordance with standard operation proe,edure ofKOEl Tcehno·ServiceCo.,Lid.' "... .." .... ,. 

There are two periods for rctainment. One is a period of 10 years from the day when we 

, have submitted the document to Japanese Ministry of Labor, based on Clause I, Article 57·3 

, Gfthe lndustrial Sa.f~ and Health Low, and the other is a period of 10 years from the day 

when we have received a notice that the dootlmcnt is applicable to No.1, 2 or 3, Clause I, 

Article 4 of Japanese Ministry of International Trade and Industry. We shall adopt a 

longer period of them. For the test chemical, there are two periods: one is 10 years and 

the other is the period when it can be retained in safety. Unstable samples may be 

disposed of before the end ofthe period. 

Retention thereafter is to be determined by cODStlltation with Sponsor. 

Results ADd diseussion 

1. 	 Dose-finding assay 


In Dose-finding assay, the highest dose level was set at SOOOJlg/plat.e,which is the maximum 


dose recommend in the guidelines, and the assay was conducted with' 7 doses at a dilution ' 


factor oN. 


The test chemical did not show any increase in the number of colonies to at least twice as 

many as that ofthe vehicle control either with or without $9 Mix. 

The growth .in1uDition of bacteria induced by the test chemical was not observed either with 

or without 89 Mix. . ' 

:Red precipitate of the test chemical was observed at and above 313J.1glplate withS9 Mix, 

78J.1.glplate without 89 Mix.. ' 

Based on the results of Dose-finding assay. the highest dose in Main assay wu set at 

313J.1g1plate with and without 89 Mix, and Main assay was conducted with 5 doses at a 

dilution factor of 2. 

No bacterial growth was detected in the sterility test on the test chemical solution and S9 

Mix. 

8 
Study No.•••• 

,~ .. M' . L,', am assay·· , The dose levels in the main assay were as foUows. 

-------._---------------------------------------------...---_.._-------­

Bacterial strain . Without 89 Mix 	 With 89 Mix 

_""':___ ..~---~~..-_~_~--_------._-----...----.-----.'--••-_...wo..r.-:...__...______ 

Tho highest dose 	 Dilution Dose The highest dose Dilution Dose 

factor levels factor levels 

-....-----~..----------.---------...--..----.--..---..---------------..._.._--­

'FAIOO- 313 jJ.. g/plate 2 5 313 J.L g/plate 2 5

, TA1535 313 p. gfplate 2 313 /J glplate 2 5 


WP2uvrA 313 j.J. g/plate 2 5 313 p.g/plate 2 5


, TA98 313 p. glplate 2 313 /J g/plate 2 S 

TAIS37 313 J.L g/plate 2 5 313 p. g/plate 2 5 

Main assay was performed under the above test conditions. The test chemical did not 

show any increase in the number of revertant colonies to at least twice as many as that ofthe 

vehicle control with or without S9 Mix in any of'the strains. 

Based on the results of assays the roproducibility of the test results Wa,·oonfinnedin 

Dose-finding assay' and Main assay. 

The growth inhibition of bactcria induccd by the test chemical was iIot· observed either with 

or without S9 Mix. 

Red precipitate of the test chemical was observed at and above 156J:lg/plate with S9 Mix, 

78JJ.g/plate without S9 Mix. 

No bacterial growth was detected in the sterility test on the test chemical solution and S9 

Mix. 

Conclusions 

Based on these results, it is concluded that nI • is not mutagenic under the test 

condition. 

9 


" 
"'rhe outline offinal report for 

reverse r.n.ut:a:tion assay in bact:c:rial syster.n.s· 

30 

CONFIDENTIAL 

StudyNoj • 

~ 

Tester strain Obtained from Date obtained

s.typhimurtum TA98 


TAlS37 
 . Japan Bioassay Center ... ;JO't: , ... .. TA100 November'28, 1996 (Kanaaawa, Japan) TA1535 

E.coli WP2uvrA 

3. 59Mb<­

(1 ) Source of 59 ( EnciIcle 1he applicabIo number, and fill in the relevant emries) 

Made in-house or Pun::ha.se :Made in-house 

.2 ' .. Purchase (Supplier: Oriental Yeast Co.•. Ltd (fokyo, Japan) ) 

Prepared on Septmlber 28. 200 I 

Lot No.(in case ofporcbase) 0109:2808 . Storage temperature ·80 "C 

( 2) Preparatioa 6£89 (Ifpurchased mat.eri8.t. till in spaCes to extent possible) 

Animal used Inducing substance' . 

. Species, Stmin Spra.gu6-Dawley ~ Name 'PheoobatbitIl (PB) and 

S.6-BcmofIavone (BJI) 

Sex Male Administration 

method lt1traperitonea injection 

,Atf;. (m weeks) 7wecl<s 

period 

~ 

and amount 

(ma/kg-weigbt) 

day 1 day 2 day 3 

PB 30. 60, 60, 

BF 30 

day 4 

60 Weight 219.0 ± 8.8g 

(3) Compositiou. ofS9 Mix 

Constitut:nts Amount in 1mL S9 Mix. Consti.tnents Amount in 1mL S9 Mix 

89 OJ mL NADPH 4 j.t.1OO1 

MgC12 8 ,.unol NADH 4 jm10l 

KCl 33 J.LIIlOl Na-phosphate buftbo 100 pmal 

Gluc0sc-6~phospbate 5 J.U1101 Otbem ( ) ---­

2 

StudyNo...-, 

': 4. Preparation ofthe Tem: Chemical in Solution (Encircle the applicable item, and fill in tllCrelevant entriCSj -

, 

Solvent ~ed. 

,,,~,,. >,

Name Supplier Lot No. Grade Pw:ity(%) 

DMSO 

Wako Pure Chemical 

Industries. Ltd, 

Osaka, Japan 

DWL5ZS2 Biochemical 

grade 99.9 

- , 

Reason fur seled:ion of 

solvent 

.~ 

Tho reason £01: Sl/l.lecdon of this vehicle is 1hat the test olwmical is not soluble ill 

water, and is solublo in DMSO under 1~. 

DMSO at a CODCeI1fralion of50/0. 

Tho test cbemioal is ~on in,­

Appea:w:v:e oftbe1rst 

chemical preparation Solution ~;:> Others ( ) 

Suspeosionand other 

methods when test chemical 

difficult to dissolve 

The test suspension was sti.rred with tube mixer. 

Sromge time and 

t.empernture ofsolution from 

preparaticln until USC 

n.,1rst cbem.ica1 was dissolved just befure use (No visible change in the test 

chemical solution was observed until use). 

maxiunun 2 hours . 25 t 

Conversion ofpurity Yes Qlo) 

5. CondiU~ ofPn>--QUltl.Ue 

( 1) Condi:t:kms 

NutricntB(OCb 

Name Manu1ic:tum Lot No. 

Nutrient Broth No.2 OXOID 218041 

Per.Iod ofprc-culb::lnl 10 hams 

Container fur cul1me (fi:mn • volume) Ltubc . app~y 21 mL 

Amount of~,medium 10 mL Inoculated votutnc 20 J.£L 

( 2) Cell viabili:ty at1be md of:Pl re 

Testet' suam Base-pair substitution t)pe FIa1l1t!Shift type 

TAtOO TAlS35 WP2wrA TA98 TA1537 

Viable cell 

number 

(xld'/mL) 

Dose-finding assay 3.2 2.3 6.4 3.4 1.7 

Main assay 3.7 3.4 6.9 3.8 1.8 

Count method (¢ncircle the applicable 

Il1IIt1ber) 

G) Convetsion from O. D. value 2. Stepwise dilutionItle1hOO 

3. Others ( ) 

3 

3.~ 

~S 
Study No U 

.ci, ~. Minimum Glucose Agar Plate Medium (Encircle the applicable number, and:fill in the re~ Clltl'ie5) ::.' 

- cb Made in-house 

Made in-house or Purchase 2 Pu.rdJase 

(Supplier: SUMIKA TECHNOSERVICE Co. (Osaka, Japan) ) 

Prepared an ., September 12. 2061"" 

Lot No. in case ofpurchase SAN20010912 

Name magar, supplier 

lot1\0. oftl1e ae;al' used .. 

Agar powder ror bacterial cell cultivatian • Wako Pure Cbemica1 Industries, 

Lot No.SBE7704 . 

.. 

7. Test method(Enclrcle1he applicable number, and fill in the relevant entries) 

(1 ) Test method and the reason why it was sclectod 

Test method used :P Pro-incubation lnethOd 2. Plate incorporation method 

.0tb.eIs( ) 

Reason in case ofothers - . -

( 2) Test ............... 
 conditions

Bacterial suspension 0.1 mL 

Test chemical solution 0.1 mL 

Na-p~buffer 0.5 mL 

.Composition S9Mix. (m case of .met:abolio activation ' '<', .. 0.5 'mL 

Top aaar solution 2.0 mL 

Orhm ( ) 

' Temperature OC· 37 Pre-incubation method 

TJIl'le 20 Ininu.b:s 

Temperature 37 OC Incubation 

Time 48 boms 

8. Count method ofcolonies 

Count method *1 !(i)By hand .~) Colony cOunter 

Conduction of 

corredion (0 No . 

By hami: When the number of revet1ant colonies on a. plate was under 

300 

~Yes An automatic colony courxter. Area and misanmt cor:n::ation 

By hand: Area correction when the number of revertant colonies on a 

plam was over 300 

*1 When the number ofrevertaut on a plate was over 1500 aud observed precipitate, interfered 

with automa:lic cowiling, they were countcQ manually. 

4 
"!P,

'~~~. 

w 

? lr"> iI~~e2 K
 I f i i 1 ~ , f. 

,
 .I, ~ 

f ~.~ f r 

I ~. :n 

i 

f S 

f 

J 

I· JIt f 

f 

ti 

, 1 

'0 

I 

8" t 
 J 

i

F 

I 

cr-. 

I J'!. ,i 
 j § E' Wits:: Ui .. ~~ 

til 'U~t~~ [I J filii 

£\ tJ iC ~ f f JI ~ 1. ~ ft' i-i,l~[ l l

i ~I «r [t '!:II' 

I:'ft 

XlI.i H f ll~ I!. UII.J 

l S; Ii Irl~h ! r:f r t ! til: hh

I ~ll'fIt i It e:i It. s· . I II t· ".11.[ Ii" 

lO

! ~.t • '& 1"1 IIJR i"

!! rj; Ii' i f! I: I· .i f.I 

.. "~r ;; i!. ~ f.. '.f.·h i I ~ s ~ Wpl lIar ,~jt .. '~' w.. ·.~' '.. '\D., ".' ~'g ~·;~.I~·..1"rIO:" .•.1·... rI 

". "K".···14 

.~.. f,l.i... '.··•• t.. ... rl.~· . J ., .".t .' f ~,~

. if !: 

Ii '.1' It -I r~ l .r. i iI! '~t.. ,~. I '<efI ~t 

. Ui 


I ij!

~'! ~ 

. 


f 


'I" 

.. 

"

- , I~ 

it 

t 


8 f

I 


J 


; , 

111 
Table 1 

DOleMfinding Assay Results 

Name of the test chemioal ; ..-==-_______________ _.A No. 

Test dates From JIU1WIIY 23 to 2~,_ 2002 , .... 

With! flor . Number ofrevertant colonies/ plate 

vithoutH Dose level Base-pair substitution type Frameshift type 

S9 Mix ( fog/plate) TA100 TA1535 WP2uvrA TA98 TA1537 

.. - 116 108 r 7 44­ 51 i~ ( ~) 10 12 

Vc:hicle control 95 ( 106) ( 6) 54 ( 50) 7 ( 10) 

.111 7 54 25 6 

1.2 122 ( 117) 3 ( 5) 42 ( 48) 25 ( 251 3 ( 5) 

11.1 11 30 22 3 

S9Mix 4.9 97 ( 104) " ( 11) 49 ( ~l 26 ( 24-) 10 ( 7) 

103 8 39 32 3 (-~ 20 102 ( 103) 12 ( 10) 37 ( 38) 31 ' ( 32) 8 ( 6) 

114 8 41 32 8 

78 # 106 '( 110) 6 ( 7) 3S ( 4D) 31 ( 32) '7 ( 8) 

121 8 +6 39 6 

313 # 117 ( 119) 7 ( 2) 56 ( 41) 23 ( J'\.) 3 ( 5) 

118 11 SO ?:9 16 

1260 # 101 ( 110) 4 ( 8) 34 ( 42) 56 f 35) 11 ( 14) 

112 5 34 29 9 

5000 # 96 ( 104) 15 ( 10) 42 ( 36) ,27 ( 28) 7 ( 8] 

116 W,} 7 12 57 46 36 34 7 ' 13 

Vehicle c:on1rol 97 ( H1) 6 ( 8) 48 ( SO) «I ( 37) 16 ( -12) 

134­ 8 43 34 22 

1.2 165 I 150) 13 ( 11) t6 ( 45) 38:( ,36) , 12 ( 11)' 

175 13 41 37 13 

S9Mix 4.9 147 ( 161) 14 ( 141 35 ( 38) 54 ( 4:6) 18 ( 161 

135 9 34­ 44 14 

(+) 20 174 [ 155) 11 ( 10) 45 ( 40) 26 ( 351 14 ( 14) 

1«1 6 42 41 12 ' ' , 78 142 ( 141) 14 ( 10) 49 ( 46) 48 ( 4$) 19 ( 16) 

131 6 49 33 19 

313 # 123 ( 127) 11 ( 9) 49 ( 49) 37 ( 35) 12 ( 16) 

126 4­ 56 23 12 

1250 # 117 ( 122) 12 { 8l 46 ( 51) 24 ( 24) 12 ( 12) 

170 7 31 46 17 

5000 # 135 ( 153) 4 ( 6) 46 ( 39) 43 ( 45) 19 ( 18) 

Positive Name AF-2 NaN3 Af-2 Af-2 ICR-191 

control . DoI!e-level - . 

without ( p. sLPlatel G.Ol O.S 0.01 0.1 1.0 

S9Mix NlJmber of 501 450 180 752 2999 coLon'es/plate 529 ( 515) 4-14­ ( 432) 175 ( 178) 597 ( 675) 2812 (2906) 

Positive Name 2M ZAA 2M 2M 'l.AA 

cOntrol Dose Jeve1 

with ( p. g/plate) 1.0 2.0 10.0 0.5 2.0 

S9Mix NtJ/llber of 1471 160 1647 669 130 colonies/plate 1367 (1422) 197 ( 179) 1904 (1776) 580 [ (25) 127 ( 129) 

Notea~ 1#; Precipitate was ob.served. 

Positive controls 

AF·2 ; l-o(l-oFuryl)-3-(S-nitto-l-furyl)acrylamide . NaN:! ; Sodium azide 

ICR-191 ; 6~Cbloro.9.(3.(;z..C.b.loroetylamlno)-propylamiDol·1Mmethoxyacridine,dihydrochloride 

2M ; 2--Aminoanthracene 
Table 2 

Main Assay ResuIts 

~~ 

c~~": Name of the test ohemioal; ..-~__ _________ - No. 

Test dates From lanuuy 30 to Febmar.Y 1, 2002 

Withe +} or Number of'revertant colonies / plate . "

\I it nout (-) Dose'level _., _.k <0 , sase-pair substitution type Frameshtft type 

S9 Mix (p, g/pLate) TAl 00 TA1535 wP2UvrA TA98 TA1537 

lOS (1~l 7 7 fa 23 20 24 12 17 : Vehicle eonttol 98 8 ( 7) ( 27) 18 ( 211 11 ( 13) .. I­ -136 9 30 20 14 

20 116 ( 126) 6 ( 51 21 ( 26) 24 ( 22) 17 ( 16) 

W 11 24 15 9 

S9M1x 39 113 ( 106) 7 ( 9) 36 ( 30) 19 ( 17) 12 ( 11) 

121 16 34 25 5 

(-) 18 t. 129 ( 125) 7 ( 121 ?!I ( 32) 29 I 27) 11 { 8J 

14·2 1 29 21 8 

156# 104 ( 123) 2 ( 21 27 ( 28) 19 ( 20) 10 ( 9) 

103 2 37 14 19 

313 It 106 ( 105) 4 ( 3) 28 ( 33) 23 I 19) 12 ( 16) 

( ) ( ) ( ) ( .-) ( I 

( ) ( ) ( ) ( ) ( ) 

131 120 6 4 25 33 30 ~ 22 25 . Vchiclcconttol '. 111 {1211 7 ( 6) 55 ( 38) 27 ( 30) 24 ( 2~) 

150 6 43 39 16 

20 151 { 151> 10 ( S) 44­ ( 44) 31 ( 35) 13 ( 15) 

156 7 51 40 19 

S9Mlx 39 143 ( 1SO) 12 ( 10) 49 ( SO) 39 ( 4QJ '" 24 . ( 22J 

144 12 47 36 10 

(+) 78 146 ( 145) 9 ( 11) 26 ( 371 34 ( 35) 2S ( 18) 

166 # 

144­ 11 39 34 ' 21 

151 ( 148) II ( 11) 35 ( 37) 40 ( 57) 23 ( 22) 

148 6 33 38 18 

313 # 157 [ 153) 9 ( 8) 38 ( 36) 32 ( 35) 21 ( 20) 

( ) { ) ( ) ( l ( ) 

( ) ( ) ( ) ( ) ( ) 

Positive NaJne AF·2 NaN3 AF-2 AF-2 ICIH91 

control Dose level 

without (p, g/plate) 0.01 0.5 0.01 0.1 1.0 

S9Mix NUlliber of 577 478 168 649 1951 

colonies/plate 512 ( 545) 509 ( 494) 178 ( 173) 657 ( 653) 2003 (19771 

POliitive Name 2M 2M 2M 2M 2M 

control .Dose level 

with , (p. ,/plate) 1.0 2.0 10.0 O.S 2.0 

S9Mix Nlllllber of 1463 223 1792 779 111 colonies/plate 1341 !'~J 265 ( 2#) 1819 (1806) )19 ( 679) 145 ( 158) 

Notes: 	 #; Precipitate "as obsetved. 

Positive controls I 

AF-2 ; l-(l-Furyl)-3-(5-nitro-~furyl)acrylamidc 

NaN3 .j Sodium azide 

ICR·191 j (j..Ch(oro·9-[3-(~chloroetylamino)-propylaminoJ-l-methoxyacridinc dihydroc:.h\oride 

lAA ; 2--Aminoanthracene ' 

31 

Dose-finding Assay 

F 1 g . 1 Test Chamical' P 11 No.·•• 

,•.iA'(Coloniesjplate) - Dos e - res p 0 n s e cur v e ( S 9 ~) 

o 1.2 4.9 20 78 313 1260 5000C p. g/P1ate) 


Fig. 2 Tc:gt Chemical : •. No. I • 

(Colqnies/P1ate) Dos e - res p 0 n s e our v e (8 9 +) 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

o 

oTA100 

.rAl5'3S 

r:l WP2uvrA 

IIITM8 

+TA1537 

200 

180 

160 

140 

120 

oTAlOO 

.TA1535 

cWP2~ 

IIITA98 

+TA1537 

100 


80 


60 


o 1.2 4.9 20 78 313 
 1260 6000( P. g/PI ate)
• • 

Main Assay .,~ t- . 

.. F,ig. 3 -TestC~I:.. No."._

(Colonies/Plate) Dos e - res p 0 n s e cur v e ( s 9 - ) 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

o 

o 20 39 76 156 

oTAlOO 

.TA1535 

c WP2uvrA 

_TA9S -­ +TA1537 

. 313( I' g/Plate) 

Fig. 4 Teat Chemical: 7 1. No •• 

(Colonte,s/Plate) Dos e - res p 0 n s e Oll r v e ( S 9 + ) 

200 

160 oTA100' 

.TA1535 

160 c WP2uvrA 

. e __------~e~-------~*TA98 ~ +TA1537 

120 

100 

80 


60 


140 

40 


20 

0 , , ! 

I ( 

0 20 39 78 156 313( f.' g/Plate) 

7 
APPENDIX 1 

Historical control data s~

j(i_~ 

Vehicle control Positlve control 

(the number of revertant colonies/plate) lthe number of revertant colonies/plate) 

Strain S9 Mean ± SO Number of data Mean ± SO. Number of data 

TA100 104 ± 11.5 196 552 ± 46.7 196 

TA1535 10 ± 4.4 180 469 ± 75.1 180 

WP2 uvrA - 27 ± 4.3 160 181 ± 23.8 160 .~ 

TA9S 21' ± 5.8 225 629 ± 54.2 225 

TA1537 11 :I: 4.7 185 5594:1: 1249.3 185 

TAl00 + 118 :I:: 11.7 194 1343 ± 112.7 187 

TA1S35 + 11 ± 4.4 178 300 ± 41.8 178 

WP2 uvrA + 31 + 4.5 159 1337 :I:: 115.4 159 

TA9S + 28 ± 4.2 216 604 ± 84.0· 208 

TA1537 + 17± 5.6 180 206± ·46... • 179 

The above are pooled data from January, 2001 to December, 2001 inoluding soreening data. 

Positive oontrol chemicals 

Test strain Chemical Dose 

Without S9 mix 

TAl 00 

TA1535 

WP2 uvrA 

TA98 

TA1537 

.with S9 mix 

TA100 

TA1535 

WP2 uvrA 

TA9S 

TA1537 

2-(2-Furyl)-3-(S-nitro-2-furyOacrylamide 

Sodium azide 

2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide 

2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamlde 

6-Chloro-9-(3-(2-chloroetl;ylamino)-propylamino)­

2-Methoxyacridlne dlhydrochloride, 

2-Aminoanthracene 

2-Aminoahthracene 

2-Arninoanthraeene 

2-Aminoanthr'acene 

2-Aminosnthracene 

(JJ. g/plate) 

0.01 

0.5 

0.01 

0.1 

1.0 

1.0 

2.0 

10.0 

0.5 

2.0