## 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 ofSupport 

### for 

### PMN Identified ./u 

### TS-SPR 

### WAY Gentlemen: 

### On behalfofour 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 ofSupport) 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-**~L** INPOllMATlON Yo•• ut**~UM** cmredO
....cd AllIIbIIdII (CA) 

### N_. onll.' a_ cIlemicIIl.1lINItD.o.t, _If JH cIaIa tile Ict_tIIr• cUJldeaHaL ,Yn riui; aat1lod.&e lIIOIher pIaIoa to
...... c:Ia..aIkIeatIIT IaIoIllUldoa

# -,011,. bat youaaballlato. will
_ be atiIipWI
..d tIle..n- wIIl_ .,.... ..tIl'IPA ,lWCIi_thlilalo
..... AIetIU
....ppoatoi7OV

......... aIlftld -' ,ouTS_r..WnIffIcIIIoa""'" YOll
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... aU WIt
...... If,.. daJaM _, ,

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...... .taaiIb:N**~.ut** aIIo**be~,** ' 'Pad:U;"

### HuMAN EXPOStJIUI AND ENWlONMlNl'AL**~** If 111_ an--..**_ufldui,proceItIiIIl;or-~** to be**cl~** Ia Pad ll, MCIIou A u.d
.. ofthJiI 

### reproqllCl UMsadJD_
........ , ' Part m - UST OPA'ITAat'MENTs ' 

### Atu.cJa addllloUi
...... If

### tha.1a aot _ap..-to

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### -.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..-""-tfaIbox HIt 110
... ..,...,. ....... 70- cWa • c:oafld
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.. CODff4mMai. Y9P mgt aIMJPtRdd•• M.JttW ""'sa gf lit. 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 (SlplfbidNeW Va Notfca), , ::; 

# . 0 'll\GlA. (1"
..

### ¥edrado.BU.""**A~** (..)



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

# o LOlIIX

## a- I!xpoIaa 'llUiaptlal). 7'2UO(C)W

, , '..' 

# o LVI r.f,odlflatlot 

# 0 LODXMadUlaliIlll



### ... :......

. mnDSACoNSOIJDATlDPMN1 

# .0 y.' • oId"bla

.**~--** IPreDDIlca c-.1aakatIoa , mqaiDlcl, 1Dtar. oa ,....·3) 

## P9RM mo.2S, (ReY. 5-95) Replaces previoUs ed1tkms Of BP AForm 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** 

### andbelief: 1. The complnynamed in**p~** I, section A, subsectionlit 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 ofthe dat,e of submission. 

### 3. I am submittingWith this notice all test data in my possession or control 

### and. a desaiption of allotherdataknown to orreasonably ascertainable by me 

### as required by §720.50 ofthe

### Premanufa.cture Notification 

### Rule. ,', ' 

### Additional Certification**Stat~ments:** 

### If you are submitting a 

### PMN, .Intermediate 

### PMN, Consolidated 

### PMN, or

### sNtJN, check the follOwing UJetfee 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 irrPart

### 1, 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 with40CFR 723..50 (c) (2);

### ,check thefollowing , certifk:ation statements: 

### THIS INFORMATION HAS. 

### BEEN PROVIPEDBY mE SUBMITTER. 

# , 0 ;rt'temanulacturer submittinS this, 

### notice intend.

## to 

# manufacture oril'tlportthe 

### new 

### cheinicalsubstance for**cotri.m~** 

### purposes, other thanin small 

### quantities 

### solely for

## reIJea!Ch and ,development. under the teims of40 CPR 

### 723.50. «' ." 

# ·0 The manufactureris fa.miU.ar 

### With theterms ofthis sec:t:ion and will complywiththoaeterma; and



# ..0 Thetiew chemical substance:£or which thenotice is lubinitted meet:s all applicable

### ~n mnditions.



# o U this application isfor an'L VB in accordance with 40 CFR 123.50 (c)(l), the

### ma:n~ Jnteridl to mmm.enc:e 

### , .manufacture ofthe 

### exempted for 

### mmmercial 

### purpoees within 

### l.year of

### the date oftheexplration ofthe

30 day**revie~** period. '

The 

### 8.Cl:U1'icyofthe statements 

### you make in this notice should

## reftectyour best pndic:tioo of

### the mtldpailld

## f.da repdiDg the chemical

sUbstance desCribed hereiD.. 

### Any knowing and willful misinlerpfelatioaiuubject to crimiDaI 

### peaa1tJ 

### punwmtto 18USC 1001. r-eo-nl-ld-en-tlal--I 

### Date

Date 

### Signature**~f** 

### alent. (lhppllcable)

. 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) ofCA nomenclature rules and COt\ventloN. Mark item 

## you. claim as 

## ","'_.n""., or 2 (Polymen) 1.lpproprLate. Complete IIIothu'iteml. H

another 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 ofthat In a continuation sheet.
. .... 1. Cla
..I or 

### 2chemicallllbatancea (for defl,.nitions of class 1 and clasIl2 substances, see theIn.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 buedon CA 9CI nomenclature rules and**~ventlons.)** .

. ,,;

c. Please identify wh1cb method you used to deVelOp orobtain the specUIed c:hemicaJ.lndent1tyinformation 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 ReptryNumber 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. due2subltaDce

- (1) lJIttheimmectiate precursor with their respective CAS RegiJay Numben,. (2) DeIc:rihe theJiatuN ofthe r.c:tlcia orpiocea (3) Indkatethe
, range ofcompoiitlon andthe typical compoaltlon (where app.ropdatel. (4)**~** oorrect**~latlye** ot" partiald,\emic:,aI

.strUctUre diagram, as complete as can be known..Ifone can bereuon.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 "'. Maximum Impurity and CAS Registry Number Confi­ percent dential (a) (b) 

# " 

# X**~** 

# "

- 

# "

, % % 'Jr.
. 

# " .**~** 

# o Mark 00 this box. 1.£ you

### attaCh a amttnuatlon sheet. 4. Synonymt

- Enter any .clIem1calsynOllyms for the 

### new dtem1calsubstanc:e _. __ in
. 101'2. 

# -­ . 

# :.

5. Tnde -LIst IrIIdenameetor the new 1 or2.

HSY-2.701 : ., 

# o Mark 00 this box ifyouattadt a amtinuatiOll sheet. 6•. Ceneric chemical name - IfYOU.claIm dtemical Jd.endty
.. confldeatial. youmultprovide apnericdtem1cal name fOI' youuubetance thatrevea!I the spedfk dtemk:al1dendty 01 the new chemlcalllUbltance to the maximum extent**~le.**

. . Refer to the TSCA Otemk:al**~ce** 

### mwnay. 1985 Bdltlcxl, AppmdbtBfOI'guldance aD developing
. pneriCnam..
. • . -: 

### Pyrazolone derivative 

# D· Mark 00 this box ifyou attach. amtlnuatioliIheet.
CAS RegIatry' Number (2)



# ::J Mark 00 this box ifyou 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 andotherdata {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!!. ifapPropriate. Number consecutively the pages of the attachmentIJ. Tn the column**below,~ter** the**incl~ve** page

numbers ofeach 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 ofany attachment in Which you claim information as confidential. Attachment" Conti· 

### . Attachment name
........ -_I "**~,.,** ... ___ '" '" .;pagenumber(s) A dl!ntlal 

## Material Safety Data Sheet (MSDS) 14-15 

# lX

Spectral Data
­ 

### - 

# [Z(. Hi - 17 

### Reverse Mutation Assay 

### 1 A _ L..n 

## - 

# .X**-r~** 

# ... Phl.\SlCD..!**~ ~MI'~**  

# I**((".,~y-t,\~** c.

# ~

.

.



# -



# .



# . .

.

-



# . .

.,



### . 

# 0 Mark (X) this box ifyou 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 of 

### 1apanese 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 orinspection 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 ofthe study January 22, 2002 

### Start ofexperiment January 23, 2002 

### Finish ofexperiment 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. StaffScientist Mai Morita Safety Evaluation GrouP. 

### KOEI Techno Service Co., 

### Ltd.. StaffScientist 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 of4 

### 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 

### ofteVertan.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 ofthe vehicle: 

### control in 

### each'strain. which indicated the assays had been performed 

### properly: Based on these results. 

### it is concluded 'that is not mutagenic· 

### under the 

### test 

# a • 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

### justbefore 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 ofeach positive 

### (;()J1trol chcnrica1 were thawedjust 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 

### ofS. 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 ofrfa 

### 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 

### a CODstant-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 ofrevertant colonies on a plate was counted.

6

### Study 

### No• • ,. 

### bbsenation and measurement 1. 

### Colony count



### . The number ofrevertant 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 ofrevertant 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 of40) and tb.e precipitate ofthe 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~** 

### ofrevertant 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 ofLabor, 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 ofthem. 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 ofbefore 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 ofbacteria 

### 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 of2. 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 ofrevertant 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 

# n I • 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 •**~** 

### Date 

### obtained Obtained

### from Tester strain s.typhimurtum TA98 

### TAlS37

. 

### Japan Bioassay Center

... ;JO't: , ... .. 

### November'28, 

### 1996 TA100 (Kanaaawa, 

### Japan) TA1535 E.coli WP2uvrA


### 3. 59Mb<­ 

### (1 ) Source of59 ( 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. 200I 

### Lot 

### No.(in case ofporcbase) 

### 0109:2808


# . Storage 

### temperature ·80 

### "C
( 2) Preparatioa 6£89 (If 

### purchased

## 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 Amountin 1mL S9Mix

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 of 

### the 

### 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 aCODCeI1fralion of

### 50/0. Tho test 

### cbemioal is**~on** in,­ Appea:w:v:e oftbe 1rst chemical preparation 

### Solution

# ~;:> Others ( ) 

### Suspeosionand other 

### methods when 

### test chemical difficultto 

### dissolve The test 

### suspension 

### was sti.rred 

### with 

### tube mixer. Sromge 

### time and t.empernture of

### solution from preparaticln 

### until USC 

### n., 1rst

### cbem.ica1 was dissolvedjust befure use (No 

### visible change in the test 

### chemical solution 

### was observed until use). maxiunun 2 

### hours 

# . 25 

### t Conversion of

### purity 

### 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 Containerfur cul1me (fi:mn • 

### volume) Ltubc 

# .**app~y** 

### 21 

### mL Amount**of~,medium** 

### 10 

### mL Inoculated 

### votutnc 

### 20 J.£L ( 2) Cellviabili:ty at1be md of :Pl 

### re Testet' 

## suam Base-pair 

### substitution t)pe 

### FIa1l1t!Shifttype 

### 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 

### dilution 

### Itle1hOO 

### 3. Others ( )



### 3

# 3.~

# ~SStudy 

### 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 lot

### 1\0. oftl1e ae;al' 

### used .. 

### Agar powder 

### rorbacterial 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 of

### others 

# - - . ( 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 of

### colonies Count 

### method *1 

# !(i) Byhand

## .~) 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**~** 

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# I**~.** 

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# J 

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### I 

# i j J'!. 

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# Ui**~~** .. 

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# 'U~t~~ [I 

# J 

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# JI**~** 

# 1.**~**

# ft'**i-i,l~[** l 

# l i

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# '!:II'

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# J R 

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# ·.~' '.. '\D., ".'

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... 

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# .~

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...'.··•• 

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.. ...**rl.~·** J 

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; , 

### 111Table 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 i t nout (-) Dose 'level _., _.k <0 , sase-pair substitution type Frameshtft type 

### S9 Mix (p, g/pLate) 

### TAl00 

### 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** )-propylamino

### J-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

1260 

### 6000( P. 

### g/PI ate) 

## o 1.2 

### 4.9 20 

### 78 313

# • • Main Assay**.,~** t- . 

# No."._

.. F,ig. 3**-TestC~I:..**



### (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 TAl 00 

### + 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 TAl00 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