## RC C - CC R STUD YNUMBE R 104030 0

SALMONELL ATYPHIMURIU M AN D ESCHERICHI ACOL I REVERS E MUTATIO N ASSA Y
WIT H
TK A4036 5 (CG I 725 )

FINA LREPOR T

STUD YCOMPLETIO N DATE : SEPTEMBE R 01 , 200 6RCC-CC R Stud yNumbe r104030 0 TK A4036 5 (CG I 725 ) Fina l Repor t Pag e 2 o f3 1



# 1 COPYO F GL P CERTIFICAT E
Hessische s Ministeriu m fu rUmweit , ISndliche n Rau m un d Verbraucherschut z 

### HESSE N

Gut eLaboipiaxis/Goo dLaboraUny Practic e GLP-BescheinlgoDg/Statemen t (rfGL PCrnnplianc e (gemSfi/accordin g t o § 19b Abs

. 1 Chemikaliengesetz ) Ein eGl-P-Inq)ejctio nzu r

Obenvachun g de r

Einhaltun g de r GLP-Gnindsatz egemaO Chemikaliengesetz bsnv

. Richtlini e88/320/E Gwurd ednrchgefiJhr t i n Assessment o f conformity wit hCHJ? accordin g t o Chemikaliengeset z an dDirectiv e88/320/EE C at :
EPrflfeinrichtungTes t

fecillity nPritrstandDrtTcs t sit e RC C Cytotes t

Ce UResearc hGmb H RC C Cytotes t

Cel l Researc hGmb H I nde nLeppsteinwiese n1 9 6438 0 RoBdor

f (Utrvcm'cchselbar eBezcichmm g un dAdresse/Unequivocal nam ean dadress )

Prnfinige nnac hKategorien/Area s o f Expertis e (gemSB/accordin g cheniVwV-GL PNr

. 5 J/OEC D guidance ) 2 PraftinBe nxii r

Besttminun g de r

toxlkologische n &igenscha5te n 3 PriUUnge nztt r

Bcstimmun ede r

erbgutveranderade n Elgenschafte n(f

nvitr o un dt nvivo ) 8 Analytiscti ePnCiTunge na nbiologische nMaterialie n 9 VirusslcherhcitsprOrunge n aToxicit y studie s 3 Mutagenicit y studie s S Analytica l studie s o nblolo^ca l material s 9 Viru s validatio nstudie s - 03.06. ; 19.07.-22.0 7 J00 4 Datiu nde r

Inspektion/Dateo f

In^xctio n (Ta g Monat

}ahi/day monthyear ) Di egenaimtePrOfeinriclitun g befiiu msic hi mnatio

nale nGLP-Oberwacbimgsverfahie nun dwirdtcgel

oiSQig au f Einhaltun g de r

(H..P-GTundsatzeflberwacht. Auf de r Grundiag ede s In^jcktionsbericfate s wirdhienni t bestaiig u das s i ndiese r

PrOfeinrichtimg di eobe nge nannte nPrfiiimge ntmte r

Einhaltun g de r

GLP

- Gnmd

sStz eAnchgefiil mwerde nkOnne a Th eabov ementione dtes t facility i s include d i ntiten^tion ^ GL PComplianc eProgtaimn ean di s inspecte do nategular

basis

. Base do nth einspectio nrepor t i t canb econSmied , that thi s tes t facili ^ i s M>I

et o conduc t

th e aforementione dstudie s i ncomplianc ewithth e Principle s o f

GLP

.

Th

. I mAuftrag 

# h

.

Referent , Wiesbaden , de nWiesbaden , de n06

. Janua r (Nam eun dFtmktio nde r

veranrwortlicbe nPerson/ Nam ean dfiinctianof

responsibl eperson ) Hess

. Ministeriu mfu r

Umweit , landKcbe nRaur aan dVerforaucherscbntz , Mainze r Strafi e801>65)8 9 Wiesbade n (Nameun dAdress ede r

GLP-t}berwachungsbefaOrde/Kainean daddres s o f th eGL PMonitotiit g Aulhori^' )

[>eS18a\Aflesb8den , Mainze rStraO e 8 0 Telefor : OS11.8 1 5 0 Telefax : 0611.8 1 6 1 94 1 E-Mail : poststetle@hnmtv.hes3en.d e 0-6518 7 Wiesbaden , MIderilnstraa e 1- 3 Telefon : 0611

. 8 1 7 0 Telefax : 0611,8 1 7218 1

st4ecdr.do cRCC-CC R Stud yNumbe r104030 0 Fina l Repor tPag e 3 o f3 1 TKA4036 5 (CG I 725 ) 

# 2 CONTENT S
1 COP YO F GL P CERTIFICAT E 2 2 CONTENT S 3 3 PREFAC E 4 3. 1 Genera l 4 3. 2 Responsibilitie s 4 3. 3 Schedul e 4 3. 4 Projec t Staf fSignature s 5 3. 5 Goo d Laborator yPractic e 5 3. 6 Guideline s 5 3. 7 Archivin g 6 3. 8 Deviation s t o Stud yPla n 6 4 STATEMEN TO F COMPLIANC E 7 5 STATEMEN TO F QUALIT YASSURANC E UNI T8 6 SUMMAR YO F RESULT S 9 6. 1 Conclusio n 9 7 OBJECTIV E 1 0 7. 1 Aim s o fth e Stud y1 0 7. 2 Reason s fo rth e Stud y1 0 8 MATERIAL S AN D METHOD S 1 1 8. 1 Tes t Ite m 1 1 8. 2 Control s 1 2 8. 3 Tes t Syste m 1 3 8. 4 Mammalia n Microsoma l Fractio n S 9 Mi x1 4 8. 5 Pre-Experimen t fo rToxicit y1 5 8. 6 Dos e Selectio n 1 6 8. 7 Experimenta l Performanc e 1 6 8. 8 Dat a Recordin g 1 6 8. 9 Acceptabilit yo fth e Assa y1 7 8.1 0 Evaluatio n o fResult s 1 7 8.1 1 Biometr y1 7 9 DISCUSSIO N O F RESULT S 1 8 1 0 REFERENCE S 1 9 1 1 DISTRIBUTIO N O F TH E REPOR T1 9 1 2 SUMMAR YO F RESULT S 2 0 12. 1 Summar yo fResult s Pre-Experimen tan d Experimen tI 2 0 12. 2 Summar yo fResult s Experimen tI I 2 1 12. 3 HISTORICA LCONTRO LDAT A2 2 1 3 ANNEX : TABLE S O F RESULT S ( 8 PAGES ) 2 3

s4c2dr.do cRCC-CC R Stud yNumbe r

TK A4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 4 o f3 1



## 3 PREFAC E
3. 1 Genera l Title : Sponsor :

Stud yMonitor : Tes t Facility :

Contractin g Institute :

Referenc e Number : Salmonell a typhimuriu m an d Escherichi a col i Revers e Mutatio n Assa ywit h TK A4036 5 (CG I 725 ) Cib a SpecialtyChemical s Inc

. Segmen t C E 400 2 Base l Switzerian d Dr

. Rolan d Hammeri , WRO-1045.1.3 9 RC C Cytotes tCel l Researc h Gmb H (RCC-CCR ) I n de n Leppsteinswiese n 1 9 6438 0 Rossdor

f German y R C C Lt d 445 2 Itinge n Switzerian d A8787 3 

## 3. 2 Responsibilitie s Stud yDirector : Deput yStud yDirector : Management : Hea d ofQualityAssuranc e Unit : Dipl

. Biol

. Andre a Sokolowsk i Dr

. Hans-Eri cWolln y Dr

. Wolfgan g Volkne r Frauke Herman n 

## 3. 3 Schedul e Dat e ofth e Stud yPlan : Experimenta l Startin g Date : Experimenta l Completio n Date : Dat e ofDraftReport : Dat e ofFina l Report : Jul y Augus t Augus t Augus t Septembe r 26 , 200 6

01.200 6 14 , 200 6 18,200 6 01,200 6

s4c2dr.do c

### RCC-CC R stud yNumber104030 0 fkA4d36i6(CGi;725 ) Fina l Report s Page;5off3j 1



## 3. 4 Projeictj^aff*Signsture §

stud yDirecto r Dipl

. Biol ; Andre a Sokolowsk i

Managemen t Date:^S^ternberij^^^0i(^6 i

Dr ; Wolfgan g Volkne r

Date : Septemtier'dl , SOGje '


## ;315' 6^pCi|ab^iiitdiV''Prac|i(:^ 5 Tti e stbd ywa s peri^oim^ fi n c» m "Chemikaliengesetz " (Chemical s Act ) o fth e Federa l Republi co fGemtiany ; ^'Anhangii* ' ^^tntm : 1;) ; date d JulyS25 i 1,99 4 CBQfel

. I ; 1994" , pp
. W3)

. las trevisio n datedyuH e 27 ; 

## io| 2 "•0Ep^^erinciR!|§-ofppj ^ ^^§eptp^,Practifce'*:,| i p |997'!iiC(97j1S6^Final f



## ^^ t GiJidellhei s thi s stady/fonbvyedtigcprote^ ^ 'd0|nesi^a:rgGom m ...^.-...-..^.•....-..- ^ ^....-..-.,...-.-..^

- .- - > llsliint|iit^dendu m to ; fiEiCD^Quidellpe s ifo rtestihgs<in(5fiemiGals",

. Section ! %

. No^^;4^f k '^BacteriatReyers e MbySo n te^t | adqf^cf3u^;21^ ^ ^^G^myipr tBir^tlveiopoM ^ 1^^362000 ; ^^ne x4D'! , laated May^ 9 , ;2p0p ' ^panes g Guidejinie^ ; ^ ^ ;?Kanp,oan%;M7r^j?fEh\^ironm6nfiProfe^^ ^ ^EiseiiNo ; 127—K/!inistry.bfHealt h «W&l^re " !Heisei;09/10/33;;Kikj^ u j$|Q.)2<

-

;i4c2clr;clocRCC-CC R Stud yNumbe r104030 0 Fina l Repor tPag e 6 o f3 1 TK A4036 5 (CG I 725 )


## 3. 7 Archivin g RC C Cytotes t Cei l Researc h Gmb H wil l archiv e th e followin g dat a fo r1 5 years : Ra wdata , stud yplan , fina l report , an d a sampl e o fth e tes titem

. N o dat a wil l b e discarde d withou t th e sponsor' s consent

. 

## 3. 8 Deviation s t o Stud yPla n Ther e wer e n o deviation s t o stud yplan

.

s4c2dr.do c

### RCC-GGFtSfudyJNumberIMBOO " 

### jfina l Repor t I PageZof^ ! fKk7&0:l0s7T25) i I 

# 4 ST^EMEN TO F CdMPyANi l
StudypNumben i iM)^C©> Te^ t llemi: : tl^^40365|eG l 725 1 StudycpirectpffDipLBjoi ; /Pufidre a Sbkolovi^k i i Title : SaliTioiiell a t^himuriumlan d Escherichi a col i Flevers e Mutatio n Assaywit h TK X4036 5 (CG I 725 )
Thi&studvg^rtoirmi&Jl^i cx>mpiiancewithiGoodiyboratdryi?ra^ "Gher^'ikaliengesdtz'^

(Chemical s Act | o fth e Federa l Republi cofiQennany ; ^Knhan g 1" ^ (Anne x1<)Vdat^

d Jul y25 , 199 4 ("BGBI , 1] 1994 ^ pp
. 1703) , lis t revisio n Sate d Jun e 27

. '2002. , 5 • "OEC D Principle s o fQop d Laborator yPractice" , as revise d i h 199 7 5iC(97)186/Final ] There were n o circums^pe^^fkM^YihayeCa^ ^
©iifctpr i R:;e^.--jS^GJ^
- Dipi : BipLAndVe a Spkplowsk i



## .?^i..rr ^ ba^'?September:p^; , 200 6

f
c



### s4c2dr.cl6c !i ! RCe^GC R Stud yNumbe r104030 0

ffes^!^[m5:(C(3l^5)f ' ' Fina ! Repor t Page8trf3 1


# fSimlMEN TQFHGIUAUT YiSSUI^ ^

UNI T StiJdy^^Nurnbe K

1(^30 0 Test Item: TK^40365 (CQ I725f

. StudyitDirectbr: : DipLi^ipt : Andre a Sokolowsk i 

## Title s Salrripriell a typhimuriu m ari d Eschericffl a cpl i Revers e Mutatio n Assa ywit h TK A40365.(0 ^ 725 ) Thegeoerarjacilitife s arid^ Cei l Researc h Gmb M ar e inspecte d penodiGaHi(:andlh e result s ar e repprted;tO;fte^^ ^ ^tud yprocedure s wer e ihspeeW d periodically

. Theistud ypla n an d thi s ;repprtwer e audite d 

## •byitheyQusiity.AssurariceJU ni ( • P|iesesiand;D"ates:6f ^Study?Pian;i(braft) ; jStudi^FI'lan : Proces s Inspection : (DbftiRepdrt : QAU Inspections/Audit s

^iftjif " 2-i,^'2006 r••' : Jiil y27V200 6 pigu| t "In?

- 2tl()^ ; ^ "

_ '

.

^ ;>a^^'3^[g006r"' ' '•" ' Date s o fFlBppftgstpiOieS ^ Directo randt o i^anagemeri t jjjiy^;";2;i^,;2{i| ; ;••/;;•; •'"ZZ 2

_ 

# ' 'yz'".""".%

. rz
. "Z

[ V\ugustM200 i 3 sAugu|t^2p2pO^'y'' 1 2 \2^'_ " ^fe^etatemjen ^

pj^serit^flrialireppr^ ^ ;i|ead;ipfMii)ity^sij^eiiit!inif c Ri'auke^MenDan n

II

.. y 'i7.K-,. JV^ V .^ii^/iv^rTxl T vD^=^5§iptembeir^li^liGi 6

.s4c2dr.d6c
-

### RCC-CC R StudyNumbe r104030 0 Fina l Report ; Page 9 o f3 1 TK A40365 (CG I 725) 

# 6 SUMMAR YO F RESULT S Thi s stud ywa s performe d t o investigat e th e potentia l o fTK A4036 5 (CG I 725 ) t o induc e gen e mutation s i n th e plat e incorporatio n tes t (experimen tI ) an d th e pre-incubatio n tes t (experimen tII ) usin g th e Salmonell a typhimuriu m strain s TA1535 , T A1537 , TA98 , an d T A100 , an d th e Escherichi a col i strai n WP 2 uvrA

. Th e assaywa s performe d wit h an d withou tlive rmicrosoma l activation

. Eac h concentration , includin g th e controls , wa s teste d i n triplicate

. Th e tes tite m wa s teste d a t th e followin g concentrations : Pre-Experiment/Experimen t I an d la : 3 ; 10 ; 33 ; 100 ; 333 ; 1000 ; 2500 ; an d 500 0 pg/plat e Experimen tII : 33 ; 100 ; 333 ; 1000 ; 2500 ; an d 500 0 pg/plat e Th e plate s incubate d wit h th e tes tite m showe d norma l backgroun d growt h u p t o 500 0 pg/plat e wit h an d withou tmetaboli cactivatio n i n bot h independen t experiments

. N o toxi ceffects , eviden t a s a reductio n i n th e numbe ro frevertants , occurre d i n th e tes t group s wit h an d withou tmetaboli cactivation

. N o substantia l increas e i n revertan t colon ynumbers o fan yo fth e five teste rstrain s wa s observe d followin g treatmen twit h TK A4036 5 (CG I 725 ) at an ydos e level , neithe ri n th e presenc e no rabsenc e o fmetaboli cactivatio n (S 9 mix)

. Ther e wa s als o n o tendencyo f highe rmutatio n rate s wit h increasin g concentration s i n th e rang e belo wth e generall y acknowledge d borde ro fbiologica l relevance

. Appropriat e referenc e mutagen s wer e use d a s positiv e control s an d showe d a distinct in
creas e ofinduce d revertan tcolonies

. 

## 6. 1 Conclusio n I n conclusion , i t ca n b e state d tha t durin g th e describe d mutagenicitytes t an d unde rth e experimenta l condition s reported , th e tes t ite m di d no tinduc e gen e mutation s bybas e pai r change s o rframeshift s i n th e genom e ofth e strain s used

. Therefore , TK

A4036 5 (CG I 725 ) i s considere d t o b e non-mutageni ci n thi s Salmonell a typhimuriu m an d Escherichi a col i revers e mutatio n assay
.

s4c2dr.doc

### RCC-CC R StudyNumbe r1040300 Fina l Repor tPage 1 0 o f3 1 TK A40365 (CG I 725) 

# 7 OBJECTIV E
7. 1 Aim s o fth e Stud y Th e experiment s wer e performe d t o asses s th e potentia l o fth e tes tite m t o induc e gen e mutation s bymean s o ftw o independen t Salmonell a typhimuriu m an d Escherichi a col i revers e mutatio n assays
. Experimen tI wa s performe d a s a plat e incorporatio n assay
. Sinc e a negativ e resul t wa s obtaine d i n thi s experiment , experimen tI I wa s performe d a s a pre-incubatio n assay
. 

## 7. 2 Reason s fo rth e Stud y Th e most widel yuse d assay s fo rdetectin g gen e mutation s ar e thos e usin g bacteri a (3)

. The yar e relativel ysimpl e an d rapi d t o perform , an d giv e reliabl e dat a o n th e abilityo fa n agen t t o interactwit h DN Aan d produc e mutations

. Revers e mutatio n assay s determin e th e frequencywit h whic h a n agen treverse s o r suppresse s th e effect o fth e forwar d mutation

. Th e geneti ctarge tpresente d t o a n agen t i s therefor e small , specifi can d selective

. Severa l bacteria l strains , o ra singl e strai n wit h multipl e markers ar e necessar yt o overcom e th e effect s o fmutage n specificity

. Th e reversio n o fbacteri a fro m growth-dependenc e o n a jjarticula ramin o aci d t o growt h i n th e absenc e oftha tamin o aci d (reversio n fro m auxotroph yt o prototrophy ) i s th e mos twidel y use d marker

. Th e Salmonell a typhimuriu m histidin e (his ) an d th e E

. col i tryptopha n (trp ) reversio n sy ste m measure s his ' - > his * an d trp " - ^ trp * reversions , respectively

. Th e S

. typhimuriu m an d Escherichi a col i strain s ar e constructe d t o differentiat e betwee n bas e pai r(T A1535 , TA100 , an d WP 2 uvrA) an d frameshift(T A1537 , T

A98 ) mutations

. Accordin g t o th e direct plat e incorporatio n o rth e pre-incubatio n metho d th e bacteri a ar e expose d t o th e tes t ite m wit h an d withou t metaboli cactivatio n an d plate d o n selectiv e medium

. Afte ra suitabl e perio d o fincubation , revertan tcolonie s ar e counted

. T o establis h a dos e respons e effect atleas t6 dos e level s wit h adequatel yspace d interval s wer e tested

. Th e maximu m dos e leve l wa s 500 0 pg/plate

. T o validat e th e test , referenc e mutagen s wer e teste d i n paralle l t o th e tes t item

.

s4c2dr.doc

### RCC-CC R StudyNumbe r1040300

TK A40365 (CG I 725) Fina l Repor t Page 1 1 o f3 1



## 8 MATERIAL S AN D METHOD S
8. 1 Tes t Ite m Interna l RCC-CC R Tes t Ite m Number : S665 1 1 1 Th e tes t ite m an d th e informatio n concernin g th e tes t ite m wer e provide d b yth e sponsor
. Identity : Batc h No. : Aggregat e stat e at roo m temperature : Colour : Purity : Stabilityi n solvent : Storage : Expiratio n Date : TKA4036 5 (CG I 725 ) 3007 3 FC6A D
Soli d Yello w Confidentia l informatio n (se e analytica l certificat e o fJul y20 , 2006 ) Not indicate d byth e sponso r I n th e refrigerato ra t2 ° - 8 ° C Jul y01 , 200 8 O n th e dayofth e experiment , th e tes t ite m TK A4036 5 (CG I 725 ) wa s dissolve d i n DMS O (MERCK , D-6429 3 Darmstadt ; purity> 9 9 %)

. Th e solven twa s chose n becaus e o fit s solubilitypropertie s an d it s relativ e non-toxicityt o th e bacteri a (5)

. Th e tes t ite m precipitate d i n th e overiayaga rat250 0 an d 500 0 pg/plat e wit h an d withou t metaboli cactivatio n i n bot h experiments

. Th e undissolve d particle s ha d n o influenc e o n th e dat a recording

.

s4c2dr.docRCC-CC R Stud yNumbe r

TK A4036 5 (CG I 725 ) 

### 104030 0 Fina l Repor t Pag e 1 2 o f3 1



## 8. 2 Control s
8.2. 1 Negativ e Control s Concurren t untreate d an d solven t control s wer e performed

. 8.2. 2 Positiv e Contro l Substance s Withou t metaboli cactivatio n Strains : Name : Supplier : Catalogu e No. : Purity : Dissolve d in : Concentrafion : Strains : Name : Supplier : Catalogu e No. : Purity : Dissolve d in : Concentrafion : Strain : Name : Supplier : Catalogu e No. : Purity : Dissolve d in : Concentration : Wit h metaboli cactivatio n Strains : Name : Supplier : Catalogu e No. : Purity : Dissolve d in : Concentrafion : T A1535 , TAIO O sodiu m azide , NaN s SERVA , D-6904 2 Heidelber g 

### 3017 5 a t leas t9 9 % wate rdeionise d 1 0 pg/plat e TA1537 , TA9 8 4-nitro-o-phenylene-diamine , 4-NOP D SIGMA , D-8204 1 Deisenhofe n N 950 4 >99.9 %

. DMS O (MERCK , D-6429 3 Darmstadt ; purit y> 9 9 % ) 1 0 pg/plat e i n T A98 , 5 0 pg/plat e i n T A153 7 WP 2 uvr A methy l methan e sulfonate , MM S Merck-Schuchardt , D-8566 2 Hohenbrun n 82077 5 > 99. 0 % wate rdeionise d 3 pL/plat e

T A1535

. T A1537 , T A98 , T A100 , WP 2 uvr A 2-aminoanthracene , 2-A A SIGMA , D-8204 1 Deisenhofe n A138 1 97. 5 % DMS O (MERCK , D-6429 3 Darmstadt ; purit y> 9 9 % ) 2. 5 pg/plat e (T A1535 , T A1537 , T A98 , T A100) , 1 0 pg/plat e (WP 2 uvrA ) Th e stabilit yo fth e posifiv e contro l substance s i n solufio n i s unknow n bu ta mutageni c respons e i n th e expecte d rang e wil l b e sufficien tevidenc e o fbiologica l stability

.

s4c2dr.do cRCC-CC R Stud yNumbe r

TK A4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 1 3 o f3 1



## 8. 3 Tes t Syste m
8.3. 1 Characterisatio n o fth e Salmonell a typhimuriu m Strain s an d E

. coi l Strai n Th e hisfidin e dependen t strain s ar e derive d fro m S

. typhimuriu m strai n LT 2 throug h a mutafio n i n th e hisfidin e locus

. Additionall ydu e t o th e "dee p rough " (rfa-minus ) mutafio n the yposses s a fault ylipopolysaccharid e envelop e whic h enable s substance s t o penetrat e th e cel l wal l mor e easily

. Afurthe rmutatio n cause s a reducfio n i n th e activit yo fa n excisio n repai rsystem

. Th e latte ralterafio n include s mutationa l processe s i n th e nitrat e reductas e an d bioti n gene s produce d i n a UV-sensitiv e are a o fth e gen e name d "uvrB-minus"

. I n th e strain s TA9 8 an d T A10 0 th e R-facto rplasmi d pK M 10 1 carrie s th e ampicilli n resistanc e marke r(6)

. Strai n WP 2 (4 ) an d it s derivative s al l carr yth e sam e defec ti n on e o fth e gene s fo r tryptopha n biosynthesis

. Tryptophan-independen t(Trp* ) mutant s (revertants ) ca n aris e eithe rb ya bas e chang e a t th e sit e o fth e origina l alteratio n o rb ya bas e chang e elsewher e i n th e chromosom e s o tha t th e origina l defec ti s suppressed

. Thi s secon d possibilit yca n occu ri n severa l differen t way s s o tha tth e syste m seem s capabl e o fdetecfin g al l type s o f mutage n whic h subsfitut e on e bas e fo ranother

. Additionally , th e uvr Aderivafiv e i s deficien t i n th e DN Arepai rproces s (excisio n repai rdamage)

. Suc h a repair-deficien tstrai n ma yb e mor e readil ymutate d b yagents

. Whe n summarise d th e mutafion s o fth e T Astrain s an d th e E

. col i strain , use d i n thi s stud y ca n b e describe d a s follows :


### Strain s TA153 7 TA9 8 TA153 5 TAIO O WP 2 uvr A Salmonell a typhimuri i

Genotyp e hi s C 3076 ; rfa" ; uvrB" : hi s D 3052 ; rfa" ; uvrB";R-facto r hi s G 46 ; rfa" ; uvrB" : hi s G 46 ; rfa" ; uvrB";R-facto r

Escherichi a col i trp" ; uvrA" : j m 

### Typ e o fmutation s indicate d fram e shiftmutation s

n n 

### base-pai rsubstitution s

I I n 

### base-pai rsubstitution s an d others Regula rcheckin g o fth e propertie s o fth e strain s regardin g th e membran e permeabilit yan d ampicilli n resistanc e a s wel l a s spontaneou s mutafio n rate s i s performe d i n RC C Cytotes t Cel l Researc h accordin g t o B

. Ame s e tal

. (1 ) an d D

. Maro n an d B

. Ame s (6)

. I n thi s wa yi t wa s ensure d tha t th e experimenta l condifion s se tdow n b yAme s wer e fulfilled

. Th e bacteria l strain s T A1535 , T A1537 , T A98 , T A100 , an d WP 2 uvr Awer e obtaine d fro m Trinov a Bioche m Gmb H (3539 4 Gielien , Germany)

.

s4c2dr.do cRCC-CC R stud yNumbe r104030 0 Fina l Repor tPag e 1 4 o f3 1 TK A4036 5 (CG I 725 )

8.3. 2 Storag e Th e strai n culture s wer e store d a s stoc k culture s i n ampoule s wit h nutrien tbrot h + 5 % DMS O (MERCK , D-6429 3 Darmstadt ) i n liqui d nitrogen

. 8.3. 3 Precuiture s Fro m th e thawe d ampoule s o fth e strain s 0. 5 m Lsuspensio n wa s transferre d int o 25 0 m L Erienmeye rflasks containin g 2 0 m Lnutrien tmedium

. Asolufio n o f2 0 p Lampicilli n (2 5 pg/mL ) wa s adde d t o th e strain s T A9 8 an d T A100

. Thi s nutrien tmediu m contain s pe r litre : 8 g Merc k

Nutrien t Brot h (MERCK , D-6429 3 Darmstadt ) 5 g NaC l (MERCK , D-6429 3 Darmstadt ) Th e bacteria l culture s wer e incubate d i n a shakin g wate rbat h fo r4 hour s a t37 ° C

. 8.3. 4 Selectiv e Aga r Th e plate s wit h th e selectiv e aga rwer e obtaine d fro m E

. Merck , D-6429 3 Darmstadt

. 8.3. 5 Overla yAga r Th e pvpria yaga rcontain s pe rlitre : fo rSalmonell a strains : fo rEscherichi a coli : 6. 0 g MERC KAga rAgar * 6. 0 g MERC KAga rAgar * 6. 0 g NaCl * 6. 0 g NaCl * 10.5m g L-HistidinexHCIxH20 * 2. 5 m g Tryptophan * 12.2m g Biofin *

* (MERCK

. D-6429 3 Dannstadt ) Sterilisafion s wer e performe d a t 121 ° C i n a n autoclave

. 

## 8. 4 Mammalia n Microsoma l Fractio n S 9 Mi x Th e bacteri a use d i n thi s assa yd o no tposses s th e enzym e system s which , i n mammals , ar e know n t o conver t promutagen s int o acfiv e DN Adamagin g metabolites

. I n orde rt o overcom e thi s majo rdrawbac k a n exogenou s metaboli csyste m i s adde d i n for m o f mammalia n microsom e enzym e activafio n mixture

.

s4c2dr.do c

### RCC-CC R stud yNumbe r1040300 Fina l Repor tPage 1 5 o f3 1 TK A40365 (CG I 725)

8.4. 1 S 9 (Preparatio n b yR C C - C C R ) Phenobarbital/p-Naphthoflavon e induce d ratlive rS 9 i s use d a s th e metaboli cactivatio n system

. Th e S 9 i s prepare d fro m 8-1 2 weeks ol d mal e Wista rHanlb m rats , weigh t approx
. 22 0 - 32 0 g induce d byapplicafion s o f8 0 mg/kg b.w

. Phenobarbita l i.p

. (Desifin ; D-2233 5 Hamburg ) an d p-Naphthoflavon e p.o
. (Aldrich , D-8955 5 Steinheim ) eac h o n thre e consecutiv e days

. Th e livers ar e prepare d 2 4 hours afte rth e las ttreatment

. Th e S 9 fracfion s are produce d bydilutio n o fth e live rhomogenat e wit h a KC l isolufio n (1+3 ) followe d bycentrifugafio n at 900 0 g
. Aliquote s o fth e supematan tar e froze n an d store d i n ampoule s at -80 ° C

. Smal l numbers o fth e ampoule s ca n b e kep ta t-20° C fo ru p t o on e week

. Eac h batc h ofS 9 mi xi s roufinel yteste d wit h 2-aminoanthracen e a s wel l a s benzo(a)pyrene

. Th e protei n concentrafio n i n th e S 9 preparafio n wa s 31. 1 mg/m L(lo tno
. R 050506)

. 8.4. 2 S9MI X Before th e experimen ta n appropriat e quantityo fS 9 supernatan t wa s thawe d an d mixe d wit h S 9 co-facto rsolufion

. Th e amoun t ofS 9 supernatan twa s 10 % v/ vi n th e S 9 mix

. Cofactors are adde d t o th e S 9 mi xt o reac h th e followin g concentrafion s i n th e S 9 mix : 8m M MgCl 2 3 3 m M KC l 5 m M Glucose-6-phosphat e 5m M NAD P i n 10 0 m M sodium-ortho-phosphate-buffer , p H 7.4

. Durin g th e experimen t th e S 9 mi xwa s store d i n a n ice bath

. Th e S 9 mi xpreparafio n wa s performe d accordin g t o Ame s e tal.(1)

. 

## 8. 5 Pre-Experimen t fo rToxicit y T o evaluat e th e toxicityo fth e tes t ite m a pre-experimen twa s performe d wit h strain s TA1535 , T

A1537 , T

A98 , TA100 , an d WP 2 uvrA

. Eigh tconcentrafion s wer e teste d fo r toxicityan d mutatio n inductio n wit h thre e plate s each

. Th e experimenta l condifion s i n thi s pre-experimen twer e th e sam e a s describe d belo wfo rth e experimen t I (plat e incorporatio n test)

. Toxicityo fth e tes t ite m result s i n a reductio n i n th e numbe ro fspontaneou s revertant s o ra clearin g ofth e bacteria l backgroun d lawn

. Th e pre-experimen ti s reporte d a s mai n experimen tI , i fth e followin g criteri a ar e met : Evaluabl e plate s (> 0 colonies ) at five concentrafion s o rmore i n al l strain s used

.

s4c2dr.doc

### RCC-CC R stud yNumbe r104030 0 Fina l Repor

t Pag e 1 6 o f3 1 TK A40365 (CG I 725)


## 8. 6 Dos e Selectio n I n th e pre-experimen t th e concentratio n rang e o fth e tes tite m wa s 3 - 500 0 pg/plate

. Th e pre-experimen t i s reporte d a s experimen tI sinc e n o toxi ceffect s wer e observe d an d 500 0 pg/plat e wer e chose n a s maxima l concentrafion

. Th e concentratio n rang e include d tw o logarithmi cdecades

. Th e followin g concentrafion s wer e tested : Pre-Experiment/Experimen t I an d la : 3 ; 10 ; 33 ; 100 ; 333 ; 1000 ; 2500 ; an d 500 0 pg/plat e Experimen t II : 33 ; 100 ; 333 ; 1000 ; 2500 ; an d 500 0 pg/plat e


## 8. 7 Experimenta l Performanc e Fo reac h strai n an d dos e leve l includin g th e controls , thre e plate s wer e used

. Th e followin g material s wer e mixe d i n a tes ttub e an d poure d ont o th e selecfiv e aga r plates : 10 0 p LTes t solufio n at eac h dos e level , solven t(negativ e control ) o rreferenc e mutage n solufio n (posifiv e control) , 50 0 p LS 9 mi x(fo rtes t wit h metaboli cacfivation ) o rS 9 mi xsubstitufio n buffe r(fo rtes t withou t metaboli cacfivafion) , 10 0 p LBacteri a suspensio n (cf

. tes tsystem , pre-cultur e ofth e strains) , 200 0 p LOveriayaga r I n th e pre-incubafio n assay10 0 p Ltes tsolufion , 50 0 p LS 9 mi x/ S 9 mi xsubstitufio n buffe r an d 10 0 p Lbacteria l suspensio n were mixe d i n a tes ttub e an d shake n at37 ° C fo r6 0 minutes

. Afte rpre-incubatio n 2. 0 m Loveriayaga r(45 ° C ) wa s adde d t o eac h tube

. Th e mixture wa s poure d o n selectiv e aga rplates

. Afte rsolidificafio n th e plate s were incubate d upsid e dow n fo ratleas t4 8 hours at 37 ° C i n th e dar k (2)

. 

## 8. 8 Dat a Recordin g Th e colonie s wer e counte d usin g th e Petr i Viewe rMk 2 (Percepfiv e Instrument s Ltd , Suffol k C B 7BN , UK) wit h th e software progra m Ame s Stud yManager

. Th e counte rwa s connecte d t o a n IB M ATcompafibl e P C wit h printe rt o prin tou tth e individua l value s an d th e mean s fro m th e plate s fo reac h concentratio n togethe rwit h standard deviation s an d enhancemen t factor s a s compare d t o th e spontaneou s reversio n rate s (se e table s o f results)

. Du e t o precipitatio n o fth e tes tite m th e revertan tcolonie s wer e partl ycounte d manually

.

s4c2dr.doc

### RCC-CC R Stud yNumbe r1040300 Fina l Repor tPage 1 7 o f3 1 TK A40365 (CG I 725)


## 8. 9 Acceptabilit yo fth e Assa y Th e Salmonell a typhimuriu m an d Escherichi a col i revers e mutatio n assayi s considere d acceptabl e i fi t meet s th e followin g criteria : regula rbackgroun d growt h i n th e negativ e an d solven tcontro l th e spontaneou s reversio n rate s i n th e negativ e an d solven t contro l are i n th e rang e o f ou rhistorica l dat a th e positiv e contro l substance s shoul d produc e a significan tincreas e i n mutan t colon y frequencie s 

## 8.1 0 Evaluatio n o fResult s Ates t ite m i s considere d a s a mutage n i fa biologicall yrelevan tincreas e i n th e numbe ro f revertant s exceedin g th e threshol d o ftwic e (strain s TA98 , TA100 , an d WP 2 uvrA ) o r thric e (strain s TA153 5 an d TA1537 ) th e colon ycoun t o fth e correspondin g solven t contro l i s observe d (3)

. Ados e dependen tincreas e i s considere d biologicall yrelevan ti fth e threshol d i s exceede d at more tha n on e concentrafio n (2)

. A n increas e exceedin g th e threshol d atonl yon e concentratio n i s Judge d a s biologicall y relevan t i freproduce d i n a n independen tsecon d experiment

. Ados e dependen tincreas e i n th e numbe rofrevertan tcolonie s belo wth e threshol d i s regarde d a s a n indicafio n o fa mutageni cpotentia l i freproduce d i n a n independen tsecon d experiment

. However , wheneve rth e colon ycount s remai n withi n th e historica l rang e o f negativ e an d solven t control s suc h a n increas e i s notconsidere d biologicall yrelevant

. 

## 8.1 1 Biometr y Accordin g t o th e OEC D guidelin e 471 , a statistica l analysi s o fth e dat a i s not mandatory

.

s4c2dr.doc

### RCC-CC R Stud yNumbe r1040300 Fina l Repor tPage 1 8 o f3 1 TK A40365 (CG I 725) 

# 9 DISCUSSIO N O F RESULT S Th e tes t ite m TK

A4036 5 (CG I 725 ) wa s assesse d fo rit s potentia l t o induc e gen e mutafion s i n th e plat e incorporafio n tes t(experimen tI ) an d th e pre-incubatio n tes t (experimen t II ) usin g Salmonell a typhimuriu m strain s TA1535 , TA1537 , TA98 , an d TA100 , an d th e Escherichi a col i strai n WP 2 uvrA

. Th e assaywa s performe d wit h an d withou t live rmicrosoma l acfivation

. Eac h concentratio n an d th e control s wer e teste d i n triplicate

. Du e t o irregula rbackgroun d growth , th e analysi s o fstrai n T

A9 8 wa s impossibl e wit h an d withou t S 9 mi xi n th e pre-experiment/experimen t I

. A n additiona l experimen t wa s performe d unde ridentica l condition s (plat e incorporatio n test ) usin g strai n T

A9 8 wit h an d withou tmetaboli cactivation

. Th e result s o fthi s addifiona l experimen t are include d i n thi s repor ta s pre-experiment/experimen tI a ( Wa)

. Th e tes t ite m wa s teste d at th e followin g concentrafions : Pre-Experiment/Experimen tI an d la : 3 ; 10 ; 33 ; 100 ; 333 ; 1000 ; 2500 ; an d 500 0 pg/plat e Experimen t II : 33 ; 100 ; 333 ; 1000 ; 2500 ; an d 500 0 pg/plat e Th e plate s incubate d wit h th e tes tite m showe d norma l backgroun d growt h u p t o 500 0 pg/plat e wit h an d withou t metaboli cactivafio n i n bot h independen texperiments

. N o toxi ceffects , eviden tas a reducfio n i n th e numbe ro frevertants , occurre d i n th e tes t group s wit h an d withou t metaboli cacfivafion

. N o substanfia l increas e i n revertan tcolon ynumbers ofan yofth e five teste rstrain s wa s observe d followin g treatmen t wit h TK A4036 5 (CG I 725 ) atan ydos e level , neithe ri n th e presenc e no rabsenc e ofmetaboli cactivafio n (S 9 mix)

. There wa s als o n o tendencyo f highe rmutafio n rate s wit h increasin g concentrafion s i n th e rang e belo wth e generall y acknowledge d borde ro fbiologica l relevance

. Appropriat e referenc e mutagen s wer e use d a s positiv e controls

. The yshowe d a distinct in
creas e ofinduce d revertan t colonies

. Th e laboratory' s historica l contro l dat a wa s slightl yexceede d i n th e untreate d an d solven t contro l ofstrai n WP 2 uvr Awit h metaboli cacfivafio n i n experimen tII

. Thes e mino r deviafion s ar e judge d t o b e base d o n biologicall yirrelevan tfluctuations i n th e numbe ro f colonie s an d have n o impac t o n th e outcom e o fth e study

. I n conclusion , i t ca n b e state d tha t durin g th e describe d mutagenicitytes tan d unde rth e experimenta l condition s reported , th e tes tite m di d notinduc e gen e mutation s bybas e pai r change s o rframeshift s i n th e genom e o fth e strain s used

.

s4c2dr.docRCC-CC R stud yNumbe r

TK A4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 1 9 o f3 1


# 1 0 REFERENCE S
1

. Ames , B.N. , J

. McCann , an d E

. Yamasak i (1977 ) Method s fo rdetecfin g carcinogen s an d mutagen s wit h th e Salmonella/mammalia n microsom e mutagenicit ytes t In : B.J

. Kilbe ye t al

. (Eds.)"Handboo k o fMutagenicit yTes tProcedures " Elsevier , Amsterdam , 1-1 7 2

. d e Sen-e s F.J

. an d M.D

. Shelb y(1979 ) Recommendation s o n dat a producfio n an d analysi s usin g th e Salmonella/microsom e rnutagenicit yassa y Mutatio n Res

. 64 , 159-16 5 3

. Hollstein.M. , J

. McCann , F.A

. Angelosant o an d W.W

. Nichol s (1979 ) Short-ter m test s fo rcarcinogen s an d mutagen s Mutatio n Res

. 65 , 133-22 6 4

. Green , M.H.L

. an d W.J

. Murie l (1976 ) Mutage n Testin g Usin g TRP * Reversio n i n Escherichi a Col i Mutation

. Res

. 38 , 3
- 3 2 5

. Maro n D.M. , J

. Katzenellenboge n an d B.N

. Ames , (1981 ) Compatibilit yo forgani csolvent s wit h th e Salmonella/Microsom e Tes t Mutatio n Res

. 88 , 343-35 0

. < 6

. Maro n D.M. , Ames , B.N

. (1983 ) Revise d method s fo rth e Salmonell a mutagenicit ytes t MutationRes

. 113 , 173-21 5



# 1 1 DISTRIBUTIO N O F TH E REPOR T Sponso r Contractin g Institut e Stud yDirecto r 3 x(copie s unbound ) I x(electroni ccop y(pdf) ) 1 X(original )

s4c2dr.do cRCC-CC R Stud yNumbe r TK A4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 2 0 o f3 1


# 1 2 SUMMAR YO F RESULT S
12. 1 Summaryo fResult s Pre-Experimen t an d Experimen t I stud yName : 104030 0 Experiment : 104030 0 W+ Wa Plat e AssayConditions : Stud yCode : RCC-CC R 104030 0 Dat e Plated : 01/08/200 6 / 08/08/200 6 Dat e Counted : 04/08/200 6 / 14/08/200 6 Metaboli c Activatio n

Wittiou t Activatio n Tes t

Grou D

DMS O Untreate d TK A4036 5 (CG I 725 )

NaN 3 4-NOP D 4-NOP D MM S Dos e Leve l

(ua/Dlate )

3M g 10|j g 3 3 M g 10 0 p g 33 3 M g 100 0 M g 250 0 M g 500 0 M g 1 0 M g 1 0 M g 5 0 M g 4. 0 M L Revertan t C

TA153 5
2 5 ± 5 18± 4 16± 4 19± 5 16± 3 15± 2 16± 3 14± 1 18±3 ^ 16±3' ' 119 0 ±8 7 Colon

yCoun t
TA153 7
1 6 ± 6 1 1 ± 3 18± 4 19± 5 2 0 ± 2 1 2 ± 1 2 1 ± 1 17± 4 14±2' ' 17±4' '
10 1 ± 7 s (Mea n ±SD )

W a TA9 8
2 9 ±1 1 3 8 ± 4 3 7 ± 6 3 1 ± 5 3 1 ± 4 2 6 ± 4 3 1 ± 9 3 9 ± 7 3 1 ±2 ^ 3 9 ±5' ' 37 4 ±1 9 TA10 0

137 ±1 3 15 3 ±2 4 143 ±1 3 15 6 ± 6 13 9 ±1 2 134 ±2 5 13 4 ± 9 138 ± 5 152 ±10' ' 15 8 ±16' ' 182 2 ±6 9 WP 2 uvr

A
.6 3 ± 2 4 8 ±7 5 5 ± 3 5 5 ±5 5 9 ± 9 5 1 ±2 6 5 3 ± 9 5 3 ± 4 5 6 ±2' ' 6 0 ±9' '

1274 ± 4

Wit h Activatio n

Keyt o Pos i DMS O

Untreate d TK A4036 5 (CG I 725 )

2-A A 2-A A
itive Control s 3M g

1 0 M O 3 3 M g 10 0 M g 33 3 M O 100 0 M O 250 0 M g 500 0 M g 2. 5 M g 10. 0 M g 18± 3 2 6 ± 3 2 3 ± 5 2 3 ± 6 2 7 ± 7 2 3 ± 3 2 4 ± 2 2 3 ± 1 2 5 ±1'' " 2o±2'' " 41 1 ±2 1 2 0 ± 2

17± 2 16± 6 16± 7 2 1 ± 1 2 4 ± 4 2 2 ± 2 2 5 ± 8 2 1 ±3'"' ^ 19±6''' ' 47 1 ±3 0

Keyt o Pl a 3 8 ±1 4

4 4 ±1 3 3 8 ± 6 3 6 ±1 0 3 6 ± 8 3 5 ± 3 3 7 ± 7 4 1 ± 7 29±5'' " 28±4''' ^ 292 6 ±15 5

lte Postfi xCod ( 15 8 ±1 2

15 6 ±1 6 144±1 3 16 3 ±2 6 14 9 ±1 0 16 3 ±3 1 14 9 ±2 4 133 ±1 9 133 ±9'' " 133 ±8''' ^ 341 9 ±17 9

B S 5 8 ± 8

5 9 ± 9 6 5 ±5 6 1 ± 4 5 7 ± 5 64± 8 6 2 ± 9 6 4 ± 3 55±5'' " 48±4''' ^ 25 1 ±2 6

NdN 3 sodiu m azid e 2-A A2-aminoanthracen e 4-NOP D 4-nitro-o-phenylene-diamin e MM S methy l methan e sulfonat e P Precipitat e M Manua l coun t

s4c2dr.do cRCC-CC R Stud yNumbe r

TKA4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 2 1 o f3 1



## 12.2Summaryo fResult s Experimen t I I
stud yNam e Experiment : Assa yCon d Metaboli c Activatio n

Withou t Activatio n s : 104030 0

104030 0 HV 2 litions : Tes t Grou D

DMS O Untreate d TK A4036 5 (CG I 725 )

NaN 3 4-NOP D 4-NOP D MM S Pre
Dos e Leve l

(ua/Dlate )

3 3 M O 10 0 Mg 33 3 Mg 100 0 M O 250 0 Mg 500 0 Mg 1 0 M O 1 0 Mg 5 0 M O 4. 0 M L Revertan t

TA153 5
2 3 ± 3 2 4 ± 5 19± 6 2 0 ± 8 19± 8 19± 4 2 1 ±8' ' 2 1 ±3" " 140 9 ±7 0 Colon yCoun t

TA153 7
15± 2 19± 4 2 4 ± 7 16± 1 17± 2 19± 3 19±5' ' 2 0 ±6''' ^
14 7 ± 6 Stud y

Date l Date '
s (Mea n ±SD )
TA9 8
2 7 ± 3 3 6 ± 6 3 5 ± 5 3 2 ± 7 3 8 ± 6 34± 6 3 1 ±3' ' 29ilP M 62 0 ±2 3 Code : RCC-C

Plated : 08/08/ : Counted : 14/

0

TA10 0
136 ±1 7 146 ±1 5 148 ±1 3 13 8 ± 9 : 12 8 ±1 4 152 ±1 5 143 ±7' ' 12 8 ±11'"' ^ 215 3 ±1 3 :C R 104030 0

2006 8/200 6

WP 2 uvr A
6 2 ±8 6 2 ± 8 6 2 ±1 6 6 3 ± 8 6 1 ± 4 64± 1 5 8 ±9' ' 53±6'' "

59 1 ±6 4

Wit h Activatio n

Ke yt o P o NaN 3 2-AA 4-NOP D MM S DMS O

Untreate d TK A4036 5 (CG I 725 )

2-A A 2-A A
sitiv e Control s sodiu m azid e 2-aminoanthracen i 4-nitro-o-phenyle n methy l methan e s i 3 3 M g 10 0 M g 33 3 M g 100 0 M g 250 0 M g 500 0 M O 2. 5 M g 10. 0 M O

e e-diamin e jifonat e 2 3 ± 1

2 2 ± 4 2 1 ± 4 2 4 ± 5 2 8 ± 8 2 2 ± 4 22±2''' ^ 20±3'' " 39 3 ±2 6 2 4 ± 2

2 5 ±1 1 2 3 ± 3 17± 4 2 3 ± 4 2 4 ± 4 19±4'' " 19±5''* ' 39 5 ±2 6

Ke yt o P P M 4 0 ± 6

4 5 ± 7 3 6 ±1 0 4 5 ± 5 4 1 ± 4 4 1 ±1 5 3 4 ±3''* ' 34±4'' " 258 7 ±37 7

lat e Postfi xCo d Precipitat e Manua l coun t 175 ±1 7

183 ±2 1 17 9 ± 9 18 9 ± 4 18 0 ± 7 174 ±2 8 15 1 ±14'' " 152±5'' " 399 1 ±16 4

e s 7 6 ± 5

7 3 ±1 1 7 0 ± 5 7 3 ± 7 7 7 ±1 4 7 5 ± 8 6 1 ±6" " 7 1 ±2" " 25 5 ±4 6

s4c2dr.do cRCC-CC R Stud yNumbe r

TK A4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 2 2 o f3 1



## 12.3HISTORICA LCONTRO LDATA Thes e dat a represen t th e laboratory' s historica l contro l dat a fro m Ma y200 5 unti l Jun e 200 6 representin g approx
. 20 0 experiment s (WP 2 uvr Ath e historica l dat a ar e base d o n approx
. 10 0 experiments)

. Strai n

TA1535

TA I 53 7

TA9 8

TAIO O

WP2uvr A Solven t contro l Negativ e contro l Positiv e contro l Solven t contro l Negativ e contro l Positiv e contro l Solven t contro l Negativ e contro l Positiv e contro l Solven t contro l Negativ e contro l Positiv e contro l Solven t contro l Negativ e contro l Positiv e contro l Mea n

20. 8 20. 4 1422. 0 11. 2 11. 6 99. 8 28. 1 30. 2 439. 0 130. 7 138. 2 2083. 1 55. 8 55. 9 991. 0 with o

S D 4. 7 4. 4 464. 7 3. 7 4. 0 32. 5 6. 1 6. 6 155. 2 20. 8 21. 6 281. 3 7. 2 8. 6 522. 9 u t S 9 mi x

Mi n 9 1 1 78 1 5 4 5 3 1 5 1 6 17 6 8 7 8 6 61 6 3 1 3 6 24 9 Ma

x 3 5 3 1 490 0 2 8 2 8 42 5 4 9 6 0 181 8 19 7 21 6 287 2 7 4 7 6 181 0 Mea n

24. 7 24. 2 332. 0 16. 2 17. 1 276. 8 37. 9 39. 0 1839. 4 147. 0 150. 1 2372. 9 63. 9 65. 6 319. 4 wit h S 9

S D 5. 9 5. 5 95. 3 5. 0 5. 4 132. 6 7. 4 7. 5 898. 6 25. 5 24. 2 958. 4 9. 1 10. 4 84. 8 mi x Mi n

7 1 0 10 7 6 7 5 9 2 0 I S 40 7 84,

. 9 6 41 7 34
- 3 3 21 1 Max 4 3 3 8 69 5 3 6 3 4 74 6 5 7 6 4 489 1 25 5 21 4 523 0 8 4 9 1 93 0 Mea n = mea n valu e o frevertants/plat e S D = standard deviatio n Mi n = minima l value/Max= maxima l valu e

s4c2dr.do cRCC-CC R Stud yNumbe r104030 0 Fina l Repor tPag e 2 3 o f3 1 TKA4036 5 (CG I 725 ) 

# 1 3 ANNEX : TABLE S O F RESULT S ( 8 PAGES ) Pre-Experimen t an d Experimen t 1:104030 0 W+ Wa Plat e Incorporatio n (4 pages ) Experimen t II : 104030 0 Pre-incubatio n ( 4 pages )

s4c2dr.do cRCC-CC R Stud yNumbe r

TK A4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 2 4 o f3 1

stud yName : 104030 0 Experiment : 104030 0 W* Wa Plat e Assa yConditions : stud yCode : RCC-CC R 104030 0 Dat e Plated : 01/08/200 6 / 08/08/200 6 Dat e Counted : 04/08/200 6 / 14/08/200 6

TA153 5 Withou t metaboli cactivatio n strai n Compoun d Dos e leve l pe rplat e Mea n

revertant s pe rplat e standar d

Deviatio n Rati o

treate d / solven t Individua l revertan t

colon ycount s
TK A4036 5 (CG I 725 ) 1 0 p g 3 3 p g 10 0 p g 33 3 p g 100 0 p g 250 0 p g 500 0 p g DMS O Untreate d Contro l 15. 7 18. 7 16. 3 14. 7 15. 7 14. 0 18. 0 16. 3 25. 0 17. 7 3. 5

4. 6 3. 2 2. 1 3. 1 1. 0 2. 6 3. 2 4. 6 4. 2 0. 6

0. 7 0. 7 0. 6 0. 6 0. 6 0. 7 0. 7 19 , 16 , 1 2 24 , 16 , 1 6 15 , 20 , 1 4 14,17,1 3 15 , 19 , 1 3 13 , 15 , 1 4 20P , 15P , 19 P 14P , 20P , 15 P 26 , 20 , 2 9 13,21 , 1 9
T A I 53 7 TK A4036 5

(CG I 725 )

DMS O Untreate d Contro l 3p g 1 0 p g 3 3 p g 10 0 p g 33 3 p g 100 0 p g 250 0 p g 500 0 p g 18. 3

19. 3 19. 7 12. 3 21. 3 16. 7 13. 7 16. 7 15. 7 11. 3 3. 8

5. 0 2. 3 0. 6 0. 6 4. 0 1. 5 4. 0 5. 5 3. 1 1. 2

1. 2 1. 3 0. 8 1. 4 1. 1 0. 9 1. 1 14,21,2 0 20 , 24 , 1 4 17,21,2 1 12 , 13 , 1 2 22 , 21 , 2 1 16,13,2 1 12P , 15P , 14 P 1 9 P , 1 9 P , 1 2 P 22 , 12 , 1 3 14,8,1 2 TA9 8 W a TKA4036 5

(CG I 725 )

DMS O Untreate d Contro l 3p g 1 0 p g 3 3 p g 10 0 p g 33 3 p g 100 0 p g 250 0 p g 500 0 p g 37. 0

31. 3 31. 0 26. 0 31. 3 39. 0 31. 3 38. 7 28. 7 38. 3 5. 6

4. 6 4. 0 4. 0 9. 5 7. 2 2. 3 4. 7 10. 8 3. 5 1. 3

1. 1 1. 1 0. 9 1. 1 1. 4 1. 1 1. 3 31 , 42 , 3 8 34 , 34 , 2 6 35 , 31 , 2 7 22 , 26 , 3 0 28 , 42 , 2 4 37 , 33 , 4 7 3 0 P , 3 4 P , 3 0 P 3 5 P , 4 4 P , 3 7 P 41,24,2 1 38 , 35 , 4 2 TAIO O TKA4036 5

(CG I 725 )

DMS O Untreate d Contro l 3 p g 143. 3 12. 9 1. 0 138 , 158 , 13 4 1 0 p g ^55. 7 5. 7 1. 1 151,154,16 2 3 3 p g 139. 3 11. 9 1. 0 126,149,14 3 10 0 p g 133. 7 25. 0 1. 0 109,159,13 3 33 3 p g 134. 3 8. 6 1. 0 142,136,12 5 100 0 p g 138. 0 5. 3 1. 0 142,140,13 2 250 0 p g 151. 7 9. 6 1. 1 15 0 P , 16 2 P , 14 3 P 500 0 p g 157. 7 15. 9 1. 2 14 7 P , 15 0 P , 17 6 P

137. 0 12. 8 134,151,12 6 753. 3 24. 3 158 , 175 , 12 7 Keyt o Plat e Postfi xCode s Precipitat e

s4c2dr.do cRCC-CC R Stud yNumbe r

TK A4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 2 5 o f3 1

Stud yName : 1 Experiment : I C Assa yConditi c
Strai n

WP2uvr A

TA153 5 T A I 53 7 TA98(Wa ) TAIO O WP 2 uvr A
Ke yt o Positiv e 04030 0

)4030 0 WPlat e >ns : Compoun d

TK A4036 5 (CG I 725 )

DMS O Untreate d Contro l
NaN 3 4-NOP D 4-NOP D NaN 3 MM S
1 Control s Dos e leve l pe rplat e 3p g 1 0 p g 3 3 p g 10 0 p g 33 3 p g 100 0 p g 250 0 p g 500 0 p g

1 0 p g 5 0 p g 1 0 p g 1 0 p g 4. 0 p L Withou t m

Mea n revertant s pe rplat e 54. 7 55. 3 59. 0 50. 7 53. 0 53. 0 56. 3 59. 7 63. 0 47. 7
1190. 3 101. 3 373. 7 1822. 0 1273. 7 letaboll cact i

Standar d Deviatio n
3. 1 4. 7 8. 9 26. 0 8. 5 4. 4 1. 5 8. 7 2. 0 7. 4
87. 3 7. 2 19. 3 69. 3 4. 0 ivatio n

Rati o treate d / solven t 0. 9 0. 9 0. 9 0. 8 0. 8 0. 8 0. 9 0. 9

47. 6 6. 5 13. 0 13. 3 20. 2 Stud yCode : RCC-CC R 104030 0

Dat e Plated : 01/08/200 6 / 08/08/200 6 Dat e Counted : 04/08/200 6 /14/08/200 6

Individua l revertan t colon ycount s
52,54,5 8 50 , 57 , 5 9 49 , 66 , 6 2 76 , 24 , 5 2 45 , 62 , 5 2 55 , 48 , 5 6 5 6 P , 5 8 P , 5 5 P 6 7 P , 6 2 P , 5 0 P 63.61,6 5 42 , 56 , 4 5 1249,1232,109 0 93 , 106 , 10 5 362 , 396 , 36 3 1779 , 1902 , 178 5 1278 , 1270 , 127 3
Ke yt o Plat e Postfi xCode s NaN 3 sodiu m azid e 4-NOP D 4-nitro-o-phenylene-diamin e MM S methy l methan e sulfonat e Precipitat e

s4c2dr.do cRCC-CC R Stud yNumbe r

TK A4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 2 6 o f3 1

Stud yName : 104030 0 Experiment : 104030 0 W+ WaPlat e Assa yConditions : Stud yCode : RCC-CC R 104030 0 Dat e Plated : 01/08/200 6 / 08/08/200 6 Dat e Counted : 04/08/200 6 / 14/08/200 6

TA153 5 Wit h metaboli cactivatio n Strai n Compoun d Dos e leve l pe rplat e Mea n

revertant s pe rplat e Standard

Deviatio n Rati o

treate d / solven t Individua l revertan t

colon ycount s
TK A4036 5 (CG I 725 )

DMS O Untreate d Contro l 3p g 22. 7 5. 1 1. 2 17,27,2 4 1 0 p g 23. 0 5. 6 1. 3 17,28,2 4 3 3 p g 27. 3 6. 7 1. 5 23,24,3 5 10 0 p g 23. 0 2. 6 1. 3 22,21,2 6 33 3 p g 24. 0 2. 0 1. 3 26,24,2 2 100 0 p g 23. 3 0. 6 1. 3 23,24,2 3 250 0 p g 25. 0 1. 0 1. 4 2 4 P M , 2 5 P M , 2 6 P M 500 0 p g 20. 3 2. 1 1. 1 2 2 P M , 1 8 P M , 2 1 P M

18. 3 3. 1 21 , 15 , 1 9 26. 0 3. 5 28,22,2 8 T A I 53 7 TKA4036 5

(CG I 725 )

DMS O Untreate d Contro l 3p g 16. 0 5. 6 0. 8 21,17,1 0 1 0 p g 16. 0 7. 0 0. 8 8,19,2 1 3 3 p g 21 3 1. 2 1. 1 22,20,2 2 10 0 p g 24. 0 4. 4 1. 2 22,29,2 1 33 3 p g 21 7 2. 1 1. 1 21,24,2 0 100 0 p g 25. 0 7. 9 1. 3 22,34,1 9 250 0 p g 21. 3 3. 1 1. 1 2 2 P M , 1 8 P M , 2 4 P M 500 0 p g 18. 7 5. 7 0. 9 2 5 P M , 1 4 P M , 1 7 P M

20. 0 1. 7 22 , 19 , 1 9 16. 7 2. 1 19,15,1 6 TA9 8 W a TKA4036 5

(CG I 725 )

DMS O Untreate d Contro l 3p g 38. 0 6. 2 1. 0 43,40,3 1 1 0 p g 36. 0 9. 5 1. 0 47,30,3 1 3 3 p g 36. 3 8. 0 1. 0 28,37,4 4 10 0 p g 35. 3 2. 5 0. 9 33,35,3 8 33 3 p g 37. 0 7. 0 1. 0 30,44,3 7 100 0 p g 41. 3 6. 5 1. 1 48,35,4 1 250 0 p g 29. 0 5. 3 0. 8 2 7 P M , 2 5 P M , 3 5 P M 500 0 p g 28. 3 4. 2 0. 8 3 3 P M , 2 7 P M , 2 5 P M

37. 7 13. 7 44,22,4 7 44. 0 13. 1 38,35,5 9
TA10 0 TKA4036 5

(CG I 725 )

DMS O Untreate d Contro l 3p g 144. 3 13. 3 0. 9 159,141,13 3 1 0 p g 163. 3 25. 5 1. 0 192 , 155 , 14 3 3 3 p g 149. 3 10. 2 0. 9 145,161,14 2 10 0 p g 163. 0 30. 5 1. 0 193,164 , 132 33 3 p g 149. 0 23. 5 0. 9 176,133 , 13 8 100 0 p g 133. 3 18. 9 0. 8 112,140,14 8 250 0 p g 133. 0 9. 2 0. 8 135 P M , 12 3 P M , 14 1 P M 500 0 p g 133. 3 8. 0 0. 8 12 5 P M , 134 P M , 14 1 P M

158. 3 12. 2 145 , 161 , 16 9 755. 7 15. 8 152 , 173 , 142 Ke yt o Plat e Postfi xCode s P Precipitat e M Manua l coun t

s4c2dr.do cRCC-CC R Stud yNumbe r

TK A4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 2 7 o f3 1

stud yName : 104030 0 Experiment : 104030 0 W * Wa Plat e Assa yConditions : Wit h metaboli cactivatio n stud yCode : RCC-CC R 104030 0 Dat e Plated : 01/08/200 6 / 08/08/200 6 Dat e Counted : 04/08/200 6 /14/08/200 6
Strai n

WP2uvr A

TA I 53 5 TA I 53 7 TA98(Wa ) TA10 0 WP 2 uvr A Keyt o Positiv e Compoun d

TK A4036 5 (CG I 725 )

DMS O Untreate d Contro l
2-AA 2-AA 2-AA 2-AA 2-AA Control s Dos e leve l pe rplat e 3p g 1 0 p g 3 3 p g 10 0 p g 33 3 p g 100 0 p g 250 0 p g 500 0 p g

2. 5 p g 2. 5 p g 2. 5 p g 2. 5 p g 10. 0 p g Mea n

revertant s pe rplat e 64. 7 61. 3 57. 0 63. 7 61. 7 64. 3 55. 3 48. 3 58. 3 59. 0
410. 7 471. 0 2926. 3 3419. 3 251. 3 Standar d

Deviatio n

5. 1 3. 5 5. 0 7. 8 9. 1 2. 5 4. 6 4. 0 7. 8 8. 7
21. 1 30. 3 154. 9 179. 2 26. 1 Rati o

treate d / solven t 1. 1 1. 1 1. 0 1. 1 1. 1 1. 1 0. 9 0. 8

22. 4 23. 6 77. 7 21. 6 4. 3 Individua l revertan t colon ycount s
69 , 59 , 6 6 61,58,6 5 62 , 52 , 5 7 70 , 66 , 5 5 72 , 55 , 5 8 64 , 62 , 6 7 5 8 P M , 5 8 P M , 5 0 P M 5 2 P M , 4 9 P M , 4 4 P M 52 , 56 , 6 7 49 , 64 , 6 4 408,433 , 39 1 461,505,44 7 3048,2979,275 2 3452 , 3226 , 358 0 222.260,27 2 Keyt o Plat e Postfi xCode s 2-A A 2-aminoanthracen e P M Precipitat e Manua l coun t

s4c2dr.do cRCC-CC R Stud yNumbe r

TK A4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 2 8 o f3 1

stud yName : 104030 0 Experiment : 104030 0 HV2 Pr e Assa yConditions : Stud yCode : RCC-CC R 104030 0 Dat e Plated : 08/08/200 6 Dat e Counted : 14/08/200 6 Withou t metaboli cactivatio n Strai n Compoun d Dos e leve l pe rplat e Mea n

revertant s pe rplat e Standard

Deviatio n Rati o

treate d / solven t Individua l revertan t

colon ycount s
T A I 53 5 TKA4036 5

(CG I 725 )

DMS O Untreate d Contro l 3 3 p g 18. 7 5. 8 0. 8 22,12,2 2 10 0 p g 20. 3 8. 1 0. 9 29,19,1 3 33 3 p g 18. 7 7. 6 0. 8 10,24,2 2 100 0 p g 19. 3 3. 5 0. 8 19,16,2 3 250 0 p g 21. 0 7. 9 0. 9 12P , 27P,24 P 500 0 p g 20. 7 3. 1 0. 9 2 0 P M , 2 4 P M , 1 8 P M

23. 0 2. 6 21,22,2 6 24. 0 5. 3 20,22,3 0 TA153 7 TKA4036 5

(CG I 725 )

DMS O Untreate d Contro l 3 3 p g 24. 0 6. 9 1. 6 28 , 16.2 8 10 0 p g
. 16. 3 1 2 , 1. 1 17,17,1 5 33 3 p g 17. 0 1. 7 1. 2 16,16,1 9 100 0 p g 19. 0 3. 5 1. 3 17,17,2 3 250 0 p g 18. 7 4. 6 1. 3 1 6 P , 1 6 P , 2 4 P 500 0 p g 20. 0 5. 6 1. 4 1 4 P M , 2 5 P M , 2 1 P M

14. 7 1. 5 16 , 13 , 1 5 18. 7 4. 0 21,14,2 1 TA9 8 TKA4036 5

(CG I 725 )

DMS O Untreate d Contro l 3 3 p g 35. 0 5. 0 1. 3 40,30,3 5 10 0 p g 32. 0 7. 2 1. 2 30,26,4 0 33 3 p g 37. 7 5. 7 1. 4 33,36,4 4 100 0 p g 34. 3 5. 5 1. 3 38,37,2 8 250 0 p g 30. 7 2. 5 1. 1 3 3 P , 2 8 P , 3 1 P 500 0 p g 29. 0 1. 0 1. 1 2 8 P M , 3 0 P M , 2 9 P M

27. 0 3. 5 29,23,2 9 36. 0 5. 6 41,30,3 7 TAIO O TKA4036 5

(CG I 725 )

DMS O Untreate d Contro l 3 3 p g 148. 0 12. 8 1. 1 145,162,13 7 10 0 p g 138. 0 9. 0 1. 0 147,129,13 8 33 3 p g 728. 3 14. 4 0. 9 120,120 , 14 5 100 0 p g 151 7 15. 3 1. 1 165,135,15 5 250 0 p g 143. 3 6. 7 1. 1 14 5 P , 14 9 P , 13 6 P 500 0 p g 127. 7 11. 4 0. 9 13 7 P M , 11 5 P M , 13 1 P M

136. 3 16. 6 134 , 154 , 12 1 146. 0 14. 7 129 , 155 , 154 WP2uvr ATKA4036 5

(CG I 725 )

DMS O Untreate d Contro l 3 3 p g 62. 0 15. 7 1. 0 44,69,7 3 10 0 p g 63. 3 7. 5 1. 0 63,71,5 6 33 3 p g 61 3 4. 0 1. 0 57,62,6 5 100 0 p g 64. 0 1 0 1 0 64,63,6 5 250 0 p g 58. 3 9. 3 0. 9 6 6 P , 6 1 P , 4 8 P 500 0 p g 53. 0 6. 2 0. 9 5 1 P M , 4 8 P M , 6 0 P M

62. 3 7. 5 70,62,5 5 61 7 7. 5 69,54,6 2 Keyt o Plat e Postfi xCode s P M Precipitat e Manua l coun t

s4c2dr.do cRCC-CC R Stud yNumbe r

TKA4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 2 9 o f3 1

Stud yName : Experiment: ' Assa yCondi t
Strai n

TA153 5 TA153 7 TA9 8 TAIO O WP 2 uvr A Ke yt o Positi v 104030 0

104030 0 HV2 Pre ions : Compoun d

NaN 3 4-NOP D 4-NOP D NaN 3 MM S l e Control s Dos e leve l pe rplat e

1 0 p g 5 0 p g 1 0 p g 1 0 p g 4. 0 p L Withou t n

Mea n revertant s pe rplat e 1409. 3 146. 7 620. 0 2153. 3 591. 0 letaboli cac t

Standard Deviatio n

70. 0 6. 4 22. 6 12. 5 63. 6 ivatio n

Rati o treate d / solven t 61 3 10. 0 23. 0 15. 8 9. 5 Stud yCode : RCC-CC R 104030 0

Dat e Plated : 08/08/200 6 Dat e Counted : 14/08/200 6
Individua l revertan t colon ycount s

1479 , 1410 , 133 9 142 , 144,15 4 596 , 623 , 64 1 2153,2141,216 6 581,533,65 9

NaN3

. sodiu m azid e 4-NOP D 4-nitro-o-phenylene-diamin e MM S methy l methan e sulfonat e

s4c2dr.do cRCC-CC R Stud yNumbe r

TK A4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 3 0 o f3 1

stud yName : 104030 0 Experiment : 104030 0 HV2 Pre Assa yConditions : Stud yCode : RCC-CC R 104030 0 Dat e Plated : 08/08/200 6 Dat e Counted : 14/08/200 6 Wit h metaboli cactivatio n Strai n Compoun d Dos e leve l pe rplat e Mea n

revertant s pe rplat e Standar d

Deviatio n Rati o

treate d / solven t Individua l revertan t

colon ycount s
TA153 5 TK A4036 5

(CG I 725 )

DMS O Untreate d Contro l 3 3 p g 21 3 4. 0 0. 9 19,19,2 6 10 0 p g 24. 3 4. 9 1. 1 22,21,3 0 33 3 p g 27. 7 8. 1 1 2 19,35,2 9 100 0 p g 22. 3 4. 0 1. 0 27,20,2 0 250 0 p g 21 7 2. 1 1. 0 2 4 P M , 2 0 P M , 2 1 P M 500 0 p g 20. 0 3: 5 0. 9 2 4 P M , 1 8 P M , 1 8 P M

22. 7 1. 2 24,22,2 2 22. 0 4. 4 27,20,1 9 TA153 7 TK A4036 5

(CG I 725 )

DMS O Untreate d Contro l 3 3 p g 23. 3 2. 5 1. 0 26,23,2 1 10 0 p g 17. 3 3. 8 0. 7 13,19,2 0 33 3 p g 22. 7 4. 0 0. 9 22,19,2 7 100 0 p g 24. 3 3. 5 1. 0 21,24,2 8 250 0 p g 19. 3 4. 2 0. 8 1 8 P M , 1 6 P M , 2 4 P M 500 0 p g 19. 3 4. 7 0. 8 2 3 P M

. 1 4 P M , 2 1 P M

24. 3 2. 3 23.23,2 7 25. 3 11. 1 37,24 , 1 5 TA9 8 TK A4036 5

(CG I 725 )

DMS O Untreate d Contro l 3 3 p g 36. 3 10. 1 0. 9 27,47,3 5 10 0 p g 45. 0 5. 3 1. 1 41,51,4 3 33 3 p g 41 0 3. 6 1 0 42,37,4 4 100 0 p g 41 0 14. 5 1. 0 55,42,2 6 250 0 p g 33. 7 3. 1 0. 8 3 3 P M , 3 1 P M , 3 7 P M 500 0 p g 34. 3 4. 2 0. 9 3 1 P M , 3 3 P M , 3 9 P M

39. 7 6. 1 33,45,4 1 44. 7 6. 8 37,47,5 0 TAIO O TK A4036 5

(CG I 725 )

DMS O Untreate d Contro l 3 3 p g 179. 3 9. 3 1. 0 173,190,17 5 10 0 p g 189. 3 4. 0 1. 1 190,193,18 5 33 3 p g 180. 3 7. 2 1. 0 172,185,18 4 100 0 p g 173. 7 27. 8 1. 0 199,178,14 4 250 0 p g 150 7 14. 2 0. 9 16 7 P M

. 144 P M , 14 1 P M 500 0 p g 152. 3 4. 5 0. 9 152 P M , 148 P M , 15 7 P M

174. 7 17. 2 169,194,16 1 183. 0 21. 2 159,199 , 19 1 WP2uvr ATK A4036 5

(CG I 725 )

DMS O Untreate d Contro l 3 3 p g 69. 7 5. 1 0. 9 64,74,7 1 10 0 p g 73. 3 7. 0 1. 0 80,66,7 4 33 3 p g 76. 7 14. 0 1. 0 61,81,8 8 100 0 p g 75. 0 7. 9 1. 0 72,69.8 4 250 0 p g 61 3 6. 0 0. 8 6 2 P M , 5 5 P M , 6 7 P M 500 0 p g 71 0 1 7 0. 9 7 2 P M , 6 9 P M , 7 2 P M

75. 7 4. 6 73,81,7 3 72. 7 10. 6 71,84,6 3 Keyt o Plat e Postfi xCode s P M Precipitat e Manua l coun t

s4c2dr.do cRCC-CC R Stud yNumbe r

TK A4036 5 (CG I 725 ) 104030 0 Fina l Repor t Pag e 3 1 o f3 1

stud yName : 104030 0 Experiment : 104030 0 HV2 Pre Assa yConditions : Wit h metaboli cactivatio n Stud yCode : RCC-CC R 104030 0 Dat e Plated : 08/08/200 6 Dat e Counted : 14/08/200 6
Strai n Compoun d Dos e leve l pe rplat e Mea n

revertant s pe rplat e Standard

Deviatio n Rati o

treate d / solven t Individua l revertan t

colon ycount s

T A I 53 5 T A I 53 7 TA9 8 TAIO O WP 2 uvr A 2-AA

2-AA 2-AA 2-AA 2-AA 2. 5 p g 2. 5 p g 2. 5 p g 2. 5 p g 10. 0 p g 393. 3

394. 7 2587. 0 3990. 7 255. 0 26. 3

25. 5 376. 6 163. 9 45. 6 17. 4

16. 2 65. 2 22. 8 3. 4 410,407 , 36 3 370,421,39 3 2172 , 2682 , 290 7 3880,3913,417 9 241,218,30 6
Keyt o Positiv e Control s 2-AA 2-aminoanthracen e

s4c2dr.do c