RCC - CCR STUDY NUMBER 1040300 

SALMONELLA TYPHIMURIUM 

AND 

ESCHERICHIA COLI 

REVERSE MUTATION ASSAY 

WITH 

TKA 40365 (CGI 725) 

FINAL REPORT 

STUDY COMPLETION DATE: 

SEPTEMBER 01, 2006 
RCC-CCR Study Number 1040300 

TKA 40365 (CGI 725) 

Final Report Page 2 of 31 

1 COPY OF GLP CERTIFICATE 

Hessisches Ministerium fur Umweit, 

ISndlichen Raum und Verbraucherschutz 

HESSEN 

Gute Laboipiaxis/Good LaboraUny Practice 

GLP-BescheinlgoDg/Statement (rfGLP Crnnpliance 

(gemSfi/according to § 19b Abs. 1 Chemikaliengesetz) 

Eine Gl-P-Inq)ejction zur Obenvachung der Einhaltung 

der GLP-Gnindsatze gemaO Chemikaliengesetz bsnv. 

Richtlinie 88/320/EG wurde dnrchgefiJhrt in 

Assessment of conformity with CHJ? according to 

Chemikaliengesetz and Directive 88/320/EEC at: 

E PrflfeinrichtungTest fecillity nPritrstandDrtTcst site 

RCC Cytotest CeU Research GmbH 

RCC Cytotest Cell Research GmbH 

In den Leppsteinwiesen 19 

64380 RoBdorf 

(Utrvcm'cchselbare Bezcichmmg und Adresse/Unequivocal name and adress) 

Prnfinigen nach Kategorien/Areas of Expertise 

(gemSB/according cheniVwV-GLP Nr. 5 J/OECD guidance) 

2 PraftinBen xiir Besttminung der toxlkologischen 

&igenscha5ten 

3 PriUUngen zttr Bcstimmune der erbgutveranderaden 

Elgenschaften (fn vitro und tn vivo) 

8 Analytisctie PnCiTungen an biologischen Materialien 

9 VirusslcherhcitsprOrungen 

a Toxicity studies 

3 Mutagenicity studies 

S Analytical studies on blolo^cal materials 

9 Virus validation studies -

03.06.; 19.07.-22.07 J004 

Datiun der Inspektion/Date of In^xction 

(Tag Monat }ahi/day month year) 

Die genaimte PrOfeinriclitung befiium sich im nationalen GLP-Oberwacbimgsverfahien und wird tcgeloiSQig auf Einhaltung der (H..P-GTundsatze flberwacht. 

Auf der Grundiage des In^jcktionsbericfates wird hiennit 

bestaiigu dass in dieser PrOfeinrichtimg die oben genannten Prfiiimgen tmter Einhaltung der GLP- GnmdsStze Anchgefiilm werden kOnnea 

The above mentioned test facility is included 

in tite n^tion^ GLP Compliance Progtaimne and is 

inspected on a tegular basis. 

Based on the inspection report it can be conSmied, 

that this test facili^ is M>Ie to conduct the 

aforementioned studies in compliance with the 

Principles of GLP. 

Th. 

Im Auftrag h. 

Referent, Wiesbaden, den Wiesbaden, den 06. Januar 

(Name und Ftmktion der veranrwortlicben Person/ 

Name and fiinctian of responsible person) 

Hess. Ministerium fur Umweit, landKcben Raura and Verforaucherscbntz, 

Mainzer Strafie 801>65)89 Wiesbaden 

(Name und Adresse der GLP-t}berwachungsbefaOrde/Kaine and address of the GLP Monitotiitg Aulhori^') 

[>eS18a\Aflesb8den, Mainzer StraOe 80 

Telefor: OS11.81 50 

Telefax: 0611.81 61 941 

E-Mail: poststetle@hnmtv.hes3en.de 

0-65187 Wiesbaden, MIderilnstraae 1-3 

Telefon: 0611. 81 70 

Telefax: 0611,81 7218 1 

st4ecdr.doc 
RCC-CCR Study Number 1040300 Final Report Page 3 of 31 

TKA 40365 (CGI 725) 

2 CONTENTS 

1 COPY OF GLP CERTIFICATE 2 

2 CONTENTS 3 

3 PREFACE 4 

3.1 General 4 

3.2 Responsibilities 4 

3.3 Schedule 4 

3.4 Project Staff Signatures 5 

3.5 Good Laboratory Practice 5 

3.6 Guidelines 5 

3.7 Archiving 6 

3.8 Deviations to Study Plan 6 

4 STATEMENT OF COMPLIANCE 7 

5 STATEMENT OF QUALITY ASSURANCE UNIT 8 

6 SUMMARY OF RESULTS 9 

6.1 Conclusion 9 

7 OBJECTIVE 10 

7.1 Aims of the Study 10 

7.2 Reasons for the Study 10 

8 MATERIALS AND METHODS 11 

8.1 Test Item 11 

8.2 Controls 12 

8.3 Test System 13 

8.4 Mammalian Microsomal Fraction S9 Mix 14 

8.5 Pre-Experiment for Toxicity 15 

8.6 Dose Selection 16 

8.7 Experimental Performance 16 

8.8 Data Recording 16 

8.9 Acceptability of the Assay 17 

8.10 Evaluation of Results 17 

8.11 Biometry 17 

9 DISCUSSION OF RESULTS 18 

10 REFERENCES 19 

11 DISTRIBUTION OF THE REPORT 19 

12 SUMMARY OF RESULTS 20 

12.1 Summary of Results Pre-Experiment and Experiment I 20 

12.2 Summary of Results Experiment II 21 

12.3 HISTORICAL CONTROL DATA 22 

13 ANNEX: TABLES OF RESULTS (8 PAGES) 23 

s4c2dr.doc 
RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 4 of 31 

3 PREFACE 

3.1 General 

Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

Contracting Institute: 

Reference Number: 

Salmonella typhimurium and Escherichia coli 

Reverse Mutation Assay with TKA 40365 (CGI 725) 

Ciba Specialty Chemicals Inc. 

Segment CE 

4002 Basel 

Switzeriand 

Dr. Roland Hammeri, WRO-1045.1.39 

RCC 

Cytotest Cell Research GmbH (RCC-CCR) 

In den Leppsteinswiesen 19 

64380 Rossdorf 

Germany 

R C C Ltd 

4452 Itingen 

Switzeriand 

A87873 

3.2 Responsibilities 

Study Director: 

Deputy Study Director: 

Management: 

Head of Quality Assurance Unit: 

Dipl. Biol. Andrea Sokolowski 

Dr. Hans-Eric Wollny 

Dr. Wolfgang Volkner 

Frauke Hermann 

3.3 Schedule 

Date of the Study Plan: 

Experimental Starting Date: 

Experimental Completion Date: 

Date of Draft Report: 

Date of Final Report: 

July 

August 

August 

August 

September 

26, 2006 

01.2006 

14, 2006 

18,2006 

01,2006 

s4c2dr.doc 
RCC-CCR study Number 1040300 

fkA4d36i6(CGi;725) 

Final Reports Page;5off3j1 

3.4 Projeictj^aff*Signsture§ 

study Director Dipl. Biol; Andrea Sokolowski 

Management 

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

Dr; Wolfgang Volkner 

Date: Septemtier'dl, SOGje' 

;315' 6^pCi|ab^iiitdiV''Prac|i(:^5 

Ttie stbdy was peri^oim^f in c»m 

"Chemikaliengesetz" (Chemicals Act) of the Federal Republic of Gemtiany; ^'Anhangii*' 

^^tntm: 1;); dated JulyS25i 1,994 CBQfel. I; 1994", pp. W3). last revision datedyuHe 27; 

io|2 

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

^^ t GiJidellheis 

this stady/fonbvyedtigcprote^^ 

'd0|nesi^a:rgGomm ...^.-...-..^.•....-..-^ ^....-..-.,...-.-..^- .-- > 

llsliint|iit^dendum to; fiEiCD^Quidellpes ifor testihgs<in(5fiemiGals",. Section! %. No^^;4^fk 

'^BacteriatReyerse MbySon te^t | adqf^cf3u^;21^^ 

^^G^myiprt Bir^tlveiopoM^ 1^^362000; ^^nex 4D'!, laated May^ 9, ;2p0p' 

^panesg Guidejinie^; ^ ^ 

;?Kanp,oan%;M7r^j?fEh\^ironm6nfiProfe^^^ 

^EiseiiNo; 127—K/!inistry.bf Health «W&l^re" 

!Heisei;09/10/33;;Kikj^u j$|Q.)2<-

;i4c2clr;cloc 
RCC-CCR Study Number 1040300 Final Report Page 6 of 31 

TKA 40365 (CGI 725) 

3.7 Archiving 

RCC Cytotest Ceil Research GmbH will archive the following data for 15 years: 

Raw data, study plan, final report, and a sample of the test item. 

No data will be discarded without the sponsor's consent. 

3.8 Deviations to Study Plan 

There were no deviations to study plan. 

s4c2dr.doc 
RCC-GGFtSfudyJNumber IMBOO" jfinal Report I PageZof^! 

fKk7&0:l0s7T25)i I 

4 ST^EMENT OF CdMPyANil 

StudypNumbeni iM)^C©> 

Te^t llemi:: tl^^40365|eGl 7251 

Studycpirectpff DipLBjoi; /Pufidrea Sbkolovi^ki i 

Title: SaliTioiiella t^himuriumland Escherichia coli 

Fleverse Mutation Assay with TKX 40365 (CGI 725) 

Thi&studvg^rtoirmi&Jl^i 

cx>mpiiancewithiGoodiyboratdryi?ra^ 

"Gher^'ikaliengesdtz'^ (Chemicals Act| of the Federal Republic ofiQennany; ^Knhang 1"^ 

(Annex 1<)Vdat^d July 25, 1994 ("BGBI, 1] 1994^ pp. 1703), list revision Sated June 27. 

'2002., 5 • 

"OECD Principles of Qopd Laboratory Practice", as revised ih 1997 5iC(97)186/Final] 

There were no circums^pe^^fkM^YihayeCa^^ 

©iifctpri R:;e^.--jS^GJ^-

Dipi: BipLAndVea Spkplowski 

.?^i..rr^ 

ba^'?September:p^;, 2006 

f 

c 

s4c2dr.cl6c! 
i ! 

RCe^GCR Study Number 1040300 

ffes^!^[m5:(C(3l^5)f' ' 

Fina! Report Page8trf31 

f SimlMENT QFHGIUAUTY iSSUI^ ^ UNIT 

StiJdy^^NurnbeK 1(^300 

Test Item: TK^40365 (CQI 725f. 

StudyitDirectbr:: DipLi^ipt: Andrea Sokolowski 

Titles Salrripriella typhimurium arid Eschericffla cpli 

Reverse Mutation Assay with TKA 40365.(0^ 725) 

Thegeoerarjacilitifes arid^ Ceil Research GmbM are inspected 

penodiGaHi(:andlhe results are repprted;tO;fte^^^ 

^tudy procedures were ihspeeWd periodically. Theistudy plan and this ;repprt were audited 

•byitheyQusiity.AssurariceJUn i( • 

P|iesesiand;D"ates:6f 

^Study?Pian;i(braft); 

jStudi^FI'lan: 

Process Inspection: 

(DbftiRepdrt: 

QAU Inspections/Audits 

^iftjif" 2-i,^'2006r ••': 

Jiily 27V2006 

pigu|t "In?- 2tl()^; ^" _' .^ 

;>a^^'3^[g006r"'' '•" ' 

Dates of FlBppftgstpiOieS^ 

Director andto i^anagemerit 

jjjiy^;";2;i^,;2{i|; ;••/;;•;• '"ZZ2 _ 

' 'yz'".""".%. rz. "Z[ 

V\ugustM200i 3 

sAugu|t^2p2pO^'y''1 2 \2^'_" 

^fe^etatemjen^ pj^serit^flrialireppr^^ 

;i|ead;ipfMii)ity^sij^eiiit!inifc Ri'auke^MenDann 

I I 

..y 'i7.K-,. J V^V .^ii^/iv^rTxlT 

vD^=^5§iptembeir^li^liGi6 

.s4c2dr.d6c-
RCC-CCR Study Number 1040300 Final Report; Page 9 of 31 

TKA 40365 (CGI 725) 

6 SUMMARY OF RESULTS 

This study was performed to investigate the potential of TKA 40365 (CGI 725) to induce 

gene mutations in the plate incorporation test (experiment I) and the pre-incubation test 

(experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA98, and 

TA 100, and the Escherichia coli strain WP2 uvrA. 

The assay was performed with and without liver microsomal activation. Each 

concentration, including the controls, was tested in triplicate. The test item was tested at 

the following concentrations: 

Pre-Experiment/Experiment I and la: 3; 10; 33; 100; 333; 1000; 2500; and 5000 pg/plate 

Experiment II: 33; 100; 333; 1000; 2500; and 5000 pg/plate 

The plates incubated with the test item showed normal background growth up to 

5000 pg/plate with and without metabolic activation in both independent experiments. 

No toxic effects, evident as a reduction in the number of revertants, occurred in the test 

groups with and without metabolic activation. 

No substantial increase in revertant colony numbers of any of the five tester strains was 

observed following treatment with TKA 40365 (CGI 725) at any dose level, neither in the 

presence nor absence of metabolic activation (S9 mix). There was also no tendency of 

higher mutation rates with increasing concentrations in the range below the generally 

acknowledged border of biological relevance. 

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. 

6.1 Conclusion 

In conclusion, it can be stated that during the described mutagenicity test and under the 

experimental conditions reported, the test item did not induce gene mutations by base pair 

changes or frameshifts in the genome of the strains used. 

Therefore, TKA 40365 (CGI 725) is considered to be non-mutagenic in this Salmonella 

typhimurium and Escherichia coli reverse mutation assay. 

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RCC-CCR Study Number 1040300 Final Report Page 10 of 31 

TKA 40365 (CGI 725) 

7 OBJECTIVE 

7.1 Aims of the Study 

The experiments were performed to assess the potential of the test item to induce gene 

mutations by means of two independent Salmonella typhimurium and Escherichia coli 

reverse mutation assays. Experiment I was performed as a plate incorporation assay. 

Since a negative result was obtained in this experiment, experiment II was performed as a 

pre-incubation assay. 

7.2 Reasons for the Study 

The most widely used assays for detecting gene mutations are those using bacteria (3). 

They are relatively simple and rapid to perform, and give reliable data on the ability of an 

agent to interact with DNA and produce mutations. 

Reverse mutation assays determine the frequency with which an agent reverses or 

suppresses the effect of the forward mutation. The genetic target presented to an agent is 

therefore small, specific and selective. Several bacterial strains, or a single strain with 

multiple markers are necessary to overcome the effects of mutagen specificity. The 

reversion of bacteria from growth-dependence on a jjarticular amino acid to growth in the 

absence of that amino acid (reversion from auxotrophy to prototrophy) is the most widely 

used marker. 

The Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his' -> his* and trp" -^ trp* reversions, respectively. The S. typhimurium 

and Escherichia coli strains are constructed to differentiate between base pair (TA 1535, 

TA 100, and WP2 uvrA) and frameshift (TA 1537, TA 98) mutations. 

According to the direct plate incorporation or the pre-incubation method the bacteria are 

exposed to the test item with and without metabolic activation and plated on selective 

medium. After a suitable period of incubation, revertant colonies are counted. 

To establish a dose response effect at least 6 dose levels with adequately spaced intervals 

were tested. The maximum dose level was 5000 pg/plate. 

To validate the test, reference mutagens were tested in parallel to the test item. 

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RCC-CCR Study Number 1040300 

TKA 40365 (CGI 725) 

Final Report Page 11 of 31 

8 MATERIALS AND METHODS 

8.1 Test Item 

Internal RCC-CCR Test Item Number: S6651 11 

The test item and the information concerning the test item were provided by the 

sponsor. 

Identity: 

Batch No.: 

Aggregate state at 

room temperature: 

Colour: 

Purity: 

Stability in solvent: 

Storage: 

Expiration Date: 

TKA 40365 (CGI 725) 

30073 FC6AD 

Solid 

Yellow 

Confidential information 

(see analytical certificate of July 20, 2006) 

Not indicated by the sponsor 

In the refrigerator at 2° - 8° C 

July 01, 2008 

On the day of the experiment, the test item TKA 40365 (CGI 725) was dissolved in DMSO 

(MERCK, D-64293 Darmstadt; purity > 99 %). The solvent was chosen because of its 

solubility properties and its relative non-toxicity to the bacteria (5). 

The test item precipitated in the overiay agar at 2500 and 5000 pg/plate with and without 

metabolic activation in both experiments. The undissolved particles had no influence on 

the data recording. 

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RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 12 of 31 

8.2 Controls 

8.2.1 Negative Controls 

Concurrent untreated and solvent controls were performed. 

8.2.2 Positive Control Substances 

Without metabolic activation 

Strains: 

Name: 

Supplier: 

Catalogue No.: 

Purity: 

Dissolved in: 

Concentrafion: 

Strains: 

Name: 

Supplier: 

Catalogue No.: 

Purity: 

Dissolved in: 

Concentrafion: 

Strain: 

Name: 

Supplier: 

Catalogue No.: 

Purity: 

Dissolved in: 

Concentration: 

With metabolic activation 

Strains: 

Name: 

Supplier: 

Catalogue No.: 

Purity: 

Dissolved in: 

Concentrafion: 

TA 1535, TAIOO 

sodium azide, NaNs 

SERVA, D-69042 Heidelberg 

30175 

at least 99 % 

water deionised 

10 pg/plate 

TA1537, TA98 

4-nitro-o-phenylene-diamine, 4-NOPD 

SIGMA, D-82041 Deisenhofen 

N 9504 

>99.9% . 

DMSO (MERCK, D-64293 Darmstadt; purity > 99 %) 

10 pg/plate in TA 98, 50 pg/plate in TA 1537 

WP2 uvrA 

methyl methane sulfonate, MMS 

Merck-Schuchardt, D-85662 Hohenbrunn 

820775 

> 99.0 % 

water deionised 

3 pL/plate 

TA 1535. TA 1537, TA 98, TA 100, WP2 uvrA 

2-aminoanthracene, 2-AA 

SIGMA, D-82041 Deisenhofen 

A 1381 

97.5 % 

DMSO (MERCK, D-64293 Darmstadt; purity > 99 %) 

2.5 pg/plate (TA 1535, TA 1537, TA 98, TA 100), 

10 pg/plate (WP2 uvrA) 

The stability of the posifive control substances in solufion is unknown but a mutagenic 

response in the expected range will be sufficient evidence of biological stability. 

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RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 13 of 31 

8.3 Test System 

8.3.1 Characterisation of the Salmonella typhimuriu m Strains and E. coi l 

Strain 

The hisfidine dependent strains are derived from S. typhimurium strain LT2 through a 

mutafion in the hisfidine locus. Additionally due to the "deep rough" (rfa-minus) mutafion 

they possess a faulty lipopolysaccharide envelope which enables substances to penetrate 

the cell wall more easily. A further mutation causes a reducfion in the activity of an excision 

repair system. The latter alterafion includes mutational processes in the nitrate reductase 

and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the 

strains TA98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance 

marker (6). 

Strain WP2 (4) and its derivatives all carry the same defect in one of the genes for 

tryptophan biosynthesis. Tryptophan-independent (Trp*) mutants (revertants) can arise 

either by a base change at the site of the original alteration or by a base change elsewhere 

in the chromosome so that the original defect is suppressed. This second possibility can 

occur in several different ways so that the system seems capable of detecfing all types of 

mutagen which subsfitute one base for another. Additionally, the uvrA derivafive is 

deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain 

may be more readily mutated by agents. 

When summarised the mutafions of the TA strains and the E. coli strain, used in this study 

can be described as follows: 

Strains 

TA1537 

TA98 

TA1535 

TAIOO 

WP2 uvrA 

Salmonella typhimurii 

Genotype 

his C 3076; rfa"; uvrB": 

his D 3052; rfa"; uvrB";R-factor 

his G 46; rfa"; uvrB": 

his G 46; rfa"; uvrB";R-factor 

Escherichia coli 

trp"; uvrA": 

j m 

Type of mutations indicated 

frame shift mutations 

n n 

base-pair substitutions 

II n 

base-pair substitutions and others 

Regular checking of the properties of the strains regarding the membrane permeability and 

ampicillin resistance as well as spontaneous mutafion rates is performed in RCC Cytotest 

Cell Research according to B. Ames et al. (1) and D. Maron and B. Ames (6). In this way it 

was ensured that the experimental condifions set down by Ames were fulfilled. 

The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from 

Trinova Biochem GmbH (35394 Gielien, Germany). 

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RCC-CCR study Number 1040300 Final Report Page 14 of 31 

TKA 40365 (CGI 725) 

8.3.2 Storage 

The strain cultures were stored as stock cultures in ampoules with nutrient broth 

+ 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen. 

8.3.3 Precuitures 

From the thawed ampoules of the strains 0.5 mL suspension was transferred into 250 mL 

Erienmeyer flasks containing 20 mL nutrient medium. A solufion of 20 pL ampicillin 

(25 pg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per 

litre: 

8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt) 

5 g NaCl (MERCK, D-64293 Darmstadt) 

The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C. 

8.3.4 Selective Agar 

The plates with the selective agar were obtained from E. Merck, D-64293 Darmstadt. 

8.3.5 Overlay Agar 

The pvpriay agar contains per litre: 

for Salmonella strains: for Escherichia coli: 

6.0 g MERCK Agar Agar* 6.0 g MERCK Agar Agar* 

6.0 g NaCl* 6.0 g NaCl* 

10.5mg L-HistidinexHCIxH20* 2.5 mg Tryptophan* 

12.2mg Biofin* 

* (MERCK. D-64293 Dannstadt) 

Sterilisafions were performed at 121° C in an autoclave. 

8.4 Mammalian Microsomal Fraction S9 Mix 

The bacteria used in this assay do not possess the enzyme systems which, in mammals, 

are known to convert promutagens into acfive DNA damaging metabolites. In order to 

overcome this major drawback an exogenous metabolic system is added in form of 

mammalian microsome enzyme activafion mixture. 

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RCC-CCR study Number 1040300 Final Report Page 15 of 31 

TKA 40365 (CGI 725) 

8.4.1 S9 (Preparation by R C C - C C R) 

Phenobarbital/p-Naphthoflavone induced rat liver S9 is used as the metabolic activation 

system. The S9 is prepared from 8-1 2 weeks old male Wistar Hanlbm rats, weight 

approx. 220 - 320 g induced by applicafions of 80 mg/kg b.w. Phenobarbital i.p. (Desifin; 

D-22335 Hamburg) and p-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three 

consecutive days. The livers are prepared 24 hours after the last treatment. The S9 

fracfions are produced by dilution of the liver homogenate with a KCl isolufion (1+3) 

followed by centrifugafion at 9000 g. Aliquotes of the supematant are frozen and stored in 

ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one 

week. Each batch of S9 mix is roufinely tested with 2-aminoanthracene as well as 

benzo(a)pyrene. 

The protein concentrafion in the S9 preparafion was 31.1 mg/mL (lot no. R 050506). 

8.4.2 S9MIX 

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed 

with S9 co-factor solufion. The amount of S9 supernatant was 10% v/v in the S9 mix. 

Cofactors are added to the S9 mix to reach the following concentrafions in the S9 mix: 

8mM MgCl2 

33 mM KCl 

5 mM Glucose-6-phosphate 

5mM NADP 

in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. 

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparafion was 

performed according to Ames et al.(1). 

8.5 Pre-Experiment for Toxicity 

To evaluate the toxicity of the test item a pre-experiment was performed with strains 

TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrafions were tested for 

toxicity and mutation induction with three plates each. The experimental condifions in this 

pre-experiment were the same as described below for the experiment I (plate incorporation 

test). 

Toxicity of the test item results in a reduction in the number of spontaneous revertants or a 

clearing ofthe bacterial background lawn. 

The pre-experiment is reported as main experiment I, if the following criteria are met: 

Evaluable plates (>0 colonies) at five concentrafions or more in all strains used. 

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RCC-CCR study Number 1040300 Final Report Page 16 of 31 

TKA 40365 (CGI 725) 

8.6 Dose Selection 

In the pre-experiment the concentration range of the test item was 3 - 5000 pg/plate. The 

pre-experiment is reported as experiment I since no toxic effects were observed and 

5000 pg/plate were chosen as maximal concentrafion. 

The concentration range included two logarithmic decades. The following concentrafions 

were tested: 

Pre-Experiment/Experiment I and la: 3; 10; 33; 100; 333; 1000; 2500; and 5000 pg/plate 

Experiment II: 33; 100; 333; 1000; 2500; and 5000 pg/plate 

8.7 Experimental Performance 

For each strain and dose level including the controls, three plates were used. 

The following materials were mixed in a test tube and poured onto the selecfive agar 

plates: 

100 pL Test solufion at each dose level, solvent (negative control) or reference mutagen 

solufion (posifive control), 

500 pL S9 mix (for test with metabolic acfivation) or S9 mix substitufion buffer (for test 

without metabolic acfivafion), 

100 pL Bacteria suspension (cf. test system, pre-culture of the strains), 

2000 pL Overiay agar 

In the pre-incubafion assay 100 pL test solufion, 500 pL S9 mix / S9 mix substitufion buffer 

and 100 pL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 

minutes. After pre-incubation 2.0 mL overiay agar (45° C) was added to each tube. The 

mixture was poured on selective agar plates. 

After solidificafion the plates were incubated upside down for at least 48 hours at 37° C in 

the dark (2). 

8.8 Data Recording 

The colonies were counted using the Petri Viewer Mk2 (Percepfive Instruments Ltd, Suffolk 

CB 7BN, UK) with the software program Ames Study Manager. The counter was 

connected to an IBM AT compafible PC with printer to print out the individual values and 

the means from the plates for each concentration together with standard deviations and 

enhancement factors as compared to the spontaneous reversion rates (see tables of 

results). Due to precipitation of the test item the revertant colonies were partly counted 

manually. 

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RCC-CCR Study Number 1040300 Final Report Page 17 of 31 

TKA 40365 (CGI 725) 

8.9 Acceptability of the Assay 

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered 

acceptable if it meets the following criteria: 

regular background growth in the negative and solvent control 

the spontaneous reversion rates in the negative and solvent control are in the range of 

our historical data 

the positive control substances should produce a significant increase in mutant colony 

frequencies 

8.10 Evaluation of Results 

A test item is considered as a mutagen if a biologically relevant increase in the number of 

revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or 

thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control 

is observed (3). 

A dose dependent increase is considered biologically relevant if the threshold is exceeded 

at more than one concentrafion (2). 

An increase exceeding the threshold at only one concentration is Judged as biologically 

relevant if reproduced in an independent second experiment. 

A dose dependent increase in the number of revertant colonies below the threshold is 

regarded as an indicafion of a mutagenic potential if reproduced in an independent second 

experiment. However, whenever the colony counts remain within the historical range of 

negative and solvent controls such an increase is not considered biologically relevant. 

8.11 Biometry 

According to the OECD guideline 471, a statistical analysis of the data is not mandatory. 

s4c2dr.doc 
RCC-CCR Study Number 1040300 Final Report Page 18 of 31 

TKA 40365 (CGI 725) 

9 DISCUSSION OF RESULTS 

The test item TKA 40365 (CGI 725) was assessed for its potential to induce gene 

mutafions in the plate incorporafion test (experiment I) and the pre-incubation test 

(experiment II) using Salmonella typhimurium strains TA1535, TA1537, TA98, and 

TA 100, and the Escherichia coli strain WP2 uvrA. 

The assay was performed with and without liver microsomal acfivation. Each concentration 

and the controls were tested in triplicate. Due to irregular background growth, the analysis 

of strain TA 98 was impossible with and without S9 mix in the pre-experiment/experiment I. 

An additional experiment was performed under identical conditions (plate incorporation 

test) using strain TA 98 with and without metabolic activation. The results of this addifional 

experiment are included in this report as pre-experiment/experiment I a (W a). The test 

item was tested at the following concentrafions: 

Pre-Experiment/Experiment I and la: 3; 10; 33; 100; 333; 1000; 2500; and 5000 pg/plate 

Experiment II: 33; 100; 333; 1000; 2500; and 5000 pg/plate 

The plates incubated with the test item showed normal background growth up to 

5000 pg/plate with and without metabolic activafion in both independent experiments. 

No toxic effects, evident as a reducfion in the number of revertants, occurred in the test 

groups with and without metabolic acfivafion. 

No substanfial increase in revertant colony numbers of any of the five tester strains was 

observed following treatment with TKA 40365 (CGI 725) at any dose level, neither in the 

presence nor absence of metabolic activafion (S9 mix). There was also no tendency of 

higher mutafion rates with increasing concentrafions in the range below the generally 

acknowledged border of biological relevance. 

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. 

The laboratory's historical control data was slightly exceeded in the untreated and solvent 

control of strain WP2 uvrA with metabolic acfivafion in experiment II. These minor 

deviafions are judged to be based on biologically irrelevant fluctuations in the number of 

colonies and have no impact on the outcome of the study. 

In conclusion, it can be stated that during the described mutagenicity test and under the 

experimental conditions reported, the test item did not induce gene mutations by base pair 

changes or frameshifts in the genome of the strains used. 

s4c2dr.doc 
RCC-CCR study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 19 of 31 

10 REFERENCES 

1. Ames, B.N., J. McCann, and E. Yamasaki (1977) 

Methods for detecfing carcinogens and mutagens with the Salmonella/mammalian 

microsome mutagenicity test 

In: B.J. Kilbey et al. (Eds.)"Handbook of Mutagenicity Test Procedures" Elsevier, 

Amsterdam, 1-17 

2. de Sen-es F.J. and M.D. Shelby (1979) 

Recommendations on data producfion and analysis using the Salmonella/microsome 

rnutagenicity assay 

Mutation Res. 64, 159-165 

3. Hollstein.M., J. McCann, F.A. Angelosanto and W.W. Nichols (1979) 

Short-term tests for carcinogens and mutagens 

Mutation Res. 65, 133-226 

4. Green, M.H.L. and W.J. Muriel (1976) 

Mutagen Testing Using TRP* Reversion in Escherichia Coli 

Mutation. Res. 38, 3- 32 

5. Maron D.M., J. Katzenellenbogen and B.N. Ames, (1981) 

Compatibility of organic solvents with the Salmonella/Microsome Test 

Mutation Res. 88, 343-350 .< 

6. Maron D.M., Ames, B.N. (1983) 

Revised methods for the Salmonella mutagenicity test 

MutationRes. 113, 173-215 

11 DISTRIBUTION OF THE REPORT 

Sponsor 

Contracting Institute 

Study Director 

3x (copies unbound) 

Ix (electronic copy (pdf)) 

1X (original) 

s4c2dr.doc 
RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 20 of 31 

12 SUMMARY OF RESULTS 

12.1 Summary of Results Pre-Experiment and Experiment I 

study Name: 1040300 

Experiment: 1040300 W + W a Plate 

Assay Conditions: 

Study Code: RCC-CCR 1040300 

Date Plated: 01/08/2006 / 08/08/2006 

Date Counted: 04/08/2006 / 14/08/2006 

Metabolic 

Activation 

Wittiout 

Activation 

Test 

GrouD 

DMSO 

Untreated 

TKA 40365 

(CGI 725) 

NaN3 

4-NOPD 

4-NOPD 

MMS 

Dose Level 

(ua/Dlate) 

3Mg 

10|jg 

33 Mg 

100 pg 

333 Mg 

1000 Mg 

2500 Mg 

5000 Mg 

10 Mg 

10 Mg 

50 Mg 

4.0 ML 

Revertant C 

TA1535 

25 ±5 

18± 4 

16± 4 

19±5 

16±3 

15±2 

16±3 

14± 1 

18±3 ^ 

16±3' ' 

1190 ±87 

Colony Count 

TA1537 

16 ±6 

11 ±3 

18±4 

19±5 

20 ±2 

12 ±1 

21 ±1 

17±4 

14±2' ' 

17±4' ' 

101 ±7 

s (Mean ±SD) 

W a 

TA98 

29 ±11 

38 ±4 

37 ±6 

31 ±5 

31 ±4 

26 ±4 

31 ±9 

39 ±7 

31 ±2 ^ 

39 ±5' ' 

374 ±19 

TA 100 

137 ±13 

153 ±24 

143 ±13 

156 ±6 

139 ±12 

134 ± 25 

134 ±9 

138 ±5 

152 ±10'' 

158 ±16'' 

1822 ±69 

WP2 uvrA 

.63 ±2 

48 ± 7 

55 ±3 

55 ± 5 

59 ±9 

51 ±26 

53 ±9 

53 ±4 

56 ±2' ' 

60 ±9' ' 

1274 ±4 

With 

Activation 

Key to Posi 

DMSO 

Untreated 

TKA 40365 

(CGI 725) 

2-AA 

2-AA 

itive Controls 

3Mg 

10 MO 

33 Mg 

100 Mg 

333 MO 

1000 MO 

2500 Mg 

5000 Mg 

2.5 Mg 

10.0 Mg 

18±3 

26 ±3 

23 ±5 

23 ±6 

27 ±7 

23 ±3 

24 ±2 

23 ±1 

25 ±1'' " 

2o±2''" 

411 ±21 

20 ±2 

17±2 

16±6 

16±7 

21 ±1 

24 ±4 

22 ±2 

25 ±8 

21 ±3'"'^ 

19±6''' ' 

471 ± 30 

Key to Pla 

38 ±14 

44 ±13 

38 ±6 

36 ±10 

36 ±8 

35 ±3 

37 ±7 

41 ±7 

29±5''" 

28±4'''^ 

2926 ±155 

lte Postfix Cod( 

158 ±12 

156 ±16 

144±13 

163 ±26 

149 ±10 

163 ±31 

149 ± 24 

133 ±19 

133 ±9''" 

133 ±8'''^ 

3419 ±179 

BS 

58 ±8 

59 ±9 

65 ± 5 

61 ±4 

57 ±5 

64±8 

62 ±9 

64 ±3 

55±5''" 

48±4'''^ 

251 ± 26 

NdN3 sodium azide 

2-AA 2-aminoanthracene 

4-NOPD 4-nitro-o-phenylene-diamine 

MMS methyl methane sulfonate 

P Precipitate 

M Manual count 

s4c2dr.doc 
RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 21 of 31 

12.2Summary of Results Experiment II 

study Name 

Experiment: 

Assay Cond 

Metabolic 

Activation 

Without 

Activation 

s: 1040300 

1040300 HV2 

litions: 

Test 

GrouD 

DMSO 

Untreated 

TKA 40365 

(CGI 725) 

NaN3 

4-NOPD 

4-NOPD 

MMS 

Pre 

Dose Level 

(ua/Dlate) 

33 MO 

100 Mg 

333 Mg 

1000 MO 

2500 Mg 

5000 Mg 

10 MO 

10 Mg 

50 MO 

4.0 ML 

Revertant 

TA1535 

23 ±3 

24 ±5 

19±6 

20 ±8 

19±8 

19±4 

21 ±8' ' 

21 ±3" " 

1409 ± 70 

Colony Count 

TA 1537 

15±2 

19±4 

24 ±7 

16±1 

17±2 

19±3 

19±5' ' 

20 ±6'''^ 

147 ±6 

Study 

Datel 

Date' 

s (Mean ±SD) 

TA98 

27 ±3 

36 ±6 

35 ±5 

32 ±7 

38 ±6 

34±6 

31 ±3' ' 

29ilP M 

620 ± 23 

Code: RCC-C 

Plated: 08/08/: 

Counted: 14/0 

TA100 

136 ±17 

146 ±15 

148 ±13 

138 ±9 : 

128 ±14 

152 ±15 

143 ±7' ' 

128 ±11'"'^ 

2153 ±13 

:CR 1040300 

2006 

8/2006 

WP2 uvrA 

62 ± 8 

62 ±8 

62 ±16 

63 ±8 

61 ±4 

64±1 

58 ±9' ' 

53±6''" 

591 ± 64 

With 

Activation 

Key to Po 

NaN3 

2-AA 

4-NOPD 

MMS 

DMSO 

Untreated 

TKA 40365 

(CGI 725) 

2-AA 

2-AA 

sitive Controls 

sodium azide 

2-aminoanthraceni 

4-nitro-o-phenylen 

methyl methane si 

33 Mg 

100 Mg 

333 Mg 

1000 Mg 

2500 Mg 

5000 MO 

2.5 Mg 

10.0 MO 

e 

e-diamine 

jifonate 

23 ±1 

22 ±4 

21 ±4 

24 ±5 

28 ±8 

22 ±4 

22±2'''^ 

20±3''" 

393 ± 26 

24 ±2 

25 ±11 

23 ±3 

17±4 

23 ±4 

24 ±4 

19±4''" 

19±5''*' 

395 ± 26 

Key to P 

P 

M 

40 ±6 

45 ±7 

36 ±10 

45 ±5 

41 ±4 

41 ±15 

34 ±3''*' 

34±4''" 

2587 ± 377 

late Postfix Cod 

Precipitate 

Manual count 

175 ±17 

183 ±21 

179 ±9 

189 ±4 

180 ±7 

174 ±28 

151 ±14''" 

152±5''" 

3991 ±164 

es 

76 ±5 

73 ±11 

70 ±5 

73 ±7 

77 ±14 

75 ±8 

61 ±6" " 

71 ±2" " 

255 ± 46 

s4c2dr.doc 
RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 22 of 31 

12.3HISTORICAL CONTROL DATA 

These data represent the laboratory's historical control data from May 2005 until June 

2006 representing approx. 200 experiments (WP2 uvrA the historical data are based on 

approx. 100 experiments). 

Strain 

TA 1535 

TAI 537 

TA98 

TAIOO 

WP2uvrA 

Solvent control 

Negative control 

Positive control 

Solvent control 

Negative control 

Positive control 

Solvent control 

Negative control 

Positive control 

Solvent control 

Negative control 

Positive control 

Solvent control 

Negative control 

Positive control 

Mean 

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 

witho 

SD 

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 

ut S9 mix 

Min 

9 

11 

781 

5 

4 

53 

15 

16 

176 

87 

86 

616 

31 

36 

249 

Max 

35 

31 

4900 

28 

28 

425 

49 

60 

1818 

197 

216 

2872 

74 

76 

1810 

Mean 

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 

with S9 

SD 

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 

mix 

Min 

7 

10 

107 

6 

7 

59 

20 

IS 

407 

84,. 

96 

417 

34-

33 

211 

Max 

43 

38 

695 

36 

34 

746 

57 

64 

4891 

255 

214 

5230 

84 

91 

930 

Mean = mean value of revertants/plate 

SD = standard deviation Min = minimal value/Max = maximal value 

s4c2dr.doc 
RCC-CCR Study Number 1040300 Final Report Page 23 of 31 

TKA 40365 (CGI 725) 

13 ANNEX: TABLES OF RESULTS (8 PAGES) 

Pre-Experiment and Experiment 1:1040300 W + W a Plate Incorporation (4 pages) 

Experiment II: 1040300 Pre-incubation (4 pages) 

s4c2dr.doc 
RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 24 of 31 

study Name: 1040300 

Experiment: 1040300 W * W a Plate 

Assay Conditions: 

study Code: RCC-CCR 1040300 

Date Plated: 01/08/2006 / 08/08/2006 

Date Counted: 04/08/2006 / 14/08/2006 

TA1535 

Without metabolic activation 

strain Compound Dose level 

per plate 

Mean 

revertants 

per plate 

standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts 

TKA 40365 

(CGI 725) 10 pg 

33 pg 

100 pg 

333 pg 

1000 pg 

2500 pg 

5000 pg 

DMSO 

Untreated Control 

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, 12 

24, 16, 16 

15, 20, 14 

14,17,13 

15, 19, 13 

13, 15, 14 

20P, 15P, 19P 

14P, 20P, 15P 

26, 20, 29 

13,21, 19 

T AI 537 TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

3pg 

10 pg 

33 pg 

100 pg 

333 pg 

1000 pg 

2500 pg 

5000 pg 

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 

14,21,20 

20, 24, 14 

17,21,21 

12, 13, 12 

22, 21, 21 

16,13,21 

12P, 15P, 14P 

19 P, 19 P, 12 P 

22, 12, 13 

14,8,12 

TA98 

W a 

TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

3pg 

10 pg 

33 pg 

100 pg 

333 pg 

1000 pg 

2500 pg 

5000 pg 

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, 38 

34, 34, 26 

35, 31, 27 

22, 26, 30 

28, 42, 24 

37, 33, 47 

30 P, 34 P, 30 P 

35 P, 44 P, 37 P 

41,24,21 

38, 35, 42 

TAIOO TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

3 pg 143.3 12.9 1.0 138, 158, 134 

10 pg ^55.7 5.7 1.1 151,154,162 

33 pg 139.3 11.9 1.0 126,149,143 

100 pg 133.7 25.0 1.0 109,159,133 

333 pg 134.3 8.6 1.0 142,136,125 

1000 pg 138.0 5.3 1.0 142,140,132 

2500 pg 151.7 9.6 1.1 150 P, 162 P, 143 P 

5000 pg 157.7 15.9 1.2 147 P, 150 P, 176 P 

137.0 12.8 134,151,126 

753.3 24.3 158, 175, 127 

Key to Plate Postfix Codes 

Precipitate 

s4c2dr.doc 
RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 25 of 31 

Study Name: 1 

Experiment: IC 

Assay Conditic 

Strain 

WP2uvrA 

TA1535 

T AI 537 

TA98(Wa) 

TAIOO 

WP2 uvrA 

Key to Positive 

040300 

)40300 W Plate 

>ns: 

Compound 

TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

NaN3 

4-NOPD 

4-NOPD 

NaN3 

MMS 

1 Controls 

Dose level 

per plate 

3pg 

10 pg 

33 pg 

100 pg 

333 pg 

1000 pg 

2500 pg 

5000 pg 

10 pg 

50 pg 

10 pg 

10 pg 

4.0 pL 

Without m 

Mean 

revertants 

per plate 

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 

letabollc acti 

Standard 

Deviation 

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 

ivation 

Ratio 

treated / 

solvent 

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 

Study Code: RCC-CCR 1040300 

Date Plated: 01/08/2006 / 08/08/2006 

Date Counted: 04/08/2006 /14/08/2006 

Individual revertant 

colony counts 

52,54,58 

50, 57, 59 

49, 66, 62 

76, 24, 52 

45, 62, 52 

55, 48, 56 

56 P, 58 P, 55 P 

67 P, 62 P, 50 P 

63.61,65 

42, 56, 45 

1249,1232,1090 

93, 106, 105 

362, 396, 363 

1779, 1902, 1785 

1278, 1270, 1273 

Key to Plate Postfix Codes 

NaN3 sodium azide 

4-NOPD 4-nitro-o-phenylene-diamine 

MMS methyl methane sulfonate 

Precipitate 

s4c2dr.doc 
RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 26 of 31 

Study Name: 1040300 

Experiment: 1040300 W + W aPlate 

Assay Conditions: 

Study Code: RCC-CCR 1040300 

Date Plated: 01/08/2006 / 08/08/2006 

Date Counted: 04/08/2006 / 14/08/2006 

TA1535 

With metabolic activation 

Strain Compound Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts 

TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

3pg 22.7 5.1 1.2 17,27,24 

10 pg 23.0 5.6 1.3 17,28,24 

33 pg 27.3 6.7 1.5 23,24,35 

100 pg 23.0 2.6 1.3 22,21,26 

333 pg 24.0 2.0 1.3 26,24,22 

1000 pg 23.3 0.6 1.3 23,24,23 

2500 pg 25.0 1.0 1.4 24 P M, 25 P M, 26 P M 

5000 pg 20.3 2.1 1.1 22 P M, 18 P M, 21 P M 

18.3 3.1 21, 15, 19 

26.0 3.5 28,22,28 

T AI 537 TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

3pg 16.0 5.6 0.8 21,17,10 

10 pg 16.0 7.0 0.8 8,19,21 

33 pg 213 1.2 1.1 22,20,22 

100 pg 24.0 4.4 1.2 22,29,21 

333 pg 217 2.1 1.1 21,24,20 

1000 pg 25.0 7.9 1.3 22,34,19 

2500 pg 21.3 3.1 1.1 22 P M, 18 P M, 24 P M 

5000 pg 18.7 5.7 0.9 25 P M, 14 P M, 17 P M 

20.0 1.7 22, 19, 19 

16.7 2.1 19,15,16 

TA98 

W a 

TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

3pg 38.0 6.2 1.0 43,40,31 

10 pg 36.0 9.5 1.0 47,30,31 

33 pg 36.3 8.0 1.0 28,37,44 

100 pg 35.3 2.5 0.9 33,35,38 

333 pg 37.0 7.0 1.0 30,44,37 

1000 pg 41.3 6.5 1.1 48,35,41 

2500 pg 29.0 5.3 0.8 27 P M, 25 P M, 35 P M 

5000 pg 28.3 4.2 0.8 33 P M, 27 P M, 25 P M 

37.7 13.7 44,22,47 

44.0 13.1 38,35,59 

TA100 TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

3pg 144.3 13.3 0.9 159,141,133 

10 pg 163.3 25.5 1.0 192, 155, 143 

33 pg 149.3 10.2 0.9 145,161,142 

100 pg 163.0 30.5 1.0 193,164, 132 

333 pg 149.0 23.5 0.9 176,133, 138 

1000 pg 133.3 18.9 0.8 112,140,148 

2500 pg 133.0 9.2 0.8 135 P M, 123 P M, 141 P M 

5000 pg 133.3 8.0 0.8 125 P M, 134 P M, 141 P M 

158.3 12.2 145, 161, 169 

755.7 15.8 152, 173, 142 

Key to Plate Postfix Codes 

P Precipitate 

M Manual count 

s4c2dr.doc 
RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 27 of 31 

study Name: 1040300 

Experiment: 1040300 W * W a Plate 

Assay Conditions: 

With metabolic activation 

study Code: RCC-CCR 1040300 

Date Plated: 01/08/2006 / 08/08/2006 

Date Counted: 04/08/2006 /14/08/2006 

Strain 

WP2uvrA 

TAI 535 

TAI 537 

TA98(Wa) 

TA100 

WP2 uvrA 

Key to Positive 

Compound 

TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

2-AA 

2-AA 

2-AA 

2-AA 

2-AA 

Controls 

Dose level 

per plate 

3pg 

10 pg 

33 pg 

100 pg 

333 pg 

1000 pg 

2500 pg 

5000 pg 

2.5 pg 

2.5 pg 

2.5 pg 

2.5 pg 

10.0 pg 

Mean 

revertants 

per plate 

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 

Standard 

Deviation 

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 

Ratio 

treated / 

solvent 

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 

Individual revertant 

colony counts 

69, 59, 66 

61,58,65 

62, 52, 57 

70, 66, 55 

72, 55, 58 

64, 62, 67 

58 P M, 58 P M, 50 P M 

52 P M, 49 P M, 44 P M 

52, 56, 67 

49, 64, 64 

408,433, 391 

461,505,447 

3048,2979,2752 

3452, 3226, 3580 

222.260,272 

Key to Plate Postfix Codes 

2-AA 2-aminoanthracene P 

M 

Precipitate 

Manual count 

s4c2dr.doc 
RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 28 of 31 

study Name: 1040300 

Experiment: 1040300 HV2 Pre 

Assay Conditions: 

Study Code: RCC-CCR 1040300 

Date Plated: 08/08/2006 

Date Counted: 14/08/2006 

Without metabolic activation 

Strain Compound Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts 

T AI 535 TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

33 pg 18.7 5.8 0.8 22,12,22 

100 pg 20.3 8.1 0.9 29,19,13 

333 pg 18.7 7.6 0.8 10,24,22 

1000 pg 19.3 3.5 0.8 19,16,23 

2500 pg 21.0 7.9 0.9 12P, 27P,24P 

5000 pg 20.7 3.1 0.9 20 P M, 24 P M, 18 P M 

23.0 2.6 21,22,26 

24.0 5.3 20,22,30 

TA1537 TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

33 pg 24.0 6.9 1.6 28, 16.28 

100 pg . 16.3 12 , 1.1 17,17,15 

333 pg 17.0 1.7 1.2 16,16,19 

1000 pg 19.0 3.5 1.3 17,17,23 

2500 pg 18.7 4.6 1.3 16 P, 16 P, 24 P 

5000 pg 20.0 5.6 1.4 14 P M, 25 P M, 21 P M 

14.7 1.5 16, 13, 15 

18.7 4.0 21,14,21 

TA98 TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

33 pg 35.0 5.0 1.3 40,30,35 

100 pg 32.0 7.2 1.2 30,26,40 

333 pg 37.7 5.7 1.4 33,36,44 

1000 pg 34.3 5.5 1.3 38,37,28 

2500 pg 30.7 2.5 1.1 33 P, 28 P, 31 P 

5000 pg 29.0 1.0 1.1 28 P M, 30 P M, 29 P M 

27.0 3.5 29,23,29 

36.0 5.6 41,30,37 

TAIOO TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

33 pg 148.0 12.8 1.1 145,162,137 

100 pg 138.0 9.0 1.0 147,129,138 

333 pg 728.3 14.4 0.9 120,120, 145 

1000 pg 1517 15.3 1.1 165,135,155 

2500 pg 143.3 6.7 1.1 145 P, 149 P, 136 P 

5000 pg 127.7 11.4 0.9 137 P M, 115 P M, 131 P M 

136.3 16.6 134, 154, 121 

146.0 14.7 129, 155, 154 

WP2uvrA TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

33 pg 62.0 15.7 1.0 44,69,73 

100 pg 63.3 7.5 1.0 63,71,56 

333 pg 613 4.0 1.0 57,62,65 

1000 pg 64.0 10 10 64,63,65 

2500 pg 58.3 9.3 0.9 66 P, 61 P, 48 P 

5000 pg 53.0 6.2 0.9 51 P M, 48 P M, 60 P M 

62.3 7.5 70,62,55 

617 7.5 69,54,62 

Key to Plate Postfix Codes 

P 

M 

Precipitate 

Manual count 

s4c2dr.doc 
RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 29 of 31 

Study Name: 

Experiment:' 

Assay Condit 

Strain 

TA1535 

TA1537 

TA98 

TAIOO 

WP2 uvrA 

Key to Positiv 

1040300 

1040300 HV2 Pre 

ions: 

Compound 

NaN3 

4-NOPD 

4-NOPD 

NaN3 

MMS 

le Controls 

Dose level 

per plate 

10 pg 

50 pg 

10 pg 

10 pg 

4.0 pL 

Without n 

Mean 

revertants 

per plate 

1409.3 

146.7 

620.0 

2153.3 

591.0 

letabolic act 

Standard 

Deviation 

70.0 

6.4 

22.6 

12.5 

63.6 

ivation 

Ratio 

treated / 

solvent 

613 

10.0 

23.0 

15.8 

9.5 

Study Code: RCC-CCR 1040300 

Date Plated: 08/08/2006 

Date Counted: 14/08/2006 

Individual revertant 

colony counts 

1479, 1410, 1339 

142, 144,154 

596, 623, 641 

2153,2141,2166 

581,533,659 

NaN3. sodium azide 

4-NOPD 4-nitro-o-phenylene-diamine 

MMS methyl methane sulfonate 

s4c2dr.doc 
RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 30 of 31 

study Name: 1040300 

Experiment: 1040300 HV2 Pre 

Assay Conditions: 

Study Code: RCC-CCR 1040300 

Date Plated: 08/08/2006 

Date Counted: 14/08/2006 

With metabolic activation 

Strain Compound Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts 

TA1535 TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

33 pg 213 4.0 0.9 19,19,26 

100 pg 24.3 4.9 1.1 22,21,30 

333 pg 27.7 8.1 12 19,35,29 

1000 pg 22.3 4.0 1.0 27,20,20 

2500 pg 217 2.1 1.0 24 P M, 20 P M, 21 P M 

5000 pg 20.0 3:5 0.9 24 P M, 18 P M, 18 P M 

22.7 1.2 24,22,22 

22.0 4.4 27,20,19 

TA1537 TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

33 pg 23.3 2.5 1.0 26,23,21 

100 pg 17.3 3.8 0.7 13,19,20 

333 pg 22.7 4.0 0.9 22,19,27 

1000 pg 24.3 3.5 1.0 21,24,28 

2500 pg 19.3 4.2 0.8 18 P M, 16 P M, 24 P M 

5000 pg 19.3 4.7 0.8 23 P M. 14 P M, 21 P M 

24.3 2.3 23.23,27 

25.3 11.1 37,24, 15 

TA98 TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

33 pg 36.3 10.1 0.9 27,47,35 

100 pg 45.0 5.3 1.1 41,51,43 

333 pg 410 3.6 10 42,37,44 

1000 pg 410 14.5 1.0 55,42,26 

2500 pg 33.7 3.1 0.8 33 P M, 31 P M, 37 P M 

5000 pg 34.3 4.2 0.9 31 P M, 33 P M, 39 P M 

39.7 6.1 33,45,41 

44.7 6.8 37,47,50 

TAIOO TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

33 pg 179.3 9.3 1.0 173,190,175 

100 pg 189.3 4.0 1.1 190,193,185 

333 pg 180.3 7.2 1.0 172,185,184 

1000 pg 173.7 27.8 1.0 199,178,144 

2500 pg 1507 14.2 0.9 167 P M. 144 P M, 141 P M 

5000 pg 152.3 4.5 0.9 152 P M, 148 P M, 157 P M 

174.7 17.2 169,194,161 

183.0 21.2 159,199, 191 

WP2uvrA TKA 40365 

(CGI 725) 

DMSO 

Untreated Control 

33 pg 69.7 5.1 0.9 64,74,71 

100 pg 73.3 7.0 1.0 80,66,74 

333 pg 76.7 14.0 1.0 61,81,88 

1000 pg 75.0 7.9 1.0 72,69.84 

2500 pg 613 6.0 0.8 62 P M, 55 P M, 67 P M 

5000 pg 710 17 0.9 72 P M, 69 P M, 72 P M 

75.7 4.6 73,81,73 

72.7 10.6 71,84,63 

Key to Plate Postfix Codes 

P 

M 

Precipitate 

Manual count 

s4c2dr.doc 
RCC-CCR Study Number 

TKA 40365 (CGI 725) 

1040300 Final Report Page 31 of 31 

study Name: 1040300 

Experiment: 1040300 HV2 Pre 

Assay Conditions: 

With metabolic activation 

Study Code: RCC-CCR 1040300 

Date Plated: 08/08/2006 

Date Counted: 14/08/2006 

Strain Compound Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts 

T AI 535 

T AI 537 

TA98 

TAIOO 

WP2 uvrA 

2-AA 

2-AA 

2-AA 

2-AA 

2-AA 

2.5 pg 

2.5 pg 

2.5 pg 

2.5 pg 

10.0 pg 

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, 363 

370,421,393 

2172, 2682, 2907 

3880,3913,4179 

241,218,306 

Key to Positive Controls 

2-AA 2-aminoanthracene 

s4c2dr.doc