## CiToxLAB Hungary Ltd.

Veszprém

- Szabadságpuszta



# FINAL REPORT

In vitro Mammalian

Chromosome Aberration Test

of Ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6]

(hydrated form of ethanedioic acid [CAS RN 144

- 62

- 7])

Study code: 11/339

- 020C

Study Director: Judit Hargitai, PhD.

22 Marc h 2013Study code: 11/339

- 020C Final Report Page 5 of 66

GENERAL INFORMATION

STUDY TITLE : In vitro Mammalian Chromosome Aberration Test of

Ethanedioic acid, hydrate (1:2) [ CAS RN 6153

- 56

- 6]

(hydrated form of ethanedioic acid [CAS RN 144

- 62

- 7])

TEST ITEM : Ethanedioic acid, hydrate (1:2)

SPONSORS : Univar USA Inc.

Address: 17425 NE Union Hill Road,

Redmond, WA 98052, USA

Phone: +1 425 889 3400

Fax: +1 425 889 376 0

WEGO Chemical and Mineral Corp.

Address: 239 Great Neck Road,

Great Neck, NY 11021, USA

Phone: +1 516 487 3510

Fax: +1 516 487 3794

STUDY MONITOR : John V. Massingale

Greenwich Chemical Consulting, Inc.

Address: 3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410, USA

Phone: +1 561 578 8110

Fax: +1 561 578 8120

E

- mail: john@grcci.com

TEST FACILITY : CiToxLAB Hungary Ltd.

(Formerly known as LAB Research Ltd. before 1 st September 2011)

Address H

- 8200 Veszprém, Szabadságpu szta

Hungary

Phone: +36 88 545 300

Fax: +36 88 545 301

TES T SITE : Microptic Cytogenetic s

(Metaphase analysis) Address: 2 Langland Close, Mumbles

Swansea, SA3 4LY,

United Kingdom

Phone: +44 (0) 1792 366714

Fax: +44 (0) 1792 366714Study code: 11/339

- 020C Final Report Page 6 of 66

STUDY DI RECTOR : Judit Hargitai

, Ph D.

Phone: +36 88 545 273

E

- mail: judit.hargitai@ hu.citoxlab.com

PRINCIPAL INVESTIGATOR : Natalie Danford, B

. Sc

. , MPH, PhD

. (Metaphase analysis) Microptic Cytogenetics

E

- mail: natalie.danford@microptic.com

TEST FACILITY

MANAGEMENT : Christopher Banks

, DABT

Managing Director

Alyson Leyshon

, M.Sc.

Senior VP Business Development Europe and

Operations Hungary

LEAD

QUALITY ASSURANCE : Szabolcs Gáty

, M.Sc.

Head of QAU

Vanda Gyimesi

, M.Sc.

Deputy Head of QA U

Éva Fábiánné Makovi, B.Sc.

QA Inspector

Diána Czuczay, M.Sc.

QA Inspector

TEST SITE

QUALITY ASSURANCE : Neville Jones, B.Sc.

Microptic Cytogenetic s

RESPONSIBLE PERSONS : Annamária Blumenschein

- technica l team leader

Bernadett Dabosi

- techn ical assistant

Mónika Szabóné Oláh

- technical assistant

Lívia Lukács

- technical assistant

Zoltán Bácskay

- technical assistant

Tam á s Mész á ros, PhD

. - Central DispensaryStudy code: 11/339

- 020C Final Report Page 7 of 66

BASIS OF STUDY : OECD Guidelines for Testing of Chemicals, No. 473

Commi ssion Regulation (EC) No. 440/2008, B.10.

EPA Health Effects Test Guidelines, OPPTS 870.5375

STUDY PLAN : 23 February 2012

AM ENDMENT 1 TO THE STUDY PLAN : 30 October 2012

AMENDMENT 2 TO THE STUDY PLAN : 05 November 2012

AMENDMENT 3 TO THE STUDY PLAN : 13 November 2012

PRELIMINARY TEST : 30

- 31 October 2012

START OF EXPERIMENT : 0 6 November 2012

END OF EXPERIMENT : 1 4 February 2 013

END OF METAPHASE ANALYSIS : 15 March 201 3

DRAFT REPORT : 19 March 2013

FINAL REPORT : 22 March 2013

Detailed s tudy schedule is shown in Appendix 1.Study code: 11/339

- 020C Final Report Page 8 of 66



### CONTENTS

Page

STATEMENT OF THE STU DY DIRECTOR 2

STATEMENT OF THE MAN AGEMENT 3

QUALITY ASSURANCE S TATEMENT 4

GENE RAL INFORMATION 5

1. SUMMARY 10

2. INTROD UCTION 12

3. OBJECTIVE 12

4. GUIDELINES 12

5. MATERIALS AND METHOD S 13

5.1. TEST ITEM 13

5.2. NEGATIVE AND POSITIVE CONTROLS 14

5.3. INDICATOR CELLS 15

5.4. EXTERNAL METABOLIC A CTIVATION SYSTEM 16

5.5. TEST PROCEDURE 18

5.6. EVALUATION OF THE RESULTS 22

5.7. CHEMICALS USED IN TH E EXPERIMENTS 23

6. ARCHIVES 24

7. DEVIATION S TO THE STUDY PLAN 25

8. RESULTS AND DISCUSSI ON 25

8.1. VEHICLE AND CONCENTRATION SE LECTION 25

8.2. CHROMOSOME ABERRATIO N ASSAYS 26

8.3. VALIDITY OF THE STUD Y 27

9. CONCLUSION 28

10. DISTRIBUTION OF THE FINAL REPORT 28

11. REFERENCES 29

APPENDICES 30

APPENDIX 1 STUDY SCHEDULE 31

APPENDIX 2 COP Y OF THE CERTIFICATE OF ANALYSIS 32

APPENDIX 3 SUMMARIZ ED RESULTS OF THE CO NCENTRATION

SELECTION CYTOTOXICI TY ASSAYS 33

APPENDIX 4 CYTOTOXI CITY RESULTS OF THE MAIN EXPERIMENTS 36

APPENDIX 5 SUMMARY TABLES OF THE MAIN E XPERIMENTS 40

APPENDIX 6 HISTORIC AL CONTROL DATA 44

APPENDIX 7 REPORT O F THE METAPHASE ANAL YSIS 46

APPENDIX 8 COPIES OF THE GLP CE RTIFICATES 64Study code: 11/339

- 020C Final Report Page 9 of 66



### LIST OF TABLES

Page

Table 1:

Chemicals Used in the Experiments 2 3

- 24

Table 2:

Summarized results of the Concentration Selection Cytotoxicity Assay A

experiment without metabolic a ctivation 33

Table 3:

Summarized results of the Concentration Selection Cytotoxicity Assay A

experiment with metabolic activation 34

Table 4:

Summarized results of the Concentration Selection Cytotoxicity Assay B

experiment without metabolic activation 3 5

Table 5 :

Cytotoxicity results of Chromosome Aberration Assay 1

experiment without metabolic activation 36

Table 6 :

Cytotoxicity results of Chromosome Aberration Assay 1

experiment with metabolic activation 3 7

Table 7 :

Cytotoxicity results of Chromos ome Aberration Assay 2

experiment without metabolic activation 3 8

Table 8 :

Cytotoxicity results of Chromosome Aberration Assay 2

experiment with metabolic activation 39

Table 9 :

Summary table of Chromosome Aberration Assay 1

without metabolic activation 4 0

Table 10 :

Summary table of Chromosome Aberration Assay 1

with metabolic activation 41

Table 11:

Summary table of Chromosome Aberration Assay 2

without metabolic activation 42

Table 12:

Summary table of Chromosome Aberration Assay 2

with metabolic ac tivation 43Study code: 11/339

- 020C Final Report Page 10 of 66

1. SUMMARY

Ethanedioic acid, hydrate (1:2) was tested in vitro in a Chromosome Aberration Assay

using Chinese hamster V79 l ung cells . The test item was formulated in phosphate

buffered saline and it was examined up to the cytotoxic concen tration s according to

the OECD guideline recommendations

. In the performed independent Chromosome

Aberration Assays using duplicate cultures at least 200 well

- spread metaphase cells

(or until a clear positive response was detected) were analysed for each t est item

treated, negative ( vehicle ) and positive control sample.

In Chromosome Aberration Assay 1, a 3

- hour treatment with metabolic activation (in

the presence of S9

- mix) and a 3

- hour treatment without metabolic activation (in the

absence of S9

- mix) wer e performed. Sampling was performed 20 hours after the

beginning of the treatment in both cases. The examined concentrations of the test item

were 4 00, 200, 150, 125, 100, 75, 50, 2 5 and 12

. 5 μg/mL.

In Assay 1, no insolubility was detected at the end of t he treatment period in the final

treatment medium in any of the examined treatment concentrations. No large changes

in pH and osmolality were detected. Cytotoxicity was observed in this assay at 400,

200, 150, 125 and 100 μg/mL concentrations without metab olic activation (relative

survival values were 7, 10, 18, 28 and 3 0 %, respectively) and at 400, 200, 150 and

125 μg/mL concentrations with metabolic activation (relative survival values were 7,

6, 24 and 46 %, respectively). Therefore, concentrations of 1 00, 75, 50 and 25 μg/mL

(a total of four) were selected for evaluation in case of the experiment without

metabolic activation and concentrations of 125, 100 and 50 μg/mL (a total of three)

were selected for evaluation in case of the experiment with metabol ic activation. None

of the treatment concentrations with or without metabolic activation caused a

significant increase in the number of cells with structural chromosome, thus this assay

was considered to be negative.

In Chromosome Aberration Assay 2, a 3

- hour treatment with metabolic activation (in

the presence of S9

- mix) and a 20

- hour treatment without metabolic activation (in the

absence of S9

- mix) were performed. Sampling was performed 2 0 hours after the

beginning of the treatment in both cases. The exa mined concentrations of the test item

were 400, 200, 150, 125, 100, 75, 50, 25 and 12.5 μg/mL

.

In Assay 2, similarly to the first experiment, no insolubility was detected at the end of

the treatment period in the final treatment medium at any of the exami ned

concentrations. No large changes in pH and osmolality were detected. Cytotoxicity

was observed at 400, 200, 150, 125, 100 and 75 μg/mL concentrations without

metabolic activation (relative survival values were 4, 2, 7, 14, 12 and 24 %,

respectively) an d at 400, 200, 150, 125 and 100 μg/mL concentrations (relative

survival values were 6, 11, 14, 30 and 47%, respectively). Concentrations of 100, 75,

50 and 25 μg/mL (a total of four) were selected for evaluation in case of the

experiment without metabolic activation; and 100, 75 and 50 μg/mL (a total of three)

were selected for evaluation in case of the experiment with metabolic activation.Study code: 11/339

- 020C Final Report Page 11 of 66

None of the treatment concentrations in Assay 2 caused a significant increase in the

number of cells with structura l chromosome aberrations in either assay with or without

metabolic activation. Therefore, this assay confirmed the negative results of the first

main test.

The occurrence of polyploid and endoreduplicated metaphases was recorded in the

m ain tests. Polyplo id metaphases were found in some cases in the negative ( vehicle )

control, test item treated or positive control samples in the performed experiments.

Endoreduplicated metaphases (1

- 1 4 ) were found in some cases negative (vehicle)

control, test item treated or positive control samples in the performed experiments.

The negative ( vehicle ) control data were in good harmony with the laboratory’s

normal range for the spontaneous aberration frequency, the positive control

substances caused a statistically signific ant increase in the number of structural

aberrations excluding gaps in the experiment s with or without metabolic activation

demonstrating the sensitivity of the test system

. The evaluated concentration range

was considered to be adequate; a t least three te st item treated concentrations were

evaluated in each assay. T he tes ts were considered to be valid.

In conclusion, Ethanedioic acid, hydrate (1:2) test item did not induce a

s ignificant level of chromosome aberrations in the performed experiments with

or without metabolic activation

. Therefore, Ethanedioic acid, hydrate (1:2) is

considered not clastogenic in this test system.Study code: 11/339

- 020C Final Report Page 12 of 66

2

. INTRODUCTION

Chromosome aberration tests, which detect structural chromosome aberrations in

somatic and/or germ cells play an important role in the evaluation of genotoxicity of a

given test item [ 1 ] . Structural aberrations develop due to breaks in one or both DNA

strands, which can result in chromosome fragments (breaks, deletions). Faulty reunion

of chromosome fragments resu lts in formation of exchanges. These aberrations can be

detected and quantified using light microscope. Extensive chromosome breaks usually

cause cell death; small changes (breaks, deletions, translocations, inversions etc.) are,

however, not necessarily l ethal and can be regarded as an indication of molecular

events, which might lead to malignant cell transformation.

3

. OBJECTIVE

The objective of this study was to determine whether Ethanedioic acid, hydrate (1:2)

test item could induce structural chrom osome aberrations in cultured Chinese hamster

cells

. This study is being performed based on the U.S. Toxic Substances Control Act

(TSCA) Section 4 Test Rule and the regulations set forth under 40 CFR 799.5087.

4

. GUIDELINES

This study follow ed the proc edures indicated by the following internationally

accepted guidelines and recommendations:

- Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 473,

" In Vitro Mammalian Chromosome Aberration Test“, adopted 21 July 1997

- Commission Regulation (EC) No 440/2008 of 30 May 2008, B.10. " Mutagenicity

–

In Vitro Mammalian Chromosome Aberration Test "

- EPA Health Effects Test Guidelines, OPPTS 870.5375

, " In Vitro Mammalian

Chromosome Aberration Test“

, EPA 712

- C

- 98

- 223, August 1998Study code: 11/339

- 020C Final Report Page 13 of 66

5

. MAT ERIALS AND METHODS

5 .1. TEST ITEM

5 .1.1. Name and Data of Test Item *

Name: Ethanedioic acid, hydrate (1:2)[CAS RN 6153

- 56

- 6]

(hydrated form of ethaned i oic acid [CAS RN 144

- 62

- 7])

Short name: Ethanedioic acid, hydrate (1:2)

Chemical name: Oxalic aci d, hydrate

CAS number: 6153

- 56

- 6

Batch/Lot number: YP1

- 20120528C

Molecular formula: C 2 H 2 O 4 x 2H 2 O

Molecular weight: 126.1 (dihydrate form)

Appearance: White crystals

Purity: 99.69 %

Manufacture date: 28 May 2012

Expiry date: 27 May 2013

Storage conditions: Controlled room temperature (15

- 25 o C, < 70 RH%)

Safety Precautions: Routine safety precautions ( lab coat, gloves, safety

glasses and face mask ) for unknown materials w ere

applied to assure personnel health and safety.

*Note: Based on the Certificate of A nalysis (dated 01 June 2012 ), Test Item Data Sheet (dated 2 6

September 2012), Material Safety Data Sheet (dated 1 6 November 20 09 ) and additional information

(e

- mail 26 September 2012 ) provided by the Sponsor.

5 .1.2. Identification, Receipt

The test it em of a suitable chemical purity was supplied by the Sponsor. All

precautions required in the handling and disposal of the test item were outlined by the

Sponsor. These documents are part of the raw data. Identification of the test item was

performed in th e Central Dispensary of CiToxLAB Hungary Ltd. based on the

information provided by the Sponsor (name, batch number, appearance and colour)

. The

copy of the Certificate of Analysis is shown in Appendix 2.

5 .1.3. Formulation

Due to the acidic property of the test item, formulations w ere prepared in a sufficient

strength supplementary buffering system (phosphate buffered saline, PBS) to ensure a

suitable final pH at testing; the same vehicle w as used in the negative controls. The

weight of test item w as ca lculated based on the test material alone.Study code: 11/339

- 020C Final Report Page 14 of 66

A supplement ary pH buffering in the system wa s considered not to adversely affect

the ability of the test item to cause genotoxic effects in the test system. The pH of the

dosing solution w as measured before t reatment to prove that the amount of buffering

did not results in unphysiological conditions (ie. alkalinity).

Stock formulation s of the test item ( 1 00 mg/mL ) w ere prepared as follows

. The

necessary amount of test item was weighed into a calibrated volume tric flask. A

partial volume of vehicle ( 1X PBS ) was added and the formulation was stirred until

homogeneity wa s reached, then the volume was adjusted to the final volume

. From

the stock formulation se veral dilutions were prepared using the selected vehicl e; the

dosing solutions were prepared immediately be fore the treatment of the cells

. The

stock solution and the vehicle were filtered sterile using a 0.22 μm syringe filter

(Supplier: Millipore, Lot No.: R2AA57025, Expiry date: January 2015) before the

pre paration of the dilutions.

Purity conversion was applied in the main experiments (ie. concentration of the test

item in the dosing formulation was adjusted according to the water content of the

provided material) as agreed by the Sponsor. For the purity c onversion, the 74.0 %

purity data of the active ingredient (molecular weight of the dihydrate form: 121.6;

molecular weight of the anhydrous form : 90.0) was taken into consideration

.

5 .2. NEGATIVE AND POSITIV E CONTROLS

Negative ( vehicle ) and positive c ontrols were included in the experiments.

Furthermore, untreated controls were also included in the preliminary experiments.

Routine safety precautions (lab coat, gloves, safety glasses and face mask) were

applied to assure personnel health and safety.

5 .2.1. Negative Control

Negative ( vehicle ) control was run concurrently with treatment groups. The following

chemicals were used for preparation of the vehicle (abbreviated as 1X PBS) in the

study:

Name (Abbreviation) : Phosphate buffered saline (PBS)

Sup plier: Sigma

- Aldrich Co.

Concentration: 10X

Lot No.: SLBB3269

Appearance: C olourless liquid

Expiry date : January 201 3

Storage condition s : Room temperature , under N 2Study code: 11/339

- 020C Final Report Page 15 of 66

Name: Distilled water

Supplier: TEVA

Lot No.: 3450611

Appearance: Colourless liquid

Exp iry date: 30 June 2014

Storage conditions: Room temperature

5 .2.2. Positive Controls

Without metabolic activation

Name ( Abbreviation ) : Ethyl methanesulfonate ( EMS )

Synonym: Ethyl methanesulphonate

Supplier: Sigma

- Aldrich Co.

Lot No.: BCBG1395V

Appear ance: Colourless l iquid

Expir y date: 17 July 2013

Storage condition s : Room temperature, under N 2

Ethyl methanesulfonate, a known mutagen and clastogen, was dissolved in DMEM

and was used as a positive control for the non

- activation experiments at a final

concentration of 0.4 μL /mL (20

- hour treatment ) or 1.0 μL /mL (3

- hour treatment ) [2].

With metabolic activation

Name ( Abbreviation ) : Cyclophosphamide monohydrate ( CP )

Supplier: Sigma

- Aldrich Co.

Lot No.: 120M1253V

Appearance: White powder

Expiry date : 31 December 201 3

Storage condition s : Refrigerated (2

- 8°C)

Cyclophosphamide, a clastogenic agent that requires metabolic transformation by

microsomal enzymes, was dissolved in sterile physiological saline solution (0.9%

NaCl infusion) for treatment and was u sed as a positive control item for the activation

experiments at a final concentration of 6.0 μg/mL.

5.3. INDICATOR CELLS

V79: Chinese hamster lung

, male

ECACC Cat. No. : 86041102

Lot No.: 10H016

Supplier: ECACC (European Collection of Cells Cultures)

M orphology: FibroblastStudy code: 11/339

- 020C Final Report Page 16 of 66

The V79 cell line is well established in toxicology studies. Stability of karyotype and

morphology makes it s uitable for genetic toxicity assays with low background

aberrations. These cells are chosen because of their small number of chromosomes

(diploid number, 2n=22) and because of the high proliferation rates (doubling time

12

- 14 h). The V79 cell line was established after spontaneous transformation of cells

isolated from the lung of a normal Chinese hamster (male). This cell lin e was

purchased from ECACC (European Collection of Cells Cultures). The cell stocks

were kept in a freezer at

- 80 ± 10 **°** C. The stock wa s checked for mycoplasma

infection . No infection of mycoplasma was noted.

Trypsin

- EDTA (0.25% Trypsin, 1mM EDTA) solution w as used for cell detachment

to subculture (cells were rinsed with 1X PBS before detachment) . The laboratory

cultures w ere maintained in 150 cm 2 plastic flasks at 37 **±** 0.5 °C in a humidified

atmosphere containing approximately 5 % CO 2 in air. The V79 cell s for this study

w ere grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM

L

- glutamine, 1 (v/v) % Antibiotic

- antimycotic solution (standard content: 10000

NE/mL penicillin, 10 mg/mL streptomycin and 25 **μ** g/mL amphotericin

- B) and

10 (v/v) % he at

- inactivated f etal bovine serum (DMEM

- 10, culture medium). During

the tre a t ments , the serum content of the medium w as reduced to 5 (v/v) % (DMEM

5).

5 .4. EXTERNAL METABOLIC A CTIVATION SYSTEM

An advantage of using in vitro cell cultures is the accurat e control of the concentration

and exposure time of cells to the test item under the study. However, due to the

limited capacity of cells growing in vitro for metabolic activation of potential

mutagens, an exogenous metabolic activation system is necessary . Many substances

only develop mutagenic potential after they are metabolised. Metabolic activation of

substances can be achieved by supplementing the cell cultures with liver microsome

preparations (S9 mix).

In the experiments with metabolic activation i n this study, a cofactor

- supplemented

post

- mitochondrial S9 fraction prepared from activated rat liver was used as an

appropriate metabolic activation system.

The post

- mitochondrial fraction (S9 fraction) was prepared by the Microbiological

Laboratory of CiToxLAB Hungary Ltd. according to Ames et al. [ 3 ] and Maron and

Ames [ 4 ]. The documentation of the preparation of this post

- mitochondrial fraction is

stored in the reagent notebook in the Microbiological Laboratory which is archived

yearly.

The supplier , batch number and expiry date of the used chemicals described in this

section are summarized in Table 1 (Section 5 .7 . Chemicals Used in the Experiments).

The composition of solution refers to 1000 mL

.Study code: 11/339

- 020C Final Report Page 17 of 66

5 .4 .1. Induction of Rat Liver Enzymes

Male Wistar rats ( 323

- 419 g, animals were 9 weeks old at the initiation) were treated

with Phenobarbital (PB) and **β**

- naphthoflavone (BNF) at 80 mg/kg/day by oral gavage

for three consecutive days. Rats were given drinking water and food ad libitum until

12 h our before sacrifice when food was removed. Initiation s of the induction of liver

enzymes used for preparation S9 used in this study w ere 28 May 201 2

.

5 .4

. 2. Preparation of Rat Liver Homogenate S9 Fraction

On Day 4, the rats were euthanized (sacrifice was by asc ending concentration of CO 2, confirmed by cutting through major thoracic blood vessels) and the livers were

removed aseptically using sterile surgical tools. After excision, livers were weighed

and washed several times in 0.15 M KCl. The washed livers were transferred to a

beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized.

Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted

and retained. The freshly prepared S9 fraction was aliquoted into 1

- 3 mL portio ns,

frozen quickly and stored at

- 80 **±** 10oC.

The protein concentration of the preparation was determined by a chemical analyzer

at 540 nm in the Clinical Chemistry Laboratory of CiToxLAB Hungary Ltd. The

protein concentration of the S9 fractio n used was d etermined to be 3 6

. 0 g/L. The date

of preparation of S9 fraction used in this study 31 May 201 2 ( CiTox LAB code:

E1 1 364 )

. The sterility of the preparation was confirmed.

The biological activity in the Salmonella assay of S9 was characterized using the two

mutagens 2

- Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation

by microsomal e n zymes. The batch of S9 used in this study functioned appropriately.

5 .4

. 3. Preparation of S9

- m ix

The complete S9

- mix was freshly prepared on the day of u se a ccording to the

following ratio :

S9 fraction 3 mL

HEPES 20 mM 2 mL

KCl 330 mM 1 mL

MgCl 2 50 mM 1 mL

NADP 40 mM 1 mL

Glucose

- 6

- phosphate 50 mM 1 mL

DME medium 1 mLStudy code: 11/339

- 020C Final Report Page 18 of 66

Prior to additi on to the culture medium the S9

- m ix was kept in an ice bath.

For a ll cultures treated in the presence of S9

- mix, a 0.5 mL aliquot of the mix was

added to each cell culture (final volume: 10 mL). The final concentration of the liver

homogenate in the test system was 1.5%.

5 .5. TEST PROCEDURE

5 .5 .1. Toxic ity and Concen tration Selection

Treatment concentrations for the mutation assay were selected based on the results of

a short preliminary test. In this Preliminary Toxicity Test , two separate a ssays were

performed. In Assay A , cells were treated for 3

- hours in the pre sence and absence of

S9

- mix with a 20

- hour harvesting time . In Assay B , cells were treated for 20 hours in

the absence of S9

- mix with a 2 0

- hour harvesting time.

The assays were performed with a range of test item concentrations to determine

cytotoxicity. Treatment was performed as described for the main test. However, single

cultures were used and positive controls were not included. Visual examination of the

final culture medium was conducted at the beginning and end of the treatments.

Measurement of pH and osmolality of the treatment medium was also performed at the

end of the treatment period.

At the scheduled harvesting time, the number of surviving cells was determined using

a haemocytometer. Results are expressed compared to the negative ( vehicle ) c ontrol as

% relative survival. Tabulated results of the preliminary experiment s are given in

Appendix 3

.

5 .5 .2. Chromosome Aberration Assays

The Chromosome Aberration Assays were conducted as t wo independent experiments

( Assay 1 and Assay 2 ) in the pre sence and in the absence of metabolic activation

.Study code: 11/339

- 020C Final Report Page 19 of 66

Experimental Design of Assay 1

3

- hr treatment without S9 Mix, harvest ing: 20 hours from the beginning of treatment

Negative (vehicle) control ( 1X PBS ) 10 μL/mL

Ethanedioic acid, hydrate (1:2) 400 μg/mL

Ethanedioic acid, hydrate (1:2) 200 μg/mL

Ethanedioic acid, hydrate (1:2) 150 μg/mL

Ethanedioic acid, hydrate (1:2) 125 μg/mL

Ethanedioic acid, hydrate (1:2) 100 μg/mL

Ethanedioic acid, hydrate (1:2) 75 μg/mL

Ethanedioic acid, hydrate (1:2) 50 μg/mL

Ethanedioic acid, hydrate (1:2) 25 μg/mL

Ethanedioic acid, hydrate (1:2) 12.5 μg/mL

Positive control (Ethyl methanesulfonate) 1.0 μL/mL

3

- hr treatment with S9 Mix, harvesting: 20 hours from the beginning of treatment

Negative (vehicle) control (1X PB S) 10 μL/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 400 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 200 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 150 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 125 μg/m L + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 100 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 75 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 50 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 25 μg/mL + S9 mix 50 μ L/mL

Ethanedioic acid, hydrate (1:2) 12.5 μg/mL + S9 mix 50 μL/mL

Positive control (Cyclophosphamide) 6.0 μg/mL + S9 mix 50 μL/mL

Note: The negative (vehicle) control, positive control and at least 3 dose levels (based on the

observed solubility and/or cytotoxicity results) were selected for chromosomal aberration analysis.

Experimental Design of Assay 2

20

- hr treatment without S9 Mix, harvest ing: 2 0 hours from the beginning of treatment

Negative (vehicle) control ( 1X PBS ) 10 μL/mL

Ethanedioic acid, hydrate (1:2) 400 μg/mL

Ethanedioic acid, hydrate (1:2) 200 μg/mL

Ethanedioic acid, hydrate (1:2) 150 μg/mL

Ethanedioic acid, hydrate (1:2) 125 μg/mL

Ethanedioic acid, hydrate (1:2) 100 μg/mL

Ethanedioic acid, hydrate (1:2) 75 μg/mL

Ethanedioic acid , hydrate (1:2) 50 μg/mL

Ethanedioic acid, hydrate (1:2) 25 μg/mL

Ethanedioic acid, hydrate (1:2) 12.5 μg/mL

Positive control (Ethyl methanesulfonate) 0.4 μL/mL

3

- hr treatment with S9 Mix, harvest ing: 2 0 hours from the beginning of treatment

Negative (vehicle) control ( 1X PBS ) 10 μL/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 400 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 200 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 150 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid , hydrate (1:2) 125 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 100 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 75 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 50 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2 ) 25 μg/mL + S9 mix 50 μL/mL

Ethanedioic acid, hydrate (1:2) 12.5 μg/mL + S9 mix 50 μL/mL

Positive control (Cyclophosphamide) 6.0 μg/mL + S9 mix 50 μL/mL

Note: The negative (vehicle) control, positive control and at least 3 dose levels (based on the

obs erved solubility and/or cytotoxicity results) were selected for chromosomal aberration analysis.Study code: 11/339

- 020C Final Report Page 20 of 66

5.5.2.1. Treatment of the Cells

For the cytogenetic experiments, 1

- 3 day old cultures (more than 50 % confluen cy )

were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 10 5

cells/dish concentration and incubated for approximately 24 hours at 37 **°** C in 10 mL

of culture medium (DMEM

- 10). Duplicate cultures were used for each test item

concentration or controls. After the seeding period, the medium w as replaced with

9.9 mL treatment medium (DMEM

- 5) in case of experiments without metabolic

activation or with 9.4 mL treatm ent medium (DMEM

- 5) + 0.5 mL S9

- mix in case of

experiments with metabolic activation

.

Cells were treated with different co nc entration test item solutions, negative ( vehicle )

or positive control solution (treatment volume: 10 0 μL/dish in all cases ) for the given

period of time at 37 **°** C in the absence or presence of S9

- mix. After the exposure

period, the cultures were washed with DMEM

- 0 medium (Dulbecco’s Modified

Eagle’s Medium supplemented with 2 mM L

- glutamine and 1 v/v% Antibiotic

antimycotic solution) . Then, 10 mL of fresh culture medium were added into the

dishes and cells were incubated further until the scheduled harvesting time.

Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) from

the beginning of treatment.

Solubility of the test item in the final treatment medium w as visually examined at the

beginning and end of the treatment in each case

. Measurement of pH and osmolality

was also performed at the end of the treatment period in both main tests

.

For concurrent measurement of cytotoxicity an extra dish was plated for each sample

and treated in the same manner. At the scheduled harvesting ti me, the number of

surviving cells w as determined using a haemocytometer. Results are expressed

compared to the negative ( vehicle ) control as % relative survival.

5. 5 .2.2

. Preparation of Chromosomes

2

- 2.5 hours prior to harvesting, cell cultures were t reated with Colchicine

(0.2 μg/ mL ). The cells were swollen with 0.075 M KCl hypotonic solution, then were

washed in fixative (Methanol : Acetic

- acid 3 : 1 (v : v) mixture) until the preparation

beca me plasma free ( 4 washes). Then, a suspension of the fixed cells was dropped

onto clean microscope slides and air

- dried. The slides were stained with 5 % Giemsa

solution, air

- dried and coverslips were mounted. At least three slides were prepared

for each culture.Study code: 11/339

- 020C Final Report Page 21 of 66

5 .5 .2. 3

. Examination of Slides

The stained sl ides w ere given random unique code numbers at the Test Facility by a

person who w as n ot involved in the metaphase analysis. The code labels cover ed all

unique identification markings on the slides to ensure that they w ere scored without

bias.

The coded sl ides were sent for evaluation to the Principal Investigator to :

Natalie Danford, B.Sc., MPH, PhD.

Microptic Cytogenetics

2 Langland Close

Mumbles

Swansea, SA3 4LY, U nited Kingdom

The metaphase analysis was conducted under the control of the Principal Inv estigator

. When the metaphase analysis was completed for each test, the slide codes were broken

and the number of metaphases with aberrations (excluding gaps) and the types of

aberrations for each culture were presented in tables.

At least o ne hundred met aphases * with 22 **±** 2 chromosomes (dicentric chromosomes

were counted as two chromosomes) from each culture were examined for the presence

or absence of chromosomal aberrations (approximately 1000x magnification), where

possible. Chromatid and chromosome type aberrations (gaps, deletions and

exchanges) were recorded separately.

*Note: The examination of slides from a culture was halted when 15 or more metaphases with

aberrations (excluding gaps) have been recorded for that culture.

The aberrations are defin ed in the following way:

Gap: small unstained lesion smaller than the width of a chromatid and

with minimal misalignment of the chromatid(s)

Break: unstained lesion larger than the width of a chromatid, or with

clear misalignment

Exchange: breakage and re union of chromatids within a chromosome, or

between chromosomes

Chromatid

- type: structural chromosome damage expressed as breakage of single

chromatids or breakage and reunion between chromatids

Chromosome

- type: structural chromosome damage expressed as br eakage, or

breakage and reunion, of both chromatids at an identical site.Study code: 11/339

- 020C Final Report Page 22 of 66

Fragments could arise from breakage and exchange events. When the origin of a

fragment was clear, it was recorded under that category (e.g. a dicentric chromosome

with a fragment was recorded as one chromosome exchange event). When the origin

of the fragment was not clear, it was recorded as a chromatid break. Metaphases with

more than five aberrations (excluding gaps) were recorded as showing multiple

damage.

Additionally, the nu mber of polyploid and endoreduplicated cells was scored.

Polyploid metaphases are defined as metaphases with approximate multiples of the

haploid chromosome number (n), other than the diploid number (i.e. ca. 3n, 4n etc).

Endoreduplicated metaphases have c hromosomes with 4, 8, etc. chromatids. Marked

reductions in the numbers of cells on the slides were recorded if needed.

The Vernier co

- ordinates of at least five metaphases (with aberrations, where

possible) were recorded for each culture.

The metaphase analysis w as conducted in compliance with Good Laboratory Practice

as required by the United Kingdom GLP Compliance Regulations 1999 (SI 1999 No.

3106, as amended 2004, SI No. 0994) and which are in compliance with the OECD

Principles of Good Laboratory Pr actice (as revised in 1997). These Principles are in

conformity with other international GLP regulations.

The Quality Assurance Unit at the Test Site perform ed study and/or process

- based

inspections according to its own Standard Operating Procedures. Insp ection results

w ere reported promptly in writin g to the Principal Investigator who subsequently

informed the Study Director. The inspection results were also communicated to the

Lead QA (CiToxLAB Hungary Ltd.) and Test Facility Management. The Test Site QA

provided a QA statement for inclusion in the study report.

The Principal Investigator receive d the signed study plan and amendment as PDF

- files

by e

- mail, and distribute d them as required at the Test Site. All study related

communication between the Stud y Director, Principal Investigator and Sponsor or

Sponsor’s representative were documented and archived with the raw data.

5 .6

. EVALUATION OF THE RE SULTS

The assay i s considered valid, if the following criteria are met:

- The negative (vehicle) control data are within the laboratory’s normal range for the

spontaneous aberration frequency.

- The positive controls induce increases in the aberration fr equency, which are

significant.Study code: 11/339

- 020C Final Report Page 23 of 66

The test item is considered to have shown clastogenic activity in this study if all of the

following criteria are met:

- Increases in the frequency of metaphases with aberrant chromosomes are observed

at one or more test concentrations (only data without gaps will be considered).

- The increases are reproducible between repli cate cultures and between tests (when

treatment conditions were the same).

- The increases are statistically significant.

- The increases are not associated with large changes in pH or osmolarity of the

treated cultures (large changes are identified with a difference of >0.5 in pH

compared to the vehicle control, or a difference of >50 mmol/kg in osmolality

compared to the vehicle control)

.

The historical control data for this laboratory were al so considered in the evaluation.

Evidence of a dose

- response r elationship (if any) w as considered to support the

conclusion.

The test item is concluded to have given a negative response if no reproducible,

statistically significant increases are observed.

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for

statistical analysis was the number of cells with one or more chromosomal aberrations

excluding gaps.

Historical control data are shown in Appendix 6

.

5. 7

. CHEMICALS USED IN TH E EXPERIMENTS

The chemicals used in the independent ly performed experiments of the study are

summarized in Table 1

.

Table 1: Chemicals u sed in the e xperiments

Chemical Supplier Lot Number Expiry Date

Acetic acid Lachner PP/2012/0 6731 3 0 June 201 7

Antibiotic

- antimycotic solution (100X) SIGMA

- ALDRICH SLBB 9274 March 2014

Colchicine SIGMA

- ALDRICH 080M1474V October 2013

Consul Mount (mounting medium) Shandon 223947 May 2016

Distilled water TEVA 3450611 June 2014Study code: 11/339

- 020C Final Report Page 24 of 66

Table 1: Chemicals u sed in the e xperiments (continued)

Chemical Supplier Lot Number Expiry Date

DMEM* SIGMA

- ALDRICH RNBC 4233 June 2013

DMEM* SIGMA

- ALDRICH RNBC 4829 August 2013

DMEM* SIGMA

- ALDRICH RNBC 5039 August 2013

Fetal bovine serum (FBS)** SIGMA

- ALDRICH 11A600 March 201 6

Giemsa solution*** SIGMA

- ALDRICH SZBC082A 03 September 2015

D

- G lucose

- 6

- pho s phate Na *** * SIGMA

- ALDRICH 079K3788V August 2012

HEPES SIGMA

- ALDRICH 041M54011V April 2015

KCl MERCK K42209636 30 April 2016

MgCl 2 SIGMA

- ALDRICH 030M0060 March 2014

Methanol VWR 12A180505 January 201 7

NADP SIGMA

- ALDRICH 051M7000V June 201 4

Phosphate buffered saline (PBS), (10X) SIGMA

- ALDRICH SLBB3269 January 201 3

Physiological saline TEVA 6931211 December 2013

Trypsin

- EDTA solution SIGMA

- ALDRICH SLBC165 5 July 2014

*: Dulbecco’s Modified Eagle’s Medium (DMEM) with 4500 mg/L glucose, L

- g lutamine, sodium

pyruvate and sodium bicarbonate

**: Heat

- inactivated, USA origin, sterile filtered, cell culture tested

***: Giemsa’s azur eosin methylene blue solution

  Stock solution was prepared of the original chemical on 06 August 2012 and stor ed at

- 23±8 o C. Expiry

date of the solution: 06 February 2013.

HEPES: N

- 2

- hydroxyethylpiperazine

- N

- 2

- ethane sulphonic acid

NADP: β

- Nicotinamide adenine dinucleotide phosphate

6

. ARCHIVES

The study documents and samples:

- study plan and study plan amend ment s

,

- all raw data and specimen s

,

- retained sample of the test item,

- correspondence,

- study report and any amendments

will be retained in the archives of CiToxLAB Hungary Ltd. (H

- 8200 Veszprém,

Szabadságpuszta, Hungary) according to the Hungarian G LP regulation [1 2 ] and to

test facility SOPs.Study code: 11/339

- 020C Final Report Page 25 of 66

After the retention time has elapsed, all the archived materials listed above would be

offered to the Sponsor or retained for a further period if agreed by a contract. No raw

data or material relating to th e study will be discarded without the Sponsor's prior

written consent.

Slides and the raw data (record sheets) were shipped back to the Test Facility. The

working copy of the Study Plan and the Report were retained in the Test Site.

7

. DEVIATION S TO THE STUDY PLAN

As additional slide reading was required, the transfer of slides and the metaphase

analysis was ended later; consequently , the D raft Report was issued later than

indicated in the Study Pl an

.

Due to technical reason, cells were maintained on 150 cm 2 flask instead of 75 cm 2

flasks before the experiments.

Th e s e deviation s w ere considered not to adversely affect the results or integrity of the

study

.

8

. RESULTS AND DISCUSSION

8 .1. VEHICLE AND CONCENTRATION SELECTION

Based on the results of a short preliminary solubility test and due to the acidic nature

of the test item, 1X PBS (phosphate buffered saline) which is compatible to the test

system was selected for vehicle of the study. The highest achievable concentration was

100 mg/mL using th is vehicle . The highest examined concentration in the preliminary

test was 5000 μg/mL.

T wo C oncentration Selection Cytotoxicity A ssay s (Assay A : 3

- hour treatment with

and without metabolic activation, 20

- hour harvesti ng time; and Assay B : 20

- hour

treatmen t without metabolic activation, 2 0

- hour harvesting time) were performed as

part of th e study to establish an appropriate concentration range for the Chromosome

Aberration Assays

.

In the preliminary toxicity test

, single cultures were tested and positive c ontrols were

not included. Following treatments, cell concentrations were determined using a

haemocytometer. Precipitation of the test item in the final culture medium was

visually examined at the begin ning and end of the treatments. Measurement of pH and

osmolality was also performed at the end of the treatment period.Study code: 11/339

- 020C Final Report Page 26 of 66

A total of nine test concentrations between 5 0 00 and 0.064 μ g/mL were used to

evaluate toxicity in each cytotoxicity assay. Detailed results of the cytotoxicity assays

are presented in Tables 2 , 3 and 4 of Appendix 3

. Treatment concentrations for the

chromosome aberration assays were selected on the basis of resul ts of the performed

Concentration Selection Cytotoxicity Assays according to the OECD guideline

instructions.

8 .2. CHROMOSOME ABERRATIO N ASSAY S

In Chromosome Aberration Assay 1, a 3

- hour treatment with metabolic activation (in

the presence of S9

- mix) a nd a 3

- hour treatment without metabolic activation (in the

absence of S9

- mix) were performed. Sampling was performed 20 hours after the

beginning of the treatment in both cases. The examined concentrations of the test ite m

were 4 00, 200, 150, 125, 100, 75, 5 0, 25 and 1 2

. 5 μg/mL

.

In Assay 1, no insolubility was detected at the end of the treatment period in the final

treatment medium in any of the examined treatment concentrations

. No large changes

in pH and osmolality were detected

. C ytotoxicity was observ ed in this assay at 4 00,

200, 150, 125 and 1 00 μg/mL concentrations without metabolic activation (relative

survival values were 7

, 10, 1 8 , 28 and 3 0 %, respectively) and at 400 , 200, 150 and

125 μg/mL concentrations with metabolic activation (relative surv ival values were 7

, 6

, 24 and 4 6 %, respectively) as detailed in Table s 5 and 6 of Appendix 4

. Therefore,

c oncentrations of 1 0 0

, 75, 50 and 25 μg/mL (a total of four ) were selected for

evaluation in case of the experiment with out metabolic activation and c oncentrations

of 125, 100 and 50 μg/mL (a total of three) were selected for evaluation in case of the

experiment with metabolic activation

. None of the treatment concentrations with or

without metabolic activation caused a significant increase in the numbe r of cells with

structural chromosome, thus this assay was considered to be negative.

In Chromosome Aberration Assay 2, a 3

- hour treatment with metabolic activation (in

the presence of S9

- mix) and a 20

- hour treatment without metabolic activation (in the

a bsence of S9

- mix) were performed. Sampling was performed 2 0 hours after the

beginning of the treatment in both cases. The examined concentrations of the test item

were 4 00, 2 00, 1 50, 1 25, 1 00

, 75, 50, 25 and 1 2

. 5 μg/mL.

I n Assay 2

, similarly to the first experiment, no insolubility was detected at the end of

the treatment period in the final treatment medium at any of the examined

concentrations. No large change s in pH and osmolality were detected

. Cytotoxicity

was observed at 4 00, 200, 150, 125, 100 and 7 5 μg/mL concentrations without

metabolic activation (relative survival values were 4, 2, 7, 14, 12 and 24 %,

respectively) and at 400, 200 , 150, 125 and 100 μg/mL concentrations (relative

survival values were 6

, 11, 14, 30 and 4 7 %, respectively) . Observati ons are detailed in

Tables 7 and 8 of Appendix 4. C oncentrations of 10 0

, 7 5 , 50 and 2 5 μg/mL (a total of

four ) were selected for evaluation in case of the experiment without metabolic

activation; and 1 0 0, 75 and 50 μg/mL (a total of three ) were selected fo r evaluation in

case of the experiment with metabolic activation

.Study code: 11/339

- 020C Final Report Page 27 of 66

None of the treatment concentrations caused a significant increase in the number of

cells with structural chromosome aberrations in either assay with or without metabolic

activation. Ther efore, this assay confirmed the negative results of the first main test

.

The occurrence of polyploid and endoreduplicated metaphases is presented in Tables

7

- 8 of Appendix 7

. P olyploid metaphases (1

- 5 ) were found in some cases in the

negative ( vehicle ) co ntrol

, test item treat ed or positive control samples in the

performed experiments. En doreduplicated metaphase s (1

- 1 4 ) w ere found in some

cases in the negative (vehicle) control, test item treated or positive control samples in

the performed experiments.

T he work phase report of the metaphase analysis is shown in Appendix 7

.

8 .3. VALIDITY OF THE STUD Y

The tested concentrations in the chromosome aberration assays were selected based

on the results of the preliminary experiments

. No i nsolubility , but c yto toxicity w as

detected in the performed main experiments . The evaluated concentration range s of

Assay 1 and Assay 2 w ere considered to be adequate

, as the y covered a concentration

range from cytotoxicity (less or equals to 50 % relative survival) to little or no

cytotoxic ity

.

Historical control data are presented in Appendix 6 . The spontaneous aberration

frequencies of the negative ( vehicle ) control s in the performed experiments were in

good harmony with t he historical control range of the testing laborator y.

Note: In Assay 2, the mean aberration frequency of the negative control was 6.0 % (experiment without

metabolic activation) and 5 .5% (experiment with metabolic activation) , thus slightly above the upper

limit of the general historical control range (0

- 5 ). However, this difference was minor and was

considered to be acceptable.

In the performed experiments, the positive control substance s ( Cyclophosphamide

(CP) in the experiments with metabolic activation and Ethyl methanesulfonate (EMS)

in the experiment s without metabolic activation) caused the expected biologically

relevant and statistically significant increase in the number of cells with structural

c hromosome aberrations (Tables 9

- 12 of Appendix 5 ) in each assay demonstrating the

sensitivity of the te st system.

At least three test item concentrations were examined in each assay.

The study w as considered to be valid.Study code: 11/339

- 020C Final Report Page 28 of 66

9

. CONCLUSION

The test item Ethanedioic acid, hydrate (1:2) was tested for potentia l clastogenic

activity using the Chromosome Abe rration Assay. The study included two

Concentration Selection Cytotoxicity Assays and t wo Chromosome Aberration

Assays.

The performed experiments were considered to be valid and to reflect the real

potential of the test item to cause structural chromosoma l aberrations in the cultured

V79 Chinese hamster cells used in this study.

Tr eatment with the test item did not result in a statistically and biologically significan t,

repeatable, dose

- dependent increase in the frequency of the cells with structural

chro mosome aberrations without gaps either in the presence or absence of a metabolic

activation system which was a cofactor

- supplemented post

- mitochondrial S9 fraction

prepared from the livers of phenobarbital/ ** β**

- naphthoflavone induced rats

.

In conclusion, Eth anedioic acid, hydrate (1:2) test item did not induce a

significant level of chromosome aberrations in the performed experiments with

or without metabolic activation. Therefore, Ethanedioic acid, hydrate (1:2) is

considered not clastogenic in this test sys tem.

10

. DISTRIBUTION OF THE FINAL REPORT

Sponsor: 1x copy, bound

1x copy, unbound

1x PDF file

Archive: 1x original, boundStudy code: 11/339

- 020C Final Report Page 29 of 66

11

. REFERENCES

1. Preston, K.J., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.C., Heddle, J.A.,

McFee, A.F., Wolff, Sh., Wassom, J.S. (1981): Mammalian in vivo and vitro

cytogenetic assays. Mutation Research, 87 , 143

- 188

2. Brusick, D. (1989). Genetic Toxicology. Principles and Methods of Toxicology,

(Ed.) A. Wallace Hayes, Raven Press, Ltd., New York

3. Ames, B. N., J oyce Mccann and Edith Yamasaki: Methods for Detecting

Carcinogens and Mutagens with the Salmonella /Mammalian

- Microsome

Mutagenicity Test. Mutat ion Research, 31: 347

- 364, 1975

4. Maron, D. M. and Ames, B. N.: Revised Method for the Salmonella Mutagenicity

Test. Mutation Research, 113: 173

- 215, 1983.

5

. Galloway,S.M. Aardema, M.J., Ishidate, M.,Jr., Ivett., J.L., Kirkland, D.J.,

(1994). Report from Working Group on in Vitro Tests for Chromosomal

Aberrations. Mutation Research, 312 , 241

- 261.

6

. Ishidate, M. Jr (1988). A proposed battery of tests for the initial evaluation of the

mutagenic potential of medicinal and industrial chemicals. Mutation Research,

205 , 397

- 407

7

. Kao, F.T. and Puck, T.T. (1967). Genetics of somatic mammalian cells. IV.

Properties of s omatic Chinese hamster cell mutants with respect to the

requirement for proline. Genetics. 55, 513

- 524

8

. Natarajan, A.T., Tates, A.B., van Buul, P. W., Meijers, M. and De Vogel, N.

(1976): Cytogenetic effects of mutagens/carcinogens after activation in

mi crosomal system in vitro. Mutation Research, 37 , 83

- 90

9

. Savage, J.R.K. (1976) Annotation: Classification and relationships of induced

chromosome structural change. Journal of Medical Genetics, 13 , 103

- 122

10

. Savage, J.R.K. (1983). Some practical notes o n chromosomal aberration. Clinical

Cytogenetics Bulletin, 1, 64

- 76

1 1

. UKEMS (1990) Scott,D., Danford,N., Dean,B., Kirkland,D.J.: Metaphase

chromosome aberration assay in vitro. Report of the UKEMS Sub

- Committee on

Guidelines for mutagenicity testing. Part I. Kirkland, D. J.(Ed.) United Kingdom

Environmental Mutagen Society. Recommended Procedures, pp. 62

- 85

12

. Hungarian Good Laboratory Practice Regulations: 9/2001 (III. 30) EÜM

- FVM

joint decree of the Minister of Health and the Minister of the Agricu l ture and

Regional Development (in Hungarian) which corresponds to the OECD GLP,

ENV/MC/CHEM(98)17)

13. ISCN (1985): An International System for Human Cytogenetic Nomenclature , ed.

D: G: Harnden et al. S. Karger, Switzerland.Study code: 11/339

- 020C Final Report Page 30 of 66



## APPENDICESStudy code: 11/339

- 020C Final Report Page 31 of 66

APPENDIX 1

S TUDY S CHEDULE

Preliminary experiment

Cell seeding (Day

- 1) : 29 October 2012

Date of treatment : 30 October 2012

Evaluation : 31 October 2012

Assay 1

Cell seeding (Day

- 1) : 05 November 2012

Start of Assay 1 (Treatment) : 0 6 November 2012

End of Assay 1 : 07 November 2012

Staining of slides : 08 November 2012

Assay 2

Cell seeding (Day

- 1) : 13 November 2012

Start of Assay 2 (Treatment) : 14 November 2012

End of Assay 2 : 15 November 2012

Staining of slides : 16 November 2012

Note: Additional slide droppi ng and staining was performed on 07

- 10 December 2012

and 13

- 14 February 2013.

Transfer of slides : 1 8 February 2 01 3

Metaphase analysis : 15 March 201 3

Draft Report : 19 March 2013

Final Report : 22 March 2013Study code: 11/339

- 020C Final Report Page 32 of 66

APPENDIX 2

COPY OF THE CERTIFIC ATE OF ANALYSISStudy code: 11/339

- 020C Final Report Page 33 of 66

APPENDIX 3

SUMMARIZED RESULTS O F THE CONCENTRATION SELECTION CYTOTOXICI TY ASSAYS

Table 2 : Summarized results of the Concentration Selection Cytotoxicity Assay A experiment without metabolic activation

Test group Dose

(μg/mL) S9

- mix Treatm ent/

sampling

time Cell number

(cells/mL) Relative

Survival (%)* Observations

beginning / end of treatment

Untreated control

– – 3/20 2.35E+05 77 normal / normal (pH: 7.4; osm: 332 mmol/kg)

Negative (vehicle) control (1X PBS, 1%)

– – 3/20 3.05E+05 100 normal / normal (pH: 7.4; osm: 326 mmol/kg)

Negative (vehicle) control (1X PBS, 2.5%)

– – 3/20 2.30E+05 100 normal /normal (pH: 7.4; osm: 322 mmol/kg)

Negative (vehicle) control (1X PBS, 5%)

– – 3/20 2.20E+05 100 normal /normal (pH: 7.4; osm: 321 mmol/k g)

Ethanedioic acid, hydrate (1:2) 5000

– 3/20 0.00E+00 0 ** discoloured medium /discoloured medium

(pH: 3.0; osm: 335 mmol/kg)

2500

– 3/20 0.00E+00 0 *** discoloured medium / discoloured medium

(pH: 4.0; osm: 300 mmol/kg)

1000

– 3/20 0.00E+00 0 discolo ured medium / discoloured medium

(pH: 7.0; osm: 304 mmol/kg)

200

– 3/20 2.00E+04 7 discoloured medium / discoloured medium

(pH: 7.4; osm: 319 mmol/kg)

40

– 3/20 2.85E+05 93 normal /normal (pH: 7.4; osm: 322 mmol/kg)

8

– 3/20 2.38E+05 78 normal /norma l (pH: 7.4; osm: 323 mmol/kg)

1.6

– 3/20 2.43E+05 80 normal /normal (pH: 7.4; osm: 326 mmol/kg)

0.32

– 3/20 1.90E+05 62 normal / normal (pH: 7.4; osm: 330 mmol/kg)

0.064

– 3/20 3.35E+05 110 normal / normal (pH: 7.4; osm: 337 mmol/kg)

*: compared to the 1% negative (vehicle) control

**: compared to the 5% negative (vehicle) control

***: compared to the 2.5% negative (vehicle) control

osm: osmolality

Note: Duplicate counts were performed at each counting.Study code: 11/339

- 020C Final Report Page 34 of 66

APPENDIX 3 ( c o ntinued )

SUMMARIZED RESULTS O F THE CONCENTRATION SELECTION CYTOTOXICI TY ASSAYS

Table 3 : Summarized results of the Concentration Selection Cytotoxicity Assay A experiment with metabolic activation

Test group Dose

(μg/mL) S9

- mix Treatment/

sampling

time Cell number

(cells/mL) Relativ e

Survival (%)* Observations

beginning / end of treatment

Untreated control

– + 3/20 2.55E+05 78 normal / normal (pH: 7.2; osm: 326 mmol/kg)

Negative (vehicle) control (1X PBS, 1%)

– + 3/20 3.28E+05 100 normal / normal (pH: 7.2; osm: 325 mmol/kg)

Nega tive (vehicle) control (1X PBS, 2.5%)

– + 3/20 2.68E+05 100 normal / normal (pH: 7.2; osm: 324 mmol/kg)

Negative (vehicle) control (1X PBS, 5%)

– + 3/20 2.60E+05 100 normal / normal (pH: 7.2; osm: 325 mmol/kg)

Ethanedioic acid, hydrate (1:2) 5000 + 3/20 0.00E+00 0 ** discoloured medium / discoloured medium

(pH: 3.0; osm: 340 mmol/kg)

2500 + 3/20 0.00E+00 0 *** discoloured medium / iscoloured medium

(pH: 4.0; osm: 304 mmol/kg)

1000 + 3/20 0.00E+00 0 discoloured medium / discoloured medium

(pH: 7.0; osm: 302 mmol/kg)

200 + 3/20 3.00E+04 9 discoloured medium / discoloured medium

(pH:7.4; osm: 316 mmol/kg)

40 + 3/20 2.48E+05 76 slightly discoloured medium / slightly discoloured

medium (pH: 7.4; osm: 318 mmol/kg)

8 + 3/20 2.70E+05 82 normal / normal (pH : 7.4; osm: 323 mmol/kg)

1.6 + 3/20 2.78E+05 85 normal / normal (pH: 7.4; osm: 324 mmol/kg)

0.32 + 3/20 2.10E+05 64 normal / normal (pH: 7.4; osm: 325 mmol/kg)

0.064 + 3/20 1.98E+05 60 normal / normal (pH: 7.4; osm: 332 mmol/kg)

*: compared to the 1 % negative (vehicle) control

**: compared to the 5% negative (vehicle) control

***: compared to the 2.5% negative (vehicle) control

osm: osmolality

Note: Duplicate counts were performed at each counting.Study code: 11/339

- 020C Final Report Page 35 of 66

APPENDIX 3 ( continued )

SUMMARIZED RESULTS O F THE CONCENTRATION SELECT ION CYTOTOXICITY ASS AYS

Table 4 : Summarized results of the Concentration Selection Cytotoxicity Assay B experiment without metabolic activation

Test group Dose

(μg/mL) S9

- mix Treatment/

sampling

time Cell number

(cells/mL) Relative

Survival (%)* Observations

beginning / end of treatment

Untreated control

– – 20/2 0 2.75E+05 98 normal /normal (pH: 7.2; osm: 345 mmol/kg)

Negative (vehicle) control (1X PBS, 1%)

– – 20/20 2.80E+05 100 normal / normal (pH: 7.2; osm: 341 mmol/kg)

Negat ive (vehicle) control (1X PBS, 2.5%)

– – 20/2 0 2.50E+05 100 normal / normal (pH: 7.2; osm: 339 mmol/kg)

Negative (vehicle) control (1X PBS, 5%)

– – 20/20 2.50E+05 100 normal /normal (pH: 7.2; osm: 337 mmol/kg)

Ethanedioic acid, hydrate (1:2) 5000

– 20/20 0.00E+00 0 ** discoloured medium /discoloured medium

(pH: 3.0; osm: 352 mmol/kg)

2500

– 20/20 0.00E+00 0 *** discoloured medium / discoloured medium

(pH: 4.0; osm: 317 mmol/kg)

1000

– 20/20 0.00E+00 0 discoloured medium / discoloured medium

(pH: 7.2; os m: 320 mmol/kg)

200

– 20/20 1.50E+04 5 discoloured medium / normal

(pH: 7.4; osm: 333 mmol/kg)

40

– 20/20 2.00E+05 71 normal /normal (pH: 7.4; osm: 338 mmol/kg)

8

– 20/20 1.75E+05 63 normal /normal (pH: 7.4; osm: 340 mmol/kg)

1.6

– 20/20 1.73E+05 62 normal /normal (pH: 7.4; osm: 341 mmol/kg)

0.32

– 20/20 2.13E+05 76 normal / normal (pH: 7.4; osm: 342 mmol/kg)

0.064

– 20/20 2.00E+05 71 normal / normal (pH: 7.4; osm: 349 mmol/kg)

*: compared to the 1% negative (vehicle) control

**: compared to t he 5% negative (vehicle) control

***: compared to the 2.5% negative (vehicle) control

osm: osmolality

Note: Duplicate counts were performed at each counting.Study code: 11/339

- 020C Final Report Page 36 of 66

APPENDIX 4

CYTOTOXICITY RESULTS OF THE MAIN EXPERIME NTS

Table 5 : Cytotoxicity Results of Chr omosome Aberration Assay 1 experiment without metabolic activation

Test group Dose

μg/mL S9

- mix Treatment/

sampling

time Cell number

(cells/mL) Relative

Survival (%)* Observations

beginning / end of treatment

Negative (vehicle) control (1X PBS, 1%)

– – 3/20 3.53E+05 100 normal / normal (pH: 7.4; osm: 336 mmol/kg)

Ethanedioic acid, hydrate (1:2) 400

– 3/20 2.50E+04 7 discoloured medium / slightly discoloured medium

(pH: 7.4; osm: 324 mmol/kg)

200

– 3/20 3.50E+04 10 slightly discoloured medium / slight ly discoloured

medium (pH: 7.4; osm:327 mmol/kg)

150

– 3/20 6.25E+04 18 slightly discoloured medium / normal

(pH: 7.4; osm: 331 mmol/kg)

125

– 3/20 1.00E+05 28 slightly discoloured medium / normal

(pH: 7.4; osm: 324 mmol/kg)

100

– 3/20 1.08E+05 30 slightly discoloured medium / normal

(pH: 7.4; osm: 329 mmol/kg)

75

– 3/20 1.78E+05 50 normal / normal (pH: 7.4; osm: 335 mmol/kg)

50

– 3/20 3.15E+05 89 normal / normal (pH: 7.4; osm: 346 mmol/kg)

25

– 3/20 3.75E+05 106 normal / normal (pH: 7.4; osm : 330 mmol/kg)

12.5

– 3/20 3.48E+05 99 normal / normal (pH: 7.4; osm: 332 mmol/kg)

Positive control (1 μL/mL EMS)

– – 3/20 3.33E+05 94 normal / normal (pH: 7.4; osm: 348 mmol/kg)

*: compared to negative ( vehicle ) control

osm: osmolality

Note s : Duplicate counts were performed at each counting.Study code: 11/339

- 020C Final Report Page 37 of 66

APPENDIX 4 ( continued )

CYTOTOXICITY RESUL TS OF THE MAIN EXPER IMENTS

Table 6 : Cytotoxicity Results of Chromosome Aberration Assay 1 experiment with metabolic activation

Test group Dose

μg/mL S9

- mix Treatment/

sampling

time Cell number

(cells/mL) Relative

Survival (%)* Observations

beginning / end of treatment

Negative (vehicle) control (1X PBS, 1%)

– + 3/20 3.60E+05 100 normal / normal (pH: 7.2; osm: 331 mmol/kg)

Ethanedioic acid, hydrate (1:2) 400 + 3/20 2.50E+04 7 discoloured medium / slightly discoloured medium

(pH: 7.2; osm: 316 mmol/kg)

200 + 3/20 2.00E+04 6 slightly discoloured medium / slightly discoloured

medium (pH: 7.2; osm:323 mmol/kg)

150 + 3/20 8.50E+04 24 slightly discoloured medium / slightly discoloured

medium (pH: 7.2; osm: 324 mmol/kg)

125 + 3/20 1.65E+05 46 slightly di scoloured medium / normal

(pH: 7.2; osm: 322 mmol/kg)

100 + 3/20 2.40E+05 67 slightly discoloured medium / normal

(pH: 7.2; osm: 326 mmol/kg)

75 + 3/20 3.23E+05 90 slightly discoloured medium / normal

(pH: 7.2; osm: 328 mmol/kg)

50 + 3/20 3.4 0 E+05 94 normal / normal (pH: 7.2; osm: 330 mmol/kg)

25 + 3/20 3.75E+05 104 normal / normal (pH: 7.2; osm: 323 mmol/kg)

12.5 + 3/20 3.18E+05 88 normal / normal (pH: 7.2; osm: 331 mmol/kg)

Positive control (6 μg/mL CP)

– + 3/20 2.20E+05 61 normal / normal ( pH: 7.2; osm: 330 mmol/kg)

*: compared to negative ( vehicle ) control

osm: osmolality

Note s : Duplicate counts were performed at each counting.Study code: 11/339

- 020C Final Report Page 38 of 66

APPENDIX 4 ( continued )

CYTOTOXICITY RESULTS OF THE MAIN EXPERIME NTS

Table 7 : Cytotoxicity Results of Chromoso me Aberration Assay 2 experiment without metabolic activation

Test group Dose

μg/mL S9

- mix Treatment/

sampling

time Cell number

(cells/mL) Relative

Survival (%)* Observations

beginning / end of treatment

Negative (vehicle) control (1X PBS, 1%)

– – 20/2 0 2.88E+05 100 normal / normal (pH: 7.4; osm: 344 mmol/kg)

Ethanedioic acid, hydrate (1:2) 400

– 20/20 1.25E+04 4 discoloured medium / slightly discoloured medium

(pH: 7.4; osm: 334 mmol/kg)

200

– 20/20 5.00E+03 2 slightly discoloured medium / slightly discoloured

medium (pH: 7.4; osm:334 mmol/kg)

150

– 20/20 2.00E+04 7 slightly discoloured medium / slightly discoloured

medium (pH: 7.4; osm: 334 mmol/kg)

125

– 20/20 4.00E+04 14 slightly discoloured medium / normal

(pH: 7.4; osm: 334 mmol/kg)

100

– 20/20 3.50E+04 12 slightly discoloured medium / normal

(pH: 7.4; osm: 334 mmol/kg)

75

– 20/20 7.00E+04 24 normal / normal (pH: 7.4; osm: 340 mmol/kg)

50

– 20/20 2.55E+05 89 normal / normal (pH: 7.4; osm: 335 mmol/kg)

25

– 20/20 3.03E+05 105 normal / normal (pH: 7.4; osm: 335 mmol/kg)

12.5

– 20/20 3.55E+05 123 normal / normal (pH: 7.4; osm: 339 mmol/kg)

Positive control (0.4 μL/mL EMS)

– – 20/20 2.05E+05 71 normal / normal (pH: 7.4; osm: 350 mmol/kg)

*: compared to negative ( vehicle ) control

osm: osmolality

Note: Duplicate counts were performed at each counting.Study code: 11/339

- 020C Final Report Page 39 of 66

APPENDIX 4 ( continued )

CYTOTOXICITY RES ULTS OF THE MAIN EXP ERIMENTS

Table 8 : Cytotoxicity Results of Chromosome Aberration Assay 2 experiment with metabolic activation

Test group Dose

μg/mL S9

- mix Treatment/

sampling

time Cell number

(cells/mL) Relative

Survival (%)* Observations

beginning / end of treatment

Negative (vehicle) control (1X PBS, 1%)

– + 3/20 3.13E +05 100 normal / normal (pH: 7.4; osm: 325 mmol/kg)

Ethanedioic acid, hydrate (1:2) 400 + 3/20 2.00E +04 6 discoloured medium / slightly discoloured medium

(pH: 7.4; osm: 318 mmol/kg )

200 + 3/20 3.50E +04 11 slightly discoloured medium / slightly discoloured

medium (pH: 7.4; osm:321 mmol/kg)

150 + 3/20 4.50E +04 14 slightly discoloured medium / slightly discoloured

medium (pH: 7.4; osm: 327 mmol/kg)

125 + 3/20 9.50E +04 30 slightly discoloured medium / slightly discoloured

medium (pH: 7.4; osm: 318 mmol/kg)

100 + 3/20 1.48E +05 47 slightly discoloured medium / normal

(pH: 7.4; osm: 326 mmol/kg)

75 + 3/20 1.83E +05 58 slightly discoloured medium / normal

(pH: 7.4; osm: 326 mmol/k g)

50 + 3/20 2.45 E+05 78 normal / normal (pH: 7.4; osm: 329 mmol/kg)

25 + 3/20 3.35 E+05 107 normal / normal (pH: 7.4; osm: 319 mmol/kg)

12.5 + 3/20 2.90 E+05 93 normal / normal (pH: 7.4; osm: 323 mmol/kg)

Positive control (6 μg/mL CP)

– + 3/20 1.80 E+ 05 58 normal / normal (pH: 7.4; osm: 331 mmol/kg)

*: compared to negative ( vehicle ) control

osm: osmolality

Note: Duplicate counts were performed at each counting.Study code: 11/339

- 020C Final Report Page 40 of 66

APPENDIX 5

SUMMARY TABLES OF TH E MAIN EXPERIMENTS

Table 9 : Summary table of Chromosome Aberration Assay 1 without metabolic activation

Concentration

( μ g/mL) Time of

Treatment /

Sampling Relative

Survival #

(%) Insolubility ## Mean %

aberrant

cells ###

Ethanedioic acid, hydrate (1:2) without metabolic activation (

- S9)

Negative (vehicle) control 3h / 20h 100

– 1.5

400 μ g/mL 3h / 20h 7

– a NE

200 μ g/mL 3h / 20h 10

– a NE

150 μ g/mL 3h / 20h 18

– NE

125 μ g/mL 3h / 20h 28

– NE

100 μ g/mL 3h / 20h 30

– 2.0

75 μ g/mL 3h / 20h 50

– 1. 5

50 μ g/mL 3h / 20h 89

– 1.0

25 μ g/mL 3h / 20h 106

– 2.5

12.5 μ g/mL 3h / 20h 99

– NE

Positive control 3h / 20h 94

– 8.25***

N egative ( vehicle ) control: 1 X PBS

Positive control (

- S9): Ethyl methanesulfonate, 1 μL/mL

NE: not evaluated

#: compared to the negative ( vehicle ) control

##: in the final treatment me dium at the end of the treatment

###: excluding gaps

a : Discoloured mediu mStudy code: 11/339

- 020C Final Report Page 41 of 66

APPENDIX 5 ( continued )

SUMMARY TABLES OF TH E MAIN EXPERIMENTS

Table 1 0 : Summary table of Chromosome Aberration Assay 1 with metabolic activation

Concentration

( μ g/mL) Time of

Treatment /

Sampling Relative

Survival #

(%) Insolubility ## Mean %

aberrant

cells ###

Ethanedioic acid, hydrate (1:2) with metabolic activation (+S9)

Negative (vehicle) control 3h / 20h 100

– 2.0

400 μ g/mL 3h / 20h 7

– a NE

200 μ g/mL 3h / 2 0h 6

– a NE

150 μ g/mL 3h / 20h 24

– a NE

125 μ g/mL 3h / 20h 46

– 1.5

100 μ g/mL 3h / 20h 67

– 1.5

75 μ g/mL 3h / 20h 90

– NE

50 μ g/mL 3h / 20h 94

– 1.0

25 μ g/mL 3h / 20h 104

– NE

12.5 μ g/mL 3h / 20h 88

– NE

Positive control 3h / 20h 61

– 90.9***

Negat ive (vehicle) control: 1X PBS

Positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

a: Discoloured medium

***: p< 0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative controlStudy code: 11/339

- 020C Final Report Page 42 of 66

APPENDIX 5 ( continued )

SUMMARY TABLES OF TH E MAIN EXPERIMENTS

Table 1 1 : Summary table of Chromosome Aberration Assay 2 without metabolic activation

Concentrati on

( μ g/mL) Time of

Treatment /

Sampling Relative

Survival #

(%) Insolubility ## Mean %

aberrant

cells ###

Ethanedioic acid, hydrate (1:2) without metabolic activation (

- S9)

Negative (vehicle) control 20h / 20h 100

– 6.0

400 μ g/mL 20h / 20h 4

– a NE

200 μ g/ mL 20h / 20h 2

– a NE

150 μ g/mL 20h / 20h 7

– a NE

125 μ g/mL 20h / 20h 14

– NE

100 μ g/mL 20h / 20h 12

– 6.5

75 μ g/mL 20h / 20h 24

– 7.5

50 μ g/mL 20h / 20h 89

– 4.0

25 μ g/mL 20h / 20h 105

– 5.5

12.5 μ g/mL 20h / 20h 123

– NE

Positive control 20h / 20h 71

– 40.5***

Negative ( vehicle ) control: 1 X PBS

Positive control (

- S9): Ethyl methanesulfonate, 0.4 μL/mL

NE: not evaluated

#: compared to the negative ( vehicle ) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

a : Discoloured medium

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative controlStudy code: 11/339

- 020C Final Report Page 43 of 66

APPENDIX 5 ( continued )

SUMMARY TABLES OF TH E MAIN EXPERIMENTS

Table 1 2 : Summary table of Chromosome Aberration Assay 2 with metaboli c activation

Concentration

( μ g/mL) Time of

Treatment /

Sampling Relative

Survival #

(%) Insolubility ## Mean %

aberrant

cells ###

Ethanedioic acid, hydrate (1:2) with metabolic activation (+S9)

Negative (vehicle) control 3h / 20h 100

– 5.5

400 μ g/mL 3h / 2 0h 6

– a NE

200 μ g/mL 3h / 20h 11

– a NE

150 μ g/mL 3h / 20h 14

– a NE

125 μ g/mL 3h / 20h 30

– a NE

100 μ g/mL 3h / 20h 47

– 7.5

75 μ g/mL 3h / 20h 58

– 3.5

50 μ g/mL 3h / 20h 78

– 8.0

25 μ g/mL 3h / 20h 107

– NE

12.5 μ g/mL 3h / 20h 93

– NE

Positive contro l 3h / 20h 58

– 85.7 ***

Negative (vehicle) control: 1X PBS

Positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

a: Discoloured medium

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative controlStudy code: 11/339

- 020C Final Report Page 44 of 66

APPENDIX 6

HISTORICAL CONTROL D ATA

(updated on 13 February 2013)

3/20h treatment/sampling time without S9

- mix

aberration rat e

(phases with aberration in %)

negative control positive control

(EMS)

incl. Gaps excl. Gaps incl. Gaps excl. Gaps

Mean 2.71 1.20 22.67 18.02

SD 1.65 0.82 12.43 8.16

Range 0

- 7 0

- 3 4

- 63 4

- 40

n 46 46 40 40

3h/20h treatment/sampling time with S9

- m ix

aberration rate

(phases with aberration in %)

negative control positive control

(CP)

incl. Gaps excl. Gaps incl. Gaps incl. Gaps

Mean 3.24 1.45 73.00 69.50

SD. 1.57 0.95 23.51 25.64

Range 0

- 8 0

- 4 21

- 100 21

- 100

n 43 43 21 21

SD

= standard dev iation

Range = min.

- max. values

n = number of experiments

EMS

= Ethyl methanesulfonate

CP = Cyclophosphamide

Notes:

1. In the period of 2008

- 2009, NNDA ( N

- Nitrosodimethylamine) was used as positive control substance in the

experiments with metabolic activat ion. Mean aberration frequency for NNDA was 22.91 (including gaps)

and 18.07 (excluding gaps) in 22 experiments.

2. Counting for a positive control sample was halted when 15 aberrant cells were counted.Study code: 11/339

- 020C Final Report Page 45 of 66

APPENDIX 6 (continued)

HISTORICAL CONTROL D ATA

(updat ed on 13 February 2013)

20h/28h treatment/sampling time without S9

- mix

aberration rate

(phases with aberration in %)

negative control positive control

(EMS)

incl. Gaps excl. Gaps incl. Gaps excl. Gaps

Mean 2.58 1.13 34.97 30.96

SD. 1.81 0.86 10.94 9.80

Range 0

- 8 0

- 4 5

- 76 5

- 68.2

n 43 43 43 43

3h/28h treatment/sampling time with S9

- mix

aberration rate

(phases with aberration in %)

negative control positive control

(CP)

incl. Gaps excl. Gaps incl. Gaps excl. Gaps

Mean 3.07 1.47 52.63 48.80

SD. 1.64 1.07 23.98 24.49

Range 0

- 8 0

- 5 7

- 93.8 6

- 93.8

n 43 43 21 21

SD

= standard deviation

Range = min.

- max. values

n = number of experiments

EMS

= Ethyl methanesulfonate

CP = Cyclophosphamide

Notes:

1. In the period of 2008

- 2009, NNDA ( N

- Nitrosodim ethylamine) was used as positive control substance in the

experiments with metabolic activation. Mean aberration frequency for NNDA was 22.34 (including gaps)

and 18.36 (excluding gaps) in 22 experiments.

2. Counting for a positive control sample was halted w hen 15 aberrant cells were counted.Study code: 11/339

- 020C Final Report Page 46 of 66

APPENDIX 7

REPORT OF THE METAPH ASE ANALYSISStudy code: 11/339

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- 020C Final Report Page 64 of 66

APPENDIX 8

COPIES OF THE GLP CE RTIFICATES

The facility name until 1 st September 2011 was LAB Research Ltd. From that date, the registered name has been CiToxLAB Hungary Ltd.,

this information has been transmitted to the GLP competent authority. The above GLP certificate is valid for this facility (n ow known as

CiToxLAB Hungary Ltd. ) until the certificate e xpires (16 December 2012).Study code: 11/339

- 020C Final Report Page 65 of 66

APPENDIX 8 (continued)

COPIES OF THE GLP CE RTIFICATESStudy code: 11/339

- 020C Final Report Page 66 of 66

APPENDIX 8 (continued)

COPIES OF THE GLP CE RTIFICATES