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 March 2013



Study 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 3760

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 1st September 2011)

Address H-8200 Veszprém, Szabadságpuszta

Hungary

Phone: +36 88 545 300

Fax: +36 88 545 301

TEST SITE : Microptic Cytogenetics

(Metaphase analysis) Address: 2 Langland Close, Mumbles

Swansea, SA3 4LY,

United Kingdom

Phone: +44 (0) 1792 366714

Fax: +44 (0) 1792 366714
Study code: 11/339-020C Final Report Page 6 of 66

STUDY DIRECTOR : Judit Hargitai, PhD.

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 QAU

Éva Fábiánné Makovi, B.Sc.

QA Inspector

Diána Czuczay, M.Sc.

QA Inspector

TEST SITE

QUALITY ASSURANCE : Neville Jones, B.Sc.

Microptic Cytogenetics

RESPONSIBLE PERSONS : Annamária Blumenschein - technical team leader

Bernadett Dabosi - technical assistant

Mónika Szabóné Oláh - technical assistant

Lívia Lukács - technical assistant

Zoltán Bácskay - technical assistant 

Tamás Mészáros, PhD. - Central Dispensary
Study code: 11/339-020C Final Report Page 7 of 66

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

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

EPA Health Effects Test Guidelines, OPPTS 870.5375

STUDY PLAN : 23 February 2012

AMENDMENT 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 : 06 November 2012

END OF EXPERIMENT : 14 February 2013

END OF METAPHASE ANALYSIS : 15 March 2013

DRAFT REPORT : 19 March 2013

FINAL REPORT : 22 March 2013

Detailed study schedule is shown in Appendix 1.
Study code: 11/339-020C Final Report Page 8 of 66

CONTENTS

Page

STATEMENT OF THE STUDY DIRECTOR 2

STATEMENT OF THE MANAGEMENT 3

QUALITY ASSURANCE STATEMENT 4

GENERAL INFORMATION 5

1. SUMMARY 10

2. INTRODUCTION 12

3. OBJECTIVE 12

4. GUIDELINES 12

5. MATERIALS AND METHODS 13

5.1. TEST ITEM 13

5.2. NEGATIVE AND POSITIVE CONTROLS 14

5.3. INDICATOR CELLS 15

5.4. EXTERNAL METABOLIC ACTIVATION SYSTEM 16

5.5. TEST PROCEDURE 18

5.6. EVALUATION OF THE RESULTS 22

5.7. CHEMICALS USED IN THE EXPERIMENTS 23

6. ARCHIVES 24

7. DEVIATIONS TO THE STUDY PLAN 25

8. RESULTS AND DISCUSSION 25

8.1. VEHICLE AND CONCENTRATION SELECTION 25

8.2. CHROMOSOME ABERRATION ASSAYS 26

8.3. VALIDITY OF THE STUDY 27

9. CONCLUSION 28

10. DISTRIBUTION OF THE FINAL REPORT 28

11. REFERENCES 29

APPENDICES 30

APPENDIX 1 STUDY SCHEDULE 31

APPENDIX 2 COPY OF THE CERTIFICATE OF ANALYSIS 32

APPENDIX 3 SUMMARIZED RESULTS OF THE CONCENTRATION 

SELECTION CYTOTOXICITY ASSAYS 33

APPENDIX 4 CYTOTOXICITY RESULTS OF THE MAIN EXPERIMENTS 36

APPENDIX 5 SUMMARY TABLES OF THE MAIN EXPERIMENTS 40

APPENDIX 6 HISTORICAL CONTROL DATA 44

APPENDIX 7 REPORT OF THE METAPHASE ANALYSIS 46

APPENDIX 8 COPIES OF THE GLP CERTIFICATES 64
Study code: 11/339-020C Final Report Page 9 of 66

LIST OF TABLES

Page

Table 1:

Chemicals Used in the Experiments 23-24

Table 2:

Summarized results of the Concentration Selection Cytotoxicity Assay A

experiment without metabolic activation 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 35

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 37

Table 7:

Cytotoxicity results of Chromosome Aberration Assay 2 

experiment without metabolic activation 38

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 40

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 activation 43
Study 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 lung cells. The test item was formulated in phosphate 

buffered saline and it was examined up to the cytotoxic concentrations 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 test 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) were performed. Sampling was performed 20 hours after the 

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

were 400, 200, 150, 125, 100, 75, 50, 25 and 12.5 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. Cytotoxicity was observed in this assay at 400, 

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

survival values were 7, 10, 18, 28 and 30 %, 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 100, 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 metabolic 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 20 hours after the 

beginning of the treatment in both cases. The examined 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 examined 

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) and 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 structural 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 

main tests. Polyploid metaphases were found in some cases in the negative (vehicle) 

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

Endoreduplicated metaphases (1-14) 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 significant increase in the number of structural 

aberrations excluding gaps in the experiments with or without metabolic activation 

demonstrating the sensitivity of the test system. The evaluated concentration range 

was considered to be adequate; at least three test item treated concentrations were 

evaluated in each assay. The tests were considered to be valid.

In conclusion, Ethanedioic 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 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 results 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 lethal 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 chromosome 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 followed the procedures 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 1998
Study code: 11/339-020C Final Report Page 13 of 66

5. MATERIALS 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 ethanedioic acid [CAS RN 144-62-7])

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

Chemical name: Oxalic acid, hydrate

CAS number: 6153-56-6

Batch/Lot number: YP1-20120528C

Molecular formula: C2H2O4 x 2H2O

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 (15-25oC, < 70 RH%)

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

glasses and face mask) for unknown materials were

applied to assure personnel health and safety.

*Note: Based on the Certificate of Analysis (dated 01 June 2012), Test Item Data Sheet (dated 26

September 2012), Material Safety Data Sheet (dated 16 November 2009) and additional information 

(e-mail 26 September 2012) provided by the Sponsor.

5.1.2. Identification, Receipt

The test item 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 the 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 were prepared in a sufficient 

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

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

weight of test item was calculated based on the test material alone. 
Study code: 11/339-020C Final Report Page 14 of 66

A supplementary pH buffering in the system was 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 was measured before treatment to prove that the amount of buffering 

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

Stock formulations of the test item (100 mg/mL) were prepared as follows. The 

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

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

homogeneity was reached, then the volume was adjusted to the final volume. From 

the stock formulation several dilutions were prepared using the selected vehicle; the

dosing solutions were prepared immediately before 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 

preparation 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 conversion, 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 POSITIVE CONTROLS

Negative (vehicle) and positive controls 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)

Supplier: Sigma-Aldrich Co.

Concentration: 10X

Lot No.: SLBB3269

Appearance: Colourless liquid

Expiry date: January 2013

Storage conditions: Room temperature, under N2
Study code: 11/339-020C Final Report Page 15 of 66

Name: Distilled water

Supplier: TEVA

Lot No.: 3450611

Appearance: Colourless liquid

Expiry 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

Appearance: Colourless liquid

Expiry date: 17 July 2013

Storage conditions: Room temperature, under N2

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 2013

Storage conditions: 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 used 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)

Morphology: Fibroblast
Study 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 suitable 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 line was 

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

were kept in a freezer at -80 ± 10°C. The stock was checked for mycoplasma 

infection. No infection of mycoplasma was noted.

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

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

cultures were maintained in 150 cm

2 plastic flasks at 37 ± 0.5 °C in a humidified 

atmosphere containing approximately 5 % CO2 in air. The V79 cells for this study 

were 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 μg/mL amphotericin-B) and 

10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium). During 

the treatments, the serum content of the medium was reduced to 5 (v/v) % (DMEM5).

5.4. EXTERNAL METABOLIC ACTIVATION SYSTEM

An advantage of using in vitro cell cultures is the accurate 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 in 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 hour before sacrifice when food was removed. Initiations of the induction of liver 

enzymes used for preparation S9 used in this study were 28 May 2012.

5.4.2. Preparation of Rat Liver Homogenate S9 Fraction

On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2, 

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 portions, 

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 fraction used was determined to be 36.0 g/L. The date 

of preparation of S9 fraction used in this study 31 May 2012 (CiToxLAB code: 

E11364). 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 enzymes. The batch of S9 used in this study functioned appropriately.

5.4.3. Preparation of S9-mix

The complete S9-mix was freshly prepared on the day of use according to the 

following ratio: 

S9 fraction 3 mL

HEPES 20 mM 2 mL

KCl 330 mM 1 mL

MgCl2 50 mM 1 mL

NADP 40 mM 1 mL

Glucose-6-phosphate 50 mM 1 mL

DME medium 1 mL
Study code: 11/339-020C Final Report Page 18 of 66

Prior to addition to the culture medium the S9-mix was kept in an ice bath.

For all 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. Toxicity and Concentration 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 assays were 

performed. In Assay A, cells were treated for 3-hours in the presence 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 20-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) control as 

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

Appendix 3.

5.5.2. Chromosome Aberration Assays

The Chromosome Aberration Assays were conducted as two independent experiments

(Assay 1 and Assay 2) in the presence 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, harvesting: 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 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 

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

Experimental Design of Assay 2

20-hr treatment without S9 Mix, harvesting: 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) 0.4 μL/mL

3-hr treatment with S9 Mix, harvesting: 20 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 

observed 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 % confluency) 

were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 105 

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 was replaced with 

9.9 mL treatment medium (DMEM-5) in case of experiments without metabolic 

activation or with 9.4 mL treatment medium (DMEM-5) + 0.5 mL S9-mix in case of 

experiments with metabolic activation.

Cells were treated with different concentration test item solutions, negative (vehicle) 

or positive control solution (treatment volume: 100 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% Antibioticantimycotic 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 was 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 time, the number of 

surviving cells was 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 treated 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 

became 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 slides were given random unique code numbers at the Test Facility by a 

person who was not involved in the metaphase analysis. The code labels covered all 

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

bias.

The coded slides were sent for evaluation to the Principal Investigator to:

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

Microptic Cytogenetics

2 Langland Close

Mumbles

Swansea, SA3 4LY, United Kingdom

The metaphase analysis was conducted under the control of the Principal Investigator. 

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 one hundred metaphases* 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 defined 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 reunion 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 breakage, 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 number 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 chromosomes 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 was 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 Practice (as revised in 1997). These Principles are in 

conformity with other international GLP regulations.

The Quality Assurance Unit at the Test Site performed study and/or process-based 

inspections according to its own Standard Operating Procedures. Inspection results 

were reported promptly in writing 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 received the signed study plan and amendment as PDF-files 

by e-mail, and distributed them as required at the Test Site. All study related 

communication between the Study Director, Principal Investigator and Sponsor or 

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

5.6. EVALUATION OF THE RESULTS

The assay is 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 frequency, 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 replicate 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 also considered in the evaluation. 

Evidence of a dose-response relationship (if any) was 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 THE EXPERIMENTS

The chemicals used in the independently performed experiments of the study are 

summarized in Table 1.

Table 1: Chemicals used in the experiments

Chemical Supplier Lot Number Expiry Date

Acetic acid Lachner PP/2012/06731 30 June 2017

Antibiotic-antimycotic solution (100X) SIGMA-ALDRICH SLBB9274 March 2014

Colchicine SIGMA-ALDRICH 080M1474V October 2013

Consul Mount (mounting medium) Shandon 223947 May 2016

Distilled water TEVA 3450611 June 2014
Study code: 11/339-020C Final Report Page 24 of 66

Table 1: Chemicals used in the experiments (continued)

Chemical Supplier Lot Number Expiry Date

DMEM* SIGMA-ALDRICH RNBC4233 June 2013

DMEM* SIGMA-ALDRICH RNBC4829 August 2013

DMEM* SIGMA-ALDRICH RNBC5039 August 2013

Fetal bovine serum (FBS)** SIGMA-ALDRICH 11A600 March 2016

Giemsa solution*** SIGMA-ALDRICH SZBC082A 03 September 2015

D-Glucose-6-phosphate Na**** SIGMA-ALDRICH 079K3788V August 2012

HEPES SIGMA-ALDRICH 041M54011V April 2015

KCl MERCK K42209636 30 April 2016

MgCl2 SIGMA-ALDRICH 030M0060 March 2014

Methanol VWR 12A180505 January 2017

NADP SIGMA-ALDRICH 051M7000V June 2014

Phosphate buffered saline (PBS), (10X) SIGMA-ALDRICH SLBB3269 January 2013

Physiological saline TEVA 6931211 December 2013

Trypsin-EDTA solution SIGMA-ALDRICH SLBC1655 July 2014

*: Dulbecco’s Modified Eagle’s Medium (DMEM) with 4500 mg/L glucose, L-glutamine, 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 stored at -23±8o

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 amendments, 

- all raw data and specimens,

- 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 GLP regulation [12] 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 the 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. DEVIATIONS TO THE STUDY PLAN

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

analysis was ended later; consequently, the Draft Report was issued later than 

indicated in the Study Plan.

Due to technical reason, cells were maintained on 150 cm2 flask instead of 75 cm2

flasks before the experiments.

These deviations were 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 this vehicle. The highest examined concentration in the preliminary 

test was 5000 μg/mL.

Two Concentration Selection Cytotoxicity Assays (Assay A: 3-hour treatment with 

and without metabolic activation, 20-hour harvesting time; and Assay B: 20-hour 

treatment without metabolic activation, 20-hour harvesting time) were performed as 

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

Aberration Assays.

In the preliminary toxicity test, single cultures were tested and positive controls 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 beginning 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 5000 and 0.064 μg/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 results of the performed 

Concentration Selection Cytotoxicity Assays according to the OECD guideline 

instructions.

8.2. CHROMOSOME ABERRATION ASSAYS

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) were performed. Sampling was performed 20 hours after the 

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

were 400, 200, 150, 125, 100, 75, 50, 25 and 12.5 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. Cytotoxicity was observed in this assay at 400, 

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

survival values were 7, 10, 18, 28 and 30 %, respectively) and at 400, 200, 150 and 

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

6, 24 and 46 %, respectively) as detailed in Tables 5 and 6 of Appendix 4. Therefore, 

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 concentrations 

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 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 20 hours after the 

beginning of the treatment in both cases. The examined 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 examined 

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

survival values were 6, 11, 14, 30 and 47%, respectively). Observations are detailed in 

Tables 7 and 8 of Appendix 4. 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 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. Therefore, 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. Polyploid metaphases (1-5) were found in some cases in the 

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

performed experiments. Endoreduplicated metaphases (1-14) were found in some 

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

the performed experiments.

The work phase report of the metaphase analysis is shown in Appendix 7.

8.3. VALIDITY OF THE STUDY

The tested concentrations in the chromosome aberration assays were selected based 

on the results of the preliminary experiments. No insolubility, but cytotoxicity was 

detected in the performed main experiments. The evaluated concentration ranges of 

Assay 1 and Assay 2 were considered to be adequate, as they covered a concentration 

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

cytotoxicity.

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

frequencies of the negative (vehicle) controls in the performed experiments were in 

good harmony with the historical control range of the testing laboratory.

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 substances (Cyclophosphamide 

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

in the experiments without metabolic activation) caused the expected biologically 

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

chromosome aberrations (Tables 9-12 of Appendix 5) in each assay demonstrating the 

sensitivity of the test system. 

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

The study was 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 potential clastogenic 

activity using the Chromosome Aberration Assay. The study included two 

Concentration Selection Cytotoxicity Assays and two Chromosome Aberration 

Assays.

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

potential of the test item to cause structural chromosomal aberrations in the cultured 

V79 Chinese hamster cells used in this study.

Treatment with the test item did not result in a statistically and biologically significant, 

repeatable, dose-dependent increase in the frequency of the cells with structural 

chromosome 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, Ethanedioic 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 system.

10. DISTRIBUTION OF THE FINAL REPORT

Sponsor: 1x copy, bound

1x copy, unbound

1x PDF file

Archive: 1x original, bound
Study 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., Joyce Mccann and Edith Yamasaki: Methods for Detecting 

Carcinogens and Mutagens with the Salmonella /Mammalian-Microsome 

Mutagenicity Test. Mutation 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 somatic 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 

microsomal 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 on chromosomal aberration. Clinical 

Cytogenetics Bulletin, 1, 64-76

11. 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 Agriculture 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

APPENDICES
Study code: 11/339-020C Final Report Page 31 of 66

APPENDIX 1

STUDY SCHEDULE

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) : 06 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 dropping and staining was performed on 07-10 December 2012 

and 13-14 February 2013.

Transfer of slides : 18 February 2013

Metaphase analysis : 15 March 2013

Draft Report : 19 March 2013

Final Report : 22 March 2013
Study code: 11/339-020C Final Report Page 32 of 66

APPENDIX 2

COPY OF THE CERTIFICATE OF ANALYSIS
Study code: 11/339-020C Final Report Page 33 of 66

APPENDIX 3

SUMMARIZED RESULTS OF THE CONCENTRATION SELECTION CYTOTOXICITY ASSAYS

Table 2: Summarized results of the Concentration Selection Cytotoxicity Assay A 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 – – 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/kg)

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 discoloured 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 /normal (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 (continued)

SUMMARIZED RESULTS OF THE CONCENTRATION SELECTION CYTOTOXICITY 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)

Relative

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)

Negative (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 OF THE CONCENTRATION SELECTION CYTOTOXICITY ASSAYS

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 – – 20/20 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)

Negative (vehicle) control (1X PBS, 2.5%) – – 20/20 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; osm: 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 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 36 of 66

APPENDIX 4

CYTOTOXICITY RESULTS OF THE MAIN EXPERIMENTS

Table 5: Cytotoxicity Results of Chromosome 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 / slightly 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

Notes: Duplicate counts were performed at each counting.
Study code: 11/339-020C Final Report Page 37 of 66

APPENDIX 4 (continued)

CYTOTOXICITY RESULTS OF THE MAIN EXPERIMENTS

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 discoloured 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 3.40E+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

Notes: 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 EXPERIMENTS

Table 7: Cytotoxicity Results of Chromosome 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%) – – 20/20 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 RESULTS OF THE MAIN EXPERIMENTS

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/kg)

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

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

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

Positive control (6 μg/mL CP) – + 3/20 1.80E+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 THE MAIN EXPERIMENTS

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

Concentration

(μg/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 μg/mL 3h / 20h 7 –a NE

200 μg/mL 3h / 20h 10 –a NE

150 μg/mL 3h / 20h 18 – NE

125 μg/mL 3h / 20h 28 – NE

100 μg/mL 3h / 20h 30 – 2.0

75 μg/mL 3h / 20h 50 – 1.5

50 μg/mL 3h / 20h 89 – 1.0

25 μg/mL 3h / 20h 106 – 2.5

12.5 μg/mL 3h / 20h 99 – NE

Positive control 3h / 20h 94 – 8.25***

Negative (vehicle) control: 1X PBS

Positive control (-S9): Ethyl methanesulfonate, 1 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
Study code: 11/339-020C Final Report Page 41 of 66

APPENDIX 5 (continued)

SUMMARY TABLES OF THE MAIN EXPERIMENTS

Table 10: Summary table of Chromosome Aberration Assay 1 with metabolic activation

Concentration

(μg/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 μg/mL 3h / 20h 7 –a NE

200 μg/mL 3h / 20h 6 –a NE

150 μg/mL 3h / 20h 24 –a NE

125 μg/mL 3h / 20h 46 – 1.5

100 μg/mL 3h / 20h 67 – 1.5

75 μg/mL 3h / 20h 90 – NE

50 μg/mL 3h / 20h 94 – 1.0

25 μg/mL 3h / 20h 104 – NE

12.5 μg/mL 3h / 20h 88 – NE

Positive control 3h / 20h 61 – 90.9***

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 control
Study code: 11/339-020C Final Report Page 42 of 66

APPENDIX 5 (continued)

SUMMARY TABLES OF THE MAIN EXPERIMENTS

Table 11: Summary table of Chromosome Aberration Assay 2 without metabolic activation

Concentration

(μg/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 μg/mL 20h / 20h 4 –a NE

200 μg/mL 20h / 20h 2 –a NE

150 μg/mL 20h / 20h 7 –a NE

125 μg/mL 20h / 20h 14 – NE

100 μg/mL 20h / 20h 12 – 6.5

75 μg/mL 20h / 20h 24 – 7.5

50 μg/mL 20h / 20h 89 – 4.0

25 μg/mL 20h / 20h 105 – 5.5

12.5 μg/mL 20h / 20h 123 – NE

Positive control 20h / 20h 71 – 40.5***

Negative (vehicle) control: 1X 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 control
Study code: 11/339-020C Final Report Page 43 of 66

APPENDIX 5 (continued)

SUMMARY TABLES OF THE MAIN EXPERIMENTS

Table 12: Summary table of Chromosome Aberration Assay 2 with metabolic activation

Concentration

(μg/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 μg/mL 3h / 20h 6 –a NE

200 μg/mL 3h / 20h 11 –a NE

150 μg/mL 3h / 20h 14 –a NE

125 μg/mL 3h / 20h 30 –a NE

100 μg/mL 3h / 20h 47 – 7.5

75 μg/mL 3h / 20h 58 – 3.5

50 μg/mL 3h / 20h 78 – 8.0

25 μg/mL 3h / 20h 107 – NE

12.5 μg/mL 3h / 20h 93 – NE

Positive control 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 control
Study code: 11/339-020C Final Report Page 44 of 66

APPENDIX 6

HISTORICAL CONTROL DATA

(updated on 13 February 2013)

3/20h 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 2.71 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-mix

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 deviation

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 activation. 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 DATA

(updated 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-Nitrosodimethylamine) 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 when 15 aberrant cells were counted.
Study code: 11/339-020C Final Report Page 46 of 66

APPENDIX 7

REPORT OF THE METAPHASE ANALYSIS
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APPENDIX 8

COPIES OF THE GLP CERTIFICATES

The facility name until 1st 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 (now known as 

CiToxLAB Hungary Ltd.) until the certificate expires (16 December 2012).
Study code: 11/339-020C Final Report Page 65 of 66

APPENDIX 8 (continued)

COPIES OF THE GLP CERTIFICATES
Study code: 11/339-020C Final Report Page 66 of 66

APPENDIX 8 (continued)

COPIES OF THE GLP CERTIFICATES