## FINAL REPORT

Study Title



## EVALUATION OF THE ABILITY OF

COPS_1 TO INDUCE CHROMOSOME ABERRATIONS IN

CULTURED PERIPHERAL HUMAN LYMPHOCYTES

Author

I.A.J. Verbaan, PhD.

Study completion date

11 June 2015

Test Facility

WIL Research Europe B.V.

H ambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 508229

Substance 204240/B

- Page 1 of 30

-COPS_1 Project 508229

- Page 2 - 1. CONTENTS

1. CONTENTS

................................................................................................ ...................................... 2

2. STATEMENT OF GLP COM PLIANCE

............................................................................................. 4

3. QUALITY ASSURANCE ST ATEMENT

............................................................................................ 5

4. SUMMARY

................................................................................................ ........................................ 6

5. INTRODUCTION

.............................................................................................................................. 7

5.1. Preface

................................................................................................ ...................................... 7

5.2. Aims of the study

....................................................................................................................... 7

5.3. Guidelines

................................................................................................ ................................ . 8

5.4. Storage and retention of records and materials

........................................................................ 8

6. MATERIALS AND METHOD S

.......................................................................................................... 8

6.1. Test substance

.......................................................................................................................... 8

6.1.1. Test substance information

............................................................................................. 8

6.1.2. Study specific test substance information

...................................................................... 8

6.1.3. Test substance preparation

............................................................................................ 9

6.2. Reference substances

.............................................................................................................. 9

6.2.1. Negative control

.............................................................................................................. 9

6.2.2. Positive controls

.............................................................................................................. 9

6.3. Test system

............................................................................................................................... 9

6.4. Cell culture

................................................................................................ ................................ 9

6.5. Metabolic activation system

.................................................................................................... 10

6.5.1. Preparation of S9

- mix

................................................................................................... 10

6.6. Study design

........................................................................................................................... 10

6.6.1. Dose range finding test

................................................................................................ . 10

6.6.2. Cytogenetic assay

......................................................................................................... 11

6.6.3. Chromosome preparation

............................................................................................. 11

6.6.4. Preparation of slides

..................................................................................................... 11

6.6.5. Mitotic index/dose selection for scoring of the cytogenetic assay

................................ 12

6.6.6. Anal ysis of slides for chromosome aberrations

............................................................ 12

6.7. Electronic data capture

........................................................................................................... 12

6.8. Interpretation

........................................................................................................................... 12

6.8.1. Acceptability of the assay

............................................................................................. 12

6.8.2. Data evaluation and statistical procedures

................................................................... 12

6.9. List of deviations

..................................................................................................................... 13

6.9.1. List of protocol deviations

............................................................................................. 13

6.9.2. List of standard operating procedures deviations

......................................................... 13

7. RESULTS

................................................................................................ ....................................... 13

7.1. Dose range finding test

........................................................................................................... 13

7.2. First cytogenetic a ssay

............................................................................................................ 14

7.3. Second cytogenetic assay

...................................................................................................... 14

7.4. Evaluation of the results

.......................................................................................................... 14

8. CONCLUSION

................................................................................................ ................................ 15

9. REFERENCES

............................................................................................................................... 15COPS_1 Project 508229

- Page 3 - TABLES

Table 1 Mitotic index of human lymphocyte cultures treated with COPS_1 in the dose

range finding test

.......................................................................................................... 16

Table 2 Mitotic index of human lymphocyte cultures treated with COPS_1 in the first

cytogenetic assay

......................................................................................................... 17

Table 3 Chromosome aberrations in human lymphocyte cultures treated with COPS_1 in

the absence of S9

- mix in the first cytogenetic assay (3 h exposure time, 24 h

fixation time)

.................................................................................................................. 18

Table 4 Chromosome aberrations in human lymphocyte cultures treated with COPS_1 in

the presence of S9

- mix in the first cytogenetic assay (3 h exposure time, 24 h

fixation time)

.................................................................................................................. 19

Table 5 Mitotic index of human lymphocyte cultures treated with COPS_1 in the second

cytogenetic assay

......................................................................................................... 20

Table 6 Chromosome aberrations in human lymphocyte cultures treated with COPS _1 in

the absence of S9

- mix in the second cytogenetic assay (24 h exposure time, 24 h

fixation time)

.................................................................................................................. 21

Table 7 Chromosome aberrations in human lymphocyte cultures treated with COPS_1 in

the absence of S9

- mix in the second cytogenetic assay (48 h exposure time, 48 h

fixation time)

.................................................................................................................. 22

Table 8 Chromosome aberrations in human lymphocyte cultures treated with COPS_1 in

the presence of S9

- mix in the second cytogenetic assay (3 h exposure time, 48 h

fixation time)

.................................................................................................................. 23

APPENDICES

APPENDIX 1 TABLES

........................................................................................................................ 16

APPENDIX 2 DEFINITIONS OF CHROM OSOME ABERRATIONS SC ORED IN METAPHASE

PORTRAITS

................................................................................................................. 24

APPENDIX 3 STATISTICAL EVALUATION OF THE TEST RESULTS

............................................. 26

APPENDIX 4 HISTORICAL CONTROL DATA FOR LYMPHOCYTE CHROMOSOME

ABERRATION STUDIES

.............................................................................................. 27

APPENDIX 5 HISTORICAL POSITIVE CONTROL DATA FOR LYMPHOCYTE

CHROMOSOME ABERRATION S TUDIES

.................................................................. 28

APPENDIX 6 HISTORICAL NEGATIVE CONTROL DATA FOR NUMERICAL ABERRATIONS

FOR LYMPHOCYTE CHROMOSOME ABERRATION STUDIES

............................... 29

APPENDIX 7 CERTIFICATE OF ANALYSIS

...................................................................................... 30COPS_1 Project 508229

- Page 6 - 4. SUMMARY

Evaluation of the ability of COPS_1 to induce chromosome aberrations in cultured p eripheral human

lymphocytes (with follow up experiment).

This report describes the effect of COPS_1 on the number of chromosome aberrations in cultured

peripheral human lymphocytes in the presence and absence of a metabolic activation system

(phenobarbita l and ß

- naphthoflavone induced rat liver S9

- mix). The possible clastogenicity of COPS_1

was tested in two independent experiments.

The study procedures described in this report were based on the most recent OECD, EC and EPA

guidelines.

Batch 14L1103 of C OPS_1 was a clear colourless liquid. COPS_1 was dissolved in culture medium. A

correction factor of 2.48 was used to correct for the purity of 40.3%

.

In the first cytogenetic assay, COPS_1 was tested up to 2182 μg a.i./ml (= 0.01 M) for a 3 h exposure

ti me with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9

- fraction. This is the highest

concentration recommended by the guidelines.

In the second cytogenetic assay, COPS_1 was tested up to 2182 μg a.i. /ml for a 24 h and 48 h

continuous ex posure time with a 24 h and 48 h fixation time i n the absence of S9

- mix. In the presence

of S9

- mix COPS_1 was also tested up to 2182 μ g/ml for a 3 h exposure time with a 48 h fixation time.

The number of cells with chromosome aberrations found in the solvent control cultures was within the

laboratory historical control data range. Positive control chemicals, mitomycin C and

cyclophosphamide, both produced a statistically significant increase in the incidence of cells with

chromosome aberrations, indicating that the test conditions were adequate and that the metabolic

activation system (S9

- mix) functioned properly.

COPS_1 did not induce a statistically significant or biologically relevant increase in the number of cells

with chromosome aberrations in the absence and presence of S9

- mix, in either of the two

independently experiments.

No effects of COPS_1 on the number of polyploid cells and cells with endoreduplicated chromosomes

were observed both in the absence and presence of S9

- mix. Therefore it can be concluded that

COPS_1 does not disturb mitotic processes and cell cycle progression and does not induce numerical

chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that COPS_1 is not clastogenic in human lymphocytes

under the experimental conditions described in this report.COPS_1 Project 508229

- Page 7 - 5. INTRODUCTION

5.1. Preface

Sponsor Society of Chemical Manufacturers and Affiliates

1850 M Street, NW Suite 700

WASHINGTON, DC 20036

USA

Study Mo nitor Dr. C. T. Helmes

Test Facility WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Study Director I.A.J. Verbaan, PhD.

Study Plan Start : 16 March 2015

Completion : 18 May 2015

5.2. Aims of the study

The objective of this study was to evaluate COPS_1 for its ability to induce structural chromosome

aberrations in cultured human lymphocytes (1), either in the presence or absence of a metabolic

activation system (S9

- mix).

Background of the test system

Whole blood samples obtained from healthy male subjects w ere treated with an anti

- coagulant

(heparin) and cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human

lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range

of chemicals (1

- 5).

The stimulated lymphocytes were exposed to COPS_1 both in the absence and presence of a

metabolic activation system (S9

- mix). In combination with this metabolic activation system indirect

chemical mutagens, i.e. those requiring metabolic transformation into reactive intermediates, can be

tested for possible clastogenic effects in vitro

.

At predetermined intervals after exposure of the stimulated human lymphocytes to COPS_1 , cell

division was arrested in the metaphase stage of the cell cycle by addition of the metaphase

- arresting

chemical colchicine. Cells were harvested, stained and metaphase cells were analysed for the

presence of structural chromosome aberrations such as breaks, gaps, minutes, dicentrics a nd

exchange figures. Results from cultures treated with COPS_1 were compared with control (vehicle)

treated cultures.

Chromosome aberrations were generally evaluated in the first post

- exposure mitosis (i.e. 24 hours

after exposure). However, since the appearance of the first post

- exposure mitosis could be

considerably delayed due to toxic insult to the cells, cells were also harvested 48 hours after exposure

to cover the interval in which maximum aberration frequency was expected.

A test substance that induces a positive response in this assay is presumed to be a potential

mammalian cell clastogenic agent.COPS_1 Project 508229

- Page 8 - 5.3. Guidelines

The study procedures described in this report were based on the following guidelines:

- Organisation for Economic Co

- operation and Development (OECD), OECD Guidelines for Testing

of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted

September 26, 2014).

- European Community (EC). Co mmission regulation (EC) No. 440/2008, Part B: Methods for the

Determination of Toxicity and other health effects, Guideline B.10: "Mutagenicity

- In Vitro

Mammalian Chromosome Aberration Test". Official Journal of the European Union No. L142, 31

May 2008.

- Code of Federal Regulations, Title 40

– Protection of Environment. Section 799.9537 Toxic

Substances Control Act (TSCA) in vitro mammalian chromosome aberration test.

5.4. Storage and retention of records and materials

Records and materials pertaining to the study, including protocol, raw data, specimens (except

specimens requiring refrigeration or freezing) and the final report, will be retained in the WIL Research

Europe archives for a period of at least 2 years after finalization of the report. After this period, the

sponsor will be contacted to determine how the records and materials should be handled. WIL

Research Europe will retain information concerning decisions made.

Those specimens requiring refrigeration or freezing will be retained by WIL Research Europe for as

long as the quality of the specimens permits evaluation but no longer than three months after

finalization of the report.

WIL Research Europe will retain a test substance sample until the expiry date, but no longer than

10 years after finalization of the report. After this period the sample will be destroyed.

6. MATERIA LS AND METHODS

6.1. Test substance

6.1.1. Test substance information

Identification COPS_1 (40.3 wt. % in H 2 O)

Appearance Clear colourless liquid

Batch 14L1103

Purity/Composition See Certificate of Analysis

Test substance storage At room temperature

Stable und er storage conditions until 21 November 2015 (expiry date)

See APPENDIX 7 ; Certificate of analysis.

6.1.2. Study specific test substance information

Purity/composition correction factor Yes, correction factor 2.48

Test substance han dling No specific handling conditions required

Stability at higher temperatures Stable

Chemical name (IUPAC), synonym or

trade name 1

- Propanesulfonic acid, 2

- hydroxy

- 3

- (2

- propen

- 1

- yloxy)

- , sodium salt (1:1)

CAS Number 52556

- 42

- 0

Molecular structure



### O SO 3



### OH Na + _COPS_1 Project 508229

- Page 9 - Molecular formula C 6 H 11 NaO 5 S

Molecular weight 218. 22

Solubility in vehicle Culture medium: Not indicated

Stability in vehicle Culture medium: Not indicated

6.1.3. Test substance preparation

The test substance was corrected for the purity (40.3%). A correction factor of 2.48 was used.

COPS_1 was dissolved in culture medium

. COPS_1 concentrations were used within 2.5 hours after

preparation . The pH and the osmolarity of the culture medium containing the highest tested

concentrati on were recorded.

6.2. Reference substances

6.2.1. Negative control

The vehicle for the test substance was culture medium

.

6.2.2. Positive controls

Without metabolic activation (

- S9

- mix):

Mitomycin C (MMC

- C; CAS no. 50

- 07

- 7, Sigma, Zwijndrecht, The Netherlands) was used as a direct

acting mutagen at a final concentration of 0.5 and 0.75 μg/ml for a 3 h exposure period, 0.2 and

0.3 μg/ml for a 24 h exposure period and 0.1 and 0.15 μg/ml for a 48 h exposure period.

With metabolic activation (+S9

- mix):

Cyclophosphamide (CP; CAS no. 50

- 18

- 0. Baxter B.V., Utrecht, The Netherlands) was used as an

indirect acting mutagen, requiring metabolic activation, at a final concentration of 10 μ g/ml for a 3 h

exposure period ( protocol deviation 1 ).

Solvent for positive controls

Hanks’ Balanced Salt Solution (HBSS) (Life technologies, Bleiswijk, The Netherlands), without calcium

and magnesium.

All reference stock solutions were stored in aliquots at ≤

- 15°C in the dark. These solutions were

thawed immediately before use.

6.3. Test system

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are

recommended in international guidelines (e.g. OECD, E C).

Blood was collected from healthy adult, non

- smoking volunteers (approximately 18 to 35 years of age).

The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was

determined (December 2014) are presented below:

Dose range finding study: age 23 , AGT = 12.9 h

First cytogenetic assay: age 26 , AGT = 12.8 h

Second cytogenetic assay: age 32 , AGT = 12.8 h

6.4. Cell culture

Blood samples

Blood samples were collected by ven i puncture using the Venoject multiple sample blood collecting

system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio

- One,

Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were

started.COPS_1 Project 508229

- Page 10

- Culture medium

Culture medium consisted of RPMI 1640 medium ( Life technologies ), supplemented with 20% (v/v)

heat

- inactivated (56°C; 30 min) foetal calf serum ( Life technologies ), L

- glutamine (2 mM) ( Life

technologies ), penicillin/streptomycin (50 U/ml and 50 μ g/ml respectively) (Life technologies) and

30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).

Lymphocyte cultures

Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence

and presence of S9

- mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin ( Remel,

Europe L td.

, U nited K ingdom ) was added.

Environmental conditions

All incubations were carried out in a controlled environment, in which optimal conditions were a humid

atmosphere of 80

- 100% (actual range 53

- 91%), containing 5.0 ± 0.5% CO 2 in air in the dark at

37.0 ± 1.0°C (actual range 34.7

- 37.3°C). Temperature and humidity were continuously monitored

throughout the experiment. The CO 2 percentage was monitored once on each working day.

Temporary deviations from the temperature, humidity and CO 2 perc entage may occur due to opening

and closing of the incubator door. Based on laboratory historical data these deviations are considered

not to affect the study integrity

.

6.5. Metabolic activation system

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared

from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital

(80 mg/kg body weight) and ß

- naphthoflavone (1 00 mg/kg)

.

6.5.1. Preparation of S9

- mix

S9

- mix was prepared immediately before use and kept on ice. S9

- mix components contained per ml

physiological saline: 1.63 mg MgCl 2 .6H 2 O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose -6- phosphate

(Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom);

4 μmol HEPES (Life technologies).

The above solution was filter (0.22 μm)

- sterilized. To 0.5 ml S9

- mix components 0.5 ml S9

- fraction

was added (50% (v/v) S9

- fraction) to complete the S9

- mix.

Metabolic activation was achieved by adding 0.2 ml S9

- mix to 5.3 ml of a lymphocyte culture

(containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin). The

concentration of the S9

- fraction in the exposure medium was 1.8% (v/v).

6.6. Study design

6.6.1. Dose range finding test

In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data

were obtained in a dose range finding test. COPS_1 was tested in the absence and in the presence of

1.8% (v/v) S9

- fraction.

Lymphocytes (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium,

without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were

cultured for 48 h and thereafter exposed to selected doses of COPS_1 for 3 h, 24 h and 48 h in the

absence of S9

- mix or for 3 h in the presence of S9

- mix. A negative control was included at each

exposure time.

The highest tested concentration was 2182 μg a.i. /ml (= 0.01 M)

.COPS_1 Project 508229

- Page 11

- After 3 h exposure to COPS_1 in the absence or presence of S9

- mix, the cells were separated from

the exposure medium by centrifugation (5 min, 365 g ). The supernatant was removed and cells were

rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were

resuspended in 5 ml culture medium and incubated for another 20

- 22 h (24 h fixation time). The cells

that were exposed for 24 h and 48 h in the absence of S9

- mix were not rinsed after exposure but were

fixed immediately (24 h and 48 h fixation time).

Cytotoxicity of COPS_1 in the lymphocyte cultures was determined using the mitotic i ndex.

Based on the results of the dose range finding test an appropriate range of dose levels was chosen for

the cytogenetic assays considering the highest dose level was the recommended 0.01 M.

6.6.2. Cytogenetic assay

The cytogenetic assay was carried out as described by Evans, 1984 (2) with minor modifications.

COPS_1 was tested in the absence and presence of 1.8% (v/v) S9

- fraction in duplicate in two

independent experiments.

First cytogenetic assay

Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of

COPS_1 for 3 h in the absence and presence of S9

- mix. After 3 h exposure, the cells were separated

from the exposure medium by centrifugation (5 min, 365 g ). The supernatant was removed and the

cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and

cells were resuspended in 5 ml culture medium and incubated for another 20

- 22 h (24 h fixation

time). Appropriate negative and positive controls were included in the first cytogeneti c assay.

Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate

dose levels were selected for the second cytogenetic assay. The independent repeat was performed

with the following modifications of experimental conditions.

Second cytogenetic assay

Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of

COPS_1 for 24 h and 48 h in the absence of S9

- mix and for 3 h in the presence of S9

- mix.

After 3 h exposure, the cells exposed to COPS_1 in the presence of S9

- mix were separated from the

exposure medium by centrifugation (5 min, 365 g ). The supernatant was removed and the cells were

rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44

- 46 h (48 h

fixation time).

The cells that were treated for 24 h and 48 h in the absence of S9

- mix were not rinsed after exposure

but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time).

Appropriate negative and positive controls were include d in the second cytogenetic assay.

6.6.3. Chromosome preparation

During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle

inhibitor colchicine (0.5 μg/ml medium) ( Acros Organics, Geel, Belgium). Thereafter the ce ll cultures

were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell

pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck)

solution at 37°C. After hypotonic treatment, cells w ere fixed with 3 changes of methanol (Merck):

acetic acid (Merck) fixative (3:1 v/v).

6.6.4. Preparation of slides

Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v)

ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the WIL

Research Europe study identification number and group number. At least two slides were preparedCOPS_1 Project 508229

- Page 12

- per culture. Slides were allowed to dry and thereafter stained for 10

- 30 min with 5% (v/v) Giemsa

(Merck) solution i n Sörensenbuffer pH 6.8 . Thereafter slides were rinsed in water and allowed to dry.

The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The

Netherlands)/pertex (Histolab, Gothenburg, Sweden) and mounted with a coverslip in an automated

coverslipper ( Leica Microsystems B.V., Rijswijk, The Netherlands ).

6.6.5. Mitotic index/dose selection for scoring of the cytogenetic assay

The mitotic index of each culture was determined by counting the number of metaphases from at least

10 00 cells (with a maximum deviation of 5%). At least three analysable concentrations were used for

scoring of the cytogenetic assay. COPS_1 was not cytotoxic, the highest concentration analysed was

the recommended 0.01 M.

6.6.6. Analysis of slides for chromosome aberrations

To prevent bias, all slides were randomly coded before examination of chromosome aberrations and

scored. An adhesive label with WIL Research Europe study identification number and code was

placed over the marked slide. One hundr e d metaphase chromosome spreads per culture were

examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps

excluded, was ≥ 25 in 50 metaphases, no more metaphases were examined. Only metaphases

containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberration s

and the number of aberrations were calculated. Since the lowest concentration of MMC -C resulted in a

positive response the highest concentration was not examined for chromosome aberrations.

6.7. Electronic data capture

Observations/measurements in the study were recorded electronically using the following

programme(s):

REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ,

USA) : temperature and humidity

.

6.8. Interpretation

6.8.1. Acceptability of the assay

A chromosome aberration test is considered acceptable if it meets the following criteria:

a) The number of chromosome aberrations found in the solvent control cultures should

reasonably be within the laboratory historical control data range (see APPENDIX 4 )

. b) The positive control substances should produce a statistically significant (Chi

- square test, one

sided, p < 0.05) increase in the number of cells with chromosome aberrations.

c) A homogeneous response between the replicate cultures is observed.

d) A possible precipitate present on the slides should not interfere with the scoring of chromosome

aberrations.

6.8.2. Data evaluation and statistical procedures

A test substance is considered positive (clastogenic) in the chromosome aberration test if:

a) It induced a dose

- rela ted statistically significant (Chi

- square test, one

- sided, p < 0.05) increase in

the number of cells with chromosome aberrations.

b) A statistically significant and biologically relevant increase in the frequencies of the number of cells

with chromosome aberrations was observed in the absence of a clear dose

- response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none

of the tested concentrations induced a statistically significant (Chi

- square tes t, one

- sided, p < 0.05)

increase in the number of cells with chromosome aberrations.

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or

excluded ) for each exposure group outside the laboratory historical control data range was comparedCOPS_1 Project 508229

- Page 13

- to that of the solvent control using Chi

- square statistics:

(N

- 1) (ad

- bc) 2

X 2

= -------------------------------- (a+b) (c+d) (a+c) (b+d)

where b = the total number of aberrant cells in the control cultures.

d = the total number of non aberrant cells in the control cultures.

n 0 = the total number of cells scored in the control cultures.

a = the total number of aberrant cells in treated cultures to be compared with the control.

c = the total number of non aberrant cells in treated cultures to be compared with the

control.

n 1 = the total number of cells scored in the treated cultures.

N = sum of n 0 and n 1

(N

- 1) (ad

- bc) 2

If P X 2 >

------------------------------- (one

- tailed) is small (p< 0.0 5) the hypothesis that the

(a+b) (c+d) (a+c) (b+d)

incidence of cells with chromosome aberrations is the same for both the treated and the solvent

control group is rejected and the number of aberrant cells in the test group is considered to be

significantly different from the control group at the 95% confidence level.

6.9. List of deviations

6.9.1. List of protocol deviations

1. In the second cytogenetic assay the positive control cultures for CP were fixed after 48 hours

instead of 24 hours.

Evaluation: CP produced a statistically significant increase in the number of cells with

chromosome aberrations and therefore met the acceptability criteria of the assay.

Consequently, this d eviation has no effect on the results of the study.

The study integrity was not adversely affected by the deviation.

6.9.2. List of standard operating procedures deviations

Any deviations from standard operating procedures were evaluated and filed in the study file. There

were no deviations from standard operating procedures that affected the integrity of the study.

7. RESULTS

7.1. Dose range finding test

A concentration of 2182 μg a.i./ml (=0.01 M) showed no precipitation in the culture medium and wa s

used as the highest concentration of COPS_1

.

The pH and osmolarity of a concentration of 2182 μg a.i. /ml were 7.6 and 308 mOsm/kg respectively

(compared to 7.6 and 297 mOsm/kg in the solvent control).

In the dose range finding test blood cultures were treated with 52

, 164

, 512

, 1600 and 2182 μ g a.i.

COPS_1 /ml culture medium with and without S9

- mix.

Table 1 ( APPENDIX 1 ) shows the mitotic index of cultures treated with various COPS_1

concentrations or with the negative control substance.COPS_1 Project 508229

- Page 14

- 7.2. First cytogenetic assay

Based on the results of the d ose range finding test the following dose levels were selected for the

cytogenetic assay:

Without and with S9

- mix: 512, 1600 and 2182 μg a.i. /ml culture medium

(3 h exposure time, 24 h fixation time).

Table 2 ( APPENDIX 1 ) shows the mitotic index of cultures treated with various COPS_1

concentrations or with the positive or negative control substances.

All dose levels were selected for scoring of chromosome aberrations

. Both in the absence and

presenc e of S9

- mix, COPS_1 did not induce a statistically significant or biologically relevant increase

in the number of cells with chromosome aberrations ( APPENDIX 1 : Table 3

, Table 4 )

.

Both in the absence and presence of S9

- mix, COPS_1 did not increase the number of polyploid cells

and cells with endoreduplicated chromosomes.

7.3. Second cytogenetic assay

To obtain more information about the possible clastogenicity of COPS_1 , a second cytogenetic assay

was performed in which human lymphocytes were continuously exposed to COPS_1 in the absence of

S9

- mix for 24 or 48 hours. In the presence of S9

- mix, cells were fixed after 48 hours following a 3 hour

exposure to COPS_1 . The following dose le vels were selected for the second cytogenetic assay:

Without S9

- mix : 512, 1600 and 2182 μg a.i. /ml culture medium

(24 h and 48 h exposure time, 24 h and 48 h fixation time).

With S9

- mix : 512, 1600 and 2182 μg a.i. /ml culture medium

(3 h exposure time, 48 h fixation time).

Table 5 ( APPENDIX 1 ) shows the mitotic index of cultures treated with various COPS_1

concentrations or with the positive or negative control substances.

All doses were selected for scoring of chromosome aberrations

. Both in the absence and presence of

S9

- mix, COPS_1 did not induce a statistically significant or biologically relevant increase in the

number of cells with chromosome aberrations ( APPENDIX 1 ; Table 6

- 8).

Both in the absence and presence of S9

- mix, COPS_1 did not increase the number of polyploid cells

and cells with endoreduplicated chromosomes.

7.4. Evaluation of the results

The ability of COPS_1 to induce chromosome aberrations in human peripheral lymphocytes was

investigated in two independent experiments. The highest concentration analysed was the

recommended 0.01 M.

The mitotic indices of cultures treated with various COPS_1 concentrations or with the negative con trol

substances are presented in Table 1

, Table 2 and Table 5 ( APPENDIX 1 ) . The scores for the number

of aberrant cells (gaps included and excluded) and the number of the various types of chromosome

aberrations at the various concentrations of COPS_1 are presented in Table 3

, Table 4 and

Table 6 - 8 ( AP PENDIX 1 ) . Duplicate cultures are indicated by A and B. The criteria according to

which the aberrations were classified are outlined in APPENDIX 2

. APPENDIX 3 presents the

s t atistical evaluations of the test results.

The number of cells with chromosome aberrations found in the solvent control cultures was within the

laboratory historical control data range (see APPENDIX 4 ) . The number of polyploid cells and cells

with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historicalCOPS_1 Project 508229

- Page 15

- control data range (see APPENDIX 6 ) . The positive control chemicals (MMC

- C and CP) both

produced statistically significant increases in the frequency of aberrant cells. It was therefore

concluded that the test conditions were adequate and that the metabolic activation system (S9

- mix)

functioned properly.

Both in the absence and presence of S9

- mix COPS_1 did not induce a statistically significant or

biologically relevant increase in the number of cells with chromosome aberrations in two independent

experiments.

No effects of COPS_1 on the number of polyploid cells and cells with endoreduplicated chromosomes

were observed both in the absence and presence of S9

- mix. Therefore it can be concluded that

COPS_1 does not disturb mitotic processes and cell cycle progression and does not induce numerical

chromosome aberrations under the experimental conditions described in this report.

8. CONCLUSION

Finally, it is concluded that this test is valid and that COPS_1 is not clastogenic in human lymphocytes

under the experimental conditions described in this report.

9. REFERENCES

1. Evans H.J. (1976). Cytological methods for detecting chemical mutagens. In: Chemical

mutagens, principles and methods for their detection, Vol. 4, Ed. A. Hollaender (New York,

London, PlenumPress), 1

- 29.

2. Evans H.J. (1984). Human Peripheral Blood Lymphocytes for the Analysis of Chromosome

Aberrations in Mutagen Tests. In: Handbook of Mutagenicity Test Procedures, Eds. B.J. Kilbey,

M. Legator, W. Nichols and C. Ramel, 405

- 427, Elsevier Science Publishers B.V., Amsterdam.

3. Howard P.N., Bloom A.D., Krooth R.S. (1972). Chromosomal aberrations induced by N

- methyl

- N'

-

nitro -N- nitrosoguanidine in mammalian cells. In Vitro 7 : 359

- 365.

4. Preston R.J., Au W., Bender M.A., Brewen J.G., Carrano A.V., Heddle J.A., McFee A.F., Wolff S.,

Wassom J.S. (1981). Mammalian in vivo and in vitro cytogenetic assays: A report of the Ge ne

- tox

Program. Mutat Res 87 : 143

- 188.

5. Galloway S.M., Aardema M.J., Ishidate M.,Jr., Ivett, J.L., Kirkland D.J., Morita T., Mosesso P.,

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

Mutat Res 312 : 241

- 261

. 6. Ames B.N., McCann J. and Yamasaki, E. (1975). Methods for detecting carcinogens and

mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat Res 31 , 347

-

364.

7. Morita T., Nagaki T., Fukuda I. and Okumura K. (1992). Clastogenicity of low pH to various

cultured mammalian cells. Mutat Res 268 : 297

- 305.COPS_1 Project 508229

- Page 16

- APPENDIX 1 TABLES

Table 1 Mitotic index of human lymphocyte cultures treated with COPS_1 in the dose

range finding test

COPS_1 Number of metaphases

concentration (μg a.i./ml) Absolute Number Percentage

of cells scored of control

Without metabolic activation (

- S9

- mix)

3 h exposure time, 24 h fixation time

Control a) 68 1048 100

52 61 1040 90

164 58 1018 85

512 57 1021 84

1600 58 1026 84

2182 62 1037 91

24 h exposure time, 24 h fixation time

Control a) 68 1014 100

52 64 1024 94

164 54 1014 79

512 50 1000 74

1600 49 1007 72

2182 41 1031 60

48 h exposure time, 48 h fixation time

Control a) 57 1037 100

52 55 1012 96

164 53 1050 93

512 48 1017 84

1600 41 1016 72

2182 35 1020 61

With metabolic activation (+S9

- mix)

3 h exposure time, 24 h fixation time

Control a) 64 1024 100

52 6 0 1018 94

164 54 1009 84

512 55 1016 86

1600 66 1026 103

2182 60 1026 94

a) Culture mediumCOPS_1 Project 508229

- Page 17

- Table 2 Mitotic index of human lymphocyte cultures treated with COPS_1 in the first

cytogenetic assay

COPS_1 Numbe r of metaphases a)

concentration (μg a.i./ml) Absolute Number Percentage

of cells scored of control

Without metabolic activation (

- S9

- mix)

3 h exposure time, 24 h fixation time

Control b) 84

- 88 1000

- 1000 100

512 86

- 67 1003

- 1000 89

1600 68

- 72 1002

- 1000 81

2182 68

- 65 1004

- 1000 77

MMC

- C; 0.5 μg/ml 38

- 43 1000

- 1003 47

MMC

- C; 0. 7 5 μg/ml 33

- 30 1000

- 1000 37

With metabolic activation (+S9

- mix)

3 h exposure time, 24 h fixation time

Control b) 65

- 64 1000

- 1000 100

512 63

- 64 1000

- 1002 98

1600 56

- 61 1000

- 1001 91

2182 56

- 52 1008

- 1000 84

CP; 10 μg/ml 48

- 42 1002

- 1003 70

a) Duplicate cultures

b) Culture mediumCOPS_1 Project 508229

- Page 18

- Table 3 Chromosome aberrations in human lymphocyt e cultures treated with COPS_1 in

the absence of S9

- mix in the first cytogenetic assay (3 h exposure time, 24 h

fixation time)

Conc Culture medium 512

μg a. i. /ml 1600

μg a.i. /ml 2182

μg a.i. /ml MMC -C

0.5 μ g/ml

Culture A B A+B A B A+B A B A+B A B A+B A B A+B

Mitotic

Index (%) 100 89 81 77 47

No. of

Cells

scored 100 100 200 100 100 200 100 100 200 100 100 200 100 100 200

No. of

Cells with

aberrations

(+ gaps) a) 0 0 0 2 0 2 0 0 0 0 0 0 28 29 *** )

57

No. of

Cells with

aberrations

(

- gaps ) 0 0 0 2 0 2 0 0 0 0 0 0 28 29 *** )

57

g’ 1

g”

b’ 2 21 28

b” 3 3

m’

m”

exch. 11 10

dic

d’

misc.

total aber r

(+ gaps) 0 0 2 0 0 0 0 0 35 42

total aberr

(- gaps) 0 0 2 0 0 0 0 0 35 41

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

*) Significantly different from control group (Chi

- square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.COPS_1 Project 508229

- Page 19

- Table 4 Chromosome aberrations in human lymphocyte cultures treated with COPS_1 in

the presence of S9 -m ix in the first cytogenetic assay (3 h exposure time, 24 h

fixation time)

Conc Culture medium 512

μ g a. i. /ml 1600

μg a.i. /ml 2182

μg a.i. /ml CP

10 μ g/ml

Culture A B A+B A B A+B A B A+B A B A+B A B A+B

Mitotic

Index (%) 100 98 91 84 70

No. of

Cells

scored 100 100 200 100 100 200 100 100 200 100 100 200 100 100 200

No. of

Cells with

aberrations

(+ gaps) a) 0 3 3 0 0 0 0 1 1 0 0 0 17 23 *** )

40

No. of

Cells with

aberrations

(

- gaps) 0 3 3 0 0 0 0 1 1 0 0 0 17 23 *** )

40

g’

g”

b’ 3 1 4 14

b” 1 6 4

m’

m”

exch. 8 9

dic

d’

misc.

total aberr

(+ gaps) 0 3 0 0 0 2 0 0 18 27

total aberr

(- gaps) 0 3 0 0 0 2 0 0 18 27

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

*) Significantly different from control group (Chi

- square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.COPS_1 Project 508229

- Page 20

- Table 5 Mitotic index of human lymphocyte cultures treated with COPS_1 in the second

cytogenetic assay

COPS_1 Number of metaphases a)

concentration (μg a.i

. /ml) Absolute Number Percentage

of cells scored of control

Without metabolic activation (

- S9

- mix)

24 h exposure time, 24 h fixation time

Control b) 92

- 74 1000

- 1000 100

512 62

- 81 1000

- 1000 86

1600 67

- 77 1000

- 1000 87

2182 44

- 68 1003

- 1002 67

MMC

- C; 0.2 μg/ml 16

- 19 1000

- 1000 21

MMC

- C; 0.3 μg/ml 18

- 25 1000

- 1000 26

48 h exposure time, 48 h fixation time

Control b) 75

- 85 1000

- 1000 100

512 76

- 57 1000

- 1000 83

1600 57

- 68 1000

- 1002 78

2 182 58

- 64 1002

- 1000 76

MMC

- C; 0.1 μg/ml 30

- 34 1000

- 1000 40

MMC

- C; 0.15 μg/ml 31

- 28 1000

- 1000 37

With metabolic activation (+S9

- mix)

3 h exposure time, 48 h fixation time

Control b) 27

- 35 1000

- 1000 100

512 46

- 30 1000

- 1000 123

1600 26

- 40 1000

- 1000 106

2182 36

- 31 1000

- 1000 108

CP; 10 μg/ml 42

- 35 1000

- 1004 124

a) Duplicate cultures

b) Culture mediumCOPS_1 Project 508229

- Page 21

- Table 6 Chromosome aberrations in human lymphocyte cultures treated with COPS_1 in

the absence of S9

- mix in the second cytogenetic assay (24 h exposure time, 24

h fixation time)

Conc Culture medium 512

μ g a. i. /ml 1600

μg a.i. /ml 2182

μg a.i. /ml MMC -C

0.2 μ g/ml

Culture A B A+B A B A+B A B A+B A B A+B A B A+B

Mitotic

Ind ex (%) 100 86 87 67 21

No. of

Cells

scored 100 100 200 100 100 200 100 100 200 100 100 200 50 100 15 0

No. of

Cells with

aberrations

(+ gaps) a) 0 1 1 0 2 2 1 1 2 1 2 3 27 41 *** )

68

No. of

Cells with

aberrations

(

- gaps) 0 1 1 0 2 2 1 1 2 1 2 3 27 41 *** )

68

g’ 1 1

g”

b’ 1 2 1 1 18 21

b” 1 5 5

m’ 1

m” 1 3

exch. 10 15

dic

d’

misc. poly 2intra 6intra,

ma,

2end o

total aberr

(+ gaps) 0 1 0 2 1 1 1 2 36 61

total aberr

(- gaps) 0 1 0 2 0 1 1 1 36 61

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

The numerical variations endoreduplication (endo) and polyploidy (poly) w ere not counted as an aberration.

*) Significantly different from control group (Chi

- square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.COPS_1 Project 508229

- Page 22

- Table 7 Chromosome aberrations in human lymphocyte cultures treated with COPS_1 in

the absence of S9

- mix in the second cytogenetic assay (48 h exposure time, 48

h fixation time)

Conc Culture medium 512

μ g a. i. /ml 1600

μg a.i. /ml 2182

μg a .i. /ml MMC -C

0.1 μ g/ml

Culture A B A+B A B A+B A B A+B A B A+B A B A+B

Mitotic

Index (%) 100 83 78 76 40

No. of

Cells

scored 100 100 200 100 100 200 100 100 200 100 100 200 50 50 1 00

No. of

Cells with

aberrations

(+ gaps) a) 0 0 0 0 0 0 0 3 3 0 0 0 25 25 *** )

50

No. of

Cells with

aberrations

(

- gaps) 0 0 0 0 0 0 0 3 3 0 0 0 25 25 *** )

50

g’

g”

b’ 3 17 13

b” 4 4

m’ 1

m” 3 2

exch. 6 6

dic

d’

misc. 2p,

poly 2intra

p

total aberr

(+ gaps) 0 0 0 0 0 3 0 0 32 29

total aberr

(- gaps) 0 0 0 0 0 3 0 0 32 29

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

The numerical variation polyploidy (poly) w as not counted as an aberration.

*) Significantly different from control group (Chi

- square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.COPS_1 Project 508229

- Page 23

- Table 8 Chromosome aberrations in human lymphocyte cultures treated with COPS_1 in

the presence of S9

- mix in the second cytogenetic assay (3 h exposure time, 48 h

fixation time)

Conc Culture medium 5 12

μ g a. i. /ml 1600

μg a.i. /ml 2182

μg a.i. /ml CP

10 μ g/ml

Culture A B A+B A B A+B A B A+B A B A+B A B A+B

Mitotic

Index (%) 100 123 106 108 124

No. of

Cells

scored 100 100 200 100 100 200 100 100 200 100 100 200 100 100 200

No. of

Cells with

aberrations

(+ gaps) a) 0 0 0 0 1 1 0 0 0 0 1 1 9 10 *** )

19

No. of

Cells with

aberrations

(

- gaps) 0 0 0 0 1 1 0 0 0 0 1 1 9 10 *** )

19

g’

g”

b’ 1 3

b” 1 4 2

m’ 2

m” 3

exch. 3 3

dic 1 1

d’

misc. endo p

total aberr

(+ gaps) 0 0 0 1 0 0 0 1 10 13

total aberr

(- gaps) 0 0 0 1 0 0 0 1 10 13

a) Abbreviations used for various types of aberrations a re listed in APPENDIX 2

. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

The numerical variation endoreduplication (endo) w as not counted as an aberration.

*) Significantly different from control group (Chi

- square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.COPS_1 Project 508229

- Page 24

- APPENDIX 2 DEFINITIONS OF CHROM OSOME ABERRATIONS SCORED IN METAPHASE

PORTRAITS

Aberration Abbreviation Description

Chromatid gap g' An achromatic lesion which appears as an

unstained region in the chromatid arm, the size

of which is equal to or smaller than the width of

the chromatid and the apparently "broken"

segments of the chromatid arm are in

alignment.

Chromosome gap g" An achromatic lesion which appears as an

unstained region in both chromatids at the

same position, the size of which is equal to or

smaller than the width of the chromatid and the

apparently "broken" segments of the

chromatids are in alignment.

Chromatid break b' An achromatic lesion in a chromatid arm, the

size of which is larger than the width of the

chromatid. The broken segments of the

chromatid arm are aligned or unaligned.

Chromosome break b" An achromatic lesion in both chromatids at the

same position, the size of which is larger than

the width of the chromatid. The broken

segments of the chromatids are aligned or

unaligned.

Chromatid deletion d' Deleted material at the end of a chromatid arm.

Minute m' A single, usually circular, part of a chromatid

lacking a centromere.

Doubl e minutes m" Two, usually circular, parts of a chromatid

lacking a centromere.

Dicentric chromosome dic A chromosome containing two centromeres.

Tricentric tric A chromosome containing three

chromosome centromeres.COPS_1 Project 508229

- Page 25

- APPENDIX 2

– continued

–

Aberration Abbreviation Description

Ring chromosome r A ring structure with a distinct lumen.

Exchange figure exch. An exchange(s) between two or more

chromosomes resulting in the formation of a tri

or more

- armed configuration.

Chromosome intra A chromosome intrachange is scored

intrachange after rejoining of a lesion within one

chromosome.

Pulverized p A fragmented or pulverized chromosome

chromosomes

Multiple ma A metaphase spread containing ten or

aberrations more of the above mentioned aberrations

(chr omatid and chromosome gaps not

included). ma is counted as 10 aberrations.

Polyploidy poly A chromosome number that is a multiple of the

normal diploid number.

Endoreduplication endo A form of polyploidy in which each centromere

connects two or four pairs of chromatids instead

of the normal one pair.COPS_1 Project 508229

- Page 26

- APPENDIX 3 STATISTICAL EVALUATION OF THE TEST RESULTS

Chi

- square Test

TOTAL NUMBER OF CELLS WITH ABERRATIONS; TREATMENT/CONTROL COMPARISON,

(INCLUSIVE/EXCLUSIVE GAPS) 1)

.

EXPOS URE S9

- MIX GAPS P- VALUE DECISION AT 95%

DOSE ( μ g/ml) one

- sided CONFIDENCE LEVEL

First cytogenetic assay

24 h fixation time

Positive controls

MMC

- C (0.5)

− + ≤0.0001 significant

− − ≤0.0001 significant

CP (10) + + ≤0.0001 significant

+

− ≤0.0001 significant

Second cytogenetic assay

24 h fixation time

Positive controls

MMC

- C (0.2)

− + ≤0.0001 significant

− − ≤0.0001 significant

CP (10) + + ≤0.0001 significant

+

− ≤0.0001 significant

48 h fixation time

Positive control

MMC

- C (0.1)

− + ≤0.0001 significant

− − ≤0.0001 significant

1) Only statistically significant results are presented.COPS_1 Project 508229

- Page 27

- APPENDIX 4 HISTORICAL CON TROL DATA FOR LYMPHOCYTE CHROMOSOME

ABERRATION STUDIES

Aberrant cells per 100 metaphases

Gaps included Gaps excluded

+ S9

- mix

- S9

- mix + S9

- mix

- S9

- mix

Range 0 - 5 0 - 5 0 - 5 0 - 4

Mean 0.8 0.8 0.7 0.6

SD 1.0 1.0 0.9 0.9

n 290 449 287 442

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between December 2011 and December 2014.COPS_1 Project 508229

- Page 28

- APPENDIX 5 HISTORICAL POSITIVE CONTROL DATA FOR LYMPHOCYTE CHROMOSOME

ABERRATION STUDIES

Aberrant cells per 100 metaphases

Gaps included Gaps excluded

+ S9

- mix

- S9

- mix + S9

- mix

- S9

- mix

Range 10

- 46 10

- 46 10

- 46 9

- 43

Mean 26.8 24.9 26.0 24.3

SD 6.8 6 .9 6.7 6.9

n 284 449 274 446

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between December 2011 and December 2014.COPS_1 Project 508229

- Page 29

- APPENDIX 6 HISTORICAL NEGATIVE CONTROL DATA FOR NUM ERICAL ABERRATIONS

FOR LYMPHOCYTE CHROMOSOME ABERRATION STUDIES

Polyploid cells and endoreduplicated chromosomes per 100

metaphases

polyploidy endoreduplication

+ S9

- mix

- S9

- mix + S9

- mix

- S9

- mix

Range 0 - 2 0 - 3 0 - 1 0 - 1

Mean 0.09 0.14 0.01 0 .04

SD 0.30 0.41 0.10 0.19

n 193 288 189 286

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between December 2011 and December 2014.COPS_1 Project 508229

- Page 30

- APPENDIX 7 CERTIFICATE OF ANALYSIS