## FINAL REPORT

Study Title



## EVALUATION OF THE AB ILITY OF CASTOR OIL, SULFATED

,

SODIUM SALT, 75% TO INDUCE CHROMOSOME ABERRATIONS

IN CULTURED PERIPHER AL HUMAN LYMPHOCYTES

(WITH REPEAT EXPERIM ENT)

Author

C.A.F. Buskens, MSc.

Test Facility

WIL R esearch Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 503341

Substance 205003/A

- Page 1 of 31

-Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 2 - 1. CONTENTS

1. CONTENTS

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

2. STATEMENT OF GLP COMPLIANCE

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

3. QUALITY ASSURANCE STATEMENT

......................................................................................... 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 METHODS

...................................................................................................... 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-fraction

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

6.5.2. Preparation of S9-mix

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

6.6. Study design

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

6.6.1. Dose range finding test

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

6.6.2. Cytogenetic assay

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

6.6.3. Chromosome preparation

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

6.6.4. Preparation of slides

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

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

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

6.6.6. Analysis of slides for chromosome aberrations

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

6.7. Electronic data capture

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

6.8. Interpretation

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

6.8.1. Acceptability of the assay

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

6.8.2. Data evaluation and statistical procedures

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

6.9. List of deviations

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

6.9.1. List of protocol deviations

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

6.9.2. List of standard operating procedures deviations

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

7. RESULTS

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

7.1. Dose range finding test

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

7.2. First cytogenetic assay

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

7.3. Second cytogenetic assay

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

7.4. Evaluation of the results

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

8. CONCLUSION

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

9. REFERENCES

............................................................................................................................ 16Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 3 - TABLES

Table 1 Mitotic index of human lymphocyte cultures treated with Castor Oil, Sulfated,

Sodium Salt, 75% in the dose range finding test

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

Table 2 Mitotic index of human lymphocyte cultures treated with Castor Oil, Sulfated,

Sodium Salt, 75% in the first cytogenetic assay

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

Table 3 Chromosome aberrations in human lymphocyte cultures treated with Castor Oil,

Sulfated, Sodium Salt, 75% in the absence of S9-mix in the first cytogenetic

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

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

Table 4 Chromosome aberrations in human lymphocyte cultures treated with Castor Oil,

Sulfated, Sodium Salt, 75% in the presence of S9-mix in the first cytogenetic

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

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

Table 5 Mitotic index of human lymphocyte cultures treated with Castor Oil, Sulfated,

Sodium Salt, 75% in the second cytogenetic assay

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

Table 6 Chromosome aberrations in human lymphocyte cultures treated with Castor Oil,

Sulfated, Sodium Salt, 75% in the absence of S9-mix in the second cytogenetic

assay (24 h exposure time, 24 h fixation time)

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

Table 7 Chromosome aberrations in human lymphocyte cultures treated with Castor Oil,

Sulfated, Sodium Salt, 75% in the absence of S9-mix in the second cytogenetic

assay (48 h exposure time, 48 h fixation time)

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

Table 8 Chromosome aberrations in human lymphocyte cultures treated with Castor Oil,

Sulfated, Sodium Salt, 75% in the presence of S9-mix in the second cytogenetic

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

............................................................. 25

APPENDICES

APPENDIX 1 TABLES

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

APPENDIX 2 DEFINITIONS OF CHROMOSOME ABERRATIONS SCORED IN METAPHASE

PORTRAITS

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

APPENDIX 3 STATISTICAL EVALUATION OF THE TEST RESULTS

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

APPENDIX 4 HISTORICAL CONTROL DATA FOR LYMPHOCYTE CHROMOSOME

ABERRATION STUDIES

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

APPENDIX 5 HISTORICAL CONTROL DATA FOR NUMERICAL ABERRATIONS FOR

LYMPHOCYTE CHROMOSOME ABERRATION STUDIES

....................................... 30

APPENDIX 6 CERTIFICATE OF ANALYSIS

................................................................................... 31Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 6 - 4. SUMMARY

Evaluation of the ability of Castor Oil, Sulfated, Sodium Salt, 75% to induce chromosome aberrations

in cultured peripheral human lymphocytes (with repeat experiment).

This report describes the effect of Castor Oil, Sulfated, Sodium Salt, 75% on the number of

chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a

metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The

possible clastogenicity of Castor Oil, Sulfated, Sodium Salt, 75% was tested in two independent

experiments.

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

guidelines.

Batch R- 10135

- 064 of Castor Oil, Sulfated, Sodium Salt, 75% was a clear amber liquid with a purity of

70.7%. The test substance concentrations were corrected for the purity. A correction factor of 1.41

was used. The concentrations are expressed as active ingredient. Castor Oil, Sulfated, Sodium Salt,

75% was dissolved in ethanol at concentrations of 20 mg/ml and below. The test substance was

suspended in ethanol at concentrations of 40 mg/ml and above.

In the first cytogenetic assay, Castor Oil, Sulfated, Sodium Salt, 75% was tested up to 666 μg/ml for a

3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction.

Castor Oil, Sulfated, Sodium Salt, 75% precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Castor Oil, Sulfated, Sodium Salt, 75% was tested up to 300 μg/ml

for a 24 h continuous exposure time with a 24 h fixation time and up to 200 μg/ml for a 48 h

continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was

reached at these dose levels. In the presence of S9-mix Castor Oil, Sulfated, Sodium Salt, 75% was

tested up to 500 μg/ml for a 3 h exposure time with a 48 h fixation time. Castor Oil, Sulfated, Sodium

Salt, 75% precipitated in the culture medium at this dose level.

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.

Castor Oil, Sulfated, Sodium Salt, 75% 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 repeated experiments.

No effects of Castor Oil, Sulfated, Sodium Salt, 75% 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 Castor Oil, Sulfated, Sodium Salt, 75% 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 Castor Oil, Sulfated, Sodium Salt, 75% is not

clastogenic in human lymphocytes under the experimental conditions described in this report.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 7 - 5. INTRODUCTION

5.1. Preface

Sponsor Sulfated Castor Oil Consortium, Society of Chemical

Manufacturers & Affiliates

1850 M Street NW, Suite 700

Washington, DC 20036

USA

Study Monitor Rodney Boatman, Ph.D.

PCA Services

2704 Trail Wood Dri ve

Durham, NC 27705

USA

Test Facility WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Study Director C.A.F. Buskens, MSc.

Study Plan Start : 04 September 2013

Completion : 10 November 2013

5.2. Aims of the study

The objective of this study was to evaluate Castor Oil, Sulfated, Sodium Salt, 75% 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- mi x).

Background of the test system

Whole blood samples obtained from healthy male subjects were 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 Castor Oil, Sulfated, Sodium Salt, 75% 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 Castor Oil,

Sulfated, Sodium Salt, 75%, 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 and exchange figures. Results from cultures treated with Castor Oil,

Sulfated, Sodium Salt, 75% 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.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- 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 July

21, 1997).

- European Community (EC). Commission 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.

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. MATERIALS AND METHODS

6.1. Test substance

6.1.1. Test substance information

Identification Castor Oil, Sulfated, Sodium Salt, 75%

CAS Number 68187

- 76

- 8

Description Clear amber liquid

Batch R

- 10135

- 064

Purity/Composition 70.7%

Test substance storage At room temperature in the dark

Stable under storage conditions until 18 July 2014 (expiry date)

See APPENDIX 6; Certificate of analysis.

6.1.2. Study specific test substance information

Purity/composition correction factor

required Yes, correction factor is 1.41 (70.7% purity)

Hygro scopic No

Volatile No

Specific Gravity / Density 1.049

Stability at higher temperatures Yes, maximum temperature 100°C,

maximum duration: until dry

Stability in vehicle Ethanol: Not indicated

Solubility in vehicle Ethanol: PartialCastor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 9 - 6.1.3. Test substance preparation

The test substance concentrations were corrected for the purity (70.7%). A correction factor of 1.41

was used. Concentrations are expressed as active ingredient.

Castor Oil, Sulfated, Sodium Salt, 75% was suspended in ethanol absolute ( Extra pure, Merck,

Darmstadt, Germany) at concentrations of 40 mg/ml and above

. The test substance was dissolved in

ethanol at concentrations of 20 mg/ml and below. Castor Oil, Sulfated, Sodium Salt, 75%

concentrations were used within 2.5 hours after preparation. The final concentration of the solvent in

the culture medium was 0.5 – 1.0% (v/v).

6.2. Reference substances

6.2.1. Negative control

The vehicle for the test substance was ethanol.

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 μg/ml for a 3 h

exposure period (24 h fixation time).

Solvent for positive controls

Hanks’ Balanced Salt Solution (HBSS) (Invitrogen Corporation, Breda, 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, EC).

Blood was collec te d from healthy adult, non-smoking, male volunteers. The Average Generation Time

(AGT) of the cells and the age of the donor at the time the AGT was determined (December 2012) are

presented below:

Dose range finding study: age 25, AGT = 12.4 h

First cytogenetic assay: age 21, AGT = 12.9 h

Second cytogenetic assay: age 36, AGT = 13.5 h

6.4. Cell culture

Blood samples

Blood samples were collected by venipuncture 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.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 10

- Culture medium

Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20%

(v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM)

(Invitrogen Corporation), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (Invitrogen

Corporation) 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 Ltd., United Kingdom) was added.

Environmental conditions

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

were a humid atmosphere of 80 - 100% (actual range 48

- 90 %), containing 5.0 ± 0.5% CO 2 in air, at a

temperature of 37.0 ± 1.0°C (actual range 35.6 - 37.6°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 (in the range of 35. 6 - 36.0°C), humidity (with

a maximum of 30%) and CO 2 percentage (with a maximum of 1%) occurred that were caused by

opening and closing of the incubator door, but the time of these deviations did not exceed 2 hour.

Based on laboratory historical data these deviations are considered not to affect the study integrity.

The temporary deviations from the humidity in the dose range finding study are explained in protocol

deviation 1.

6.5. Metabolic activation system

Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats (6), which were

obtained from Charles River (Sulzfeld, Germany).

Rat liver microsomal enzymes in the second cytogenetic assay were obtained from Trinova Biochem

GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed

orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

6.5.1. Preparation of S9-fraction

The animals were housed at WIL Research Europe in a special room under standard laboratory

conditions, as described in the Standard Operating Procedures, and allowed to acclimatise for at least

5 days. The rats were orally dosed at three consecutive days with a suspension of phenobarbital

(80 mg/kg body weight; Bufa B.V., IJsselstein, The Netherlands ) and ß-naphthoflavone (100 mg/kg

body weight; Sigma) in corn oil (Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands) (they

were denied access to food for 3 to 4 hours preceding each dosing). One day after the final exposure

(24 h), the rats were sedated using oxygen/carbon dioxide and then killed by decapitation. The rats

received a limited quantity of food during the night before sacrifice. The livers of the rats were removed

aseptically, and washed in cold (0°C), sterile 0.1 M sodium phosphate buffer (pH 7.4) containing

0.1 mM Na 2 -EDTA (Merck, Darmstadt, Germany). The livers were minced in a blender and

homogenised in 3 volumes of phosphate buffer with a Potter homogeniser. The homogenate was

centrifuged for 15 min at 9000 g . The supernatant (S9) was transferred into sterile ampules, which

were stored in liquid nitrogen (-196°C) for a maximum of 1 year.

The S9 batch was characterised in a bacterial reverse mutation assay in Salmonella typhimurium

tester strain TA98 with the mutagens Benzo-(a)-pyrene (Sigma) and 2-aminoanthracene (Sigma) at

concentrations of 5 μg/plate and 1 μg/plate, respectively. These mutagens require metabolic activation

for exerting their mutagenic effects.

6.5.2. Preparation of S9-mix

S9 -mix was prepared immediately before use and kept on ice. S9-mix components contained per ml:

1.63 mg MgCl 2 .6H 2 O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim,Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 11

- Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES

(Invitrogen Corporation ).

The above solution was filter (0.22 μ m) -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

(c ontaining 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. Castor Oil, Sulfated, Sodium Salt, 75% 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 Castor Oil, Sulfated, Sodium Salt, 75%

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 5000 μg/ml.

After 3 h exposure to Castor Oil, Sulfated, Sodium Salt, 75% 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 Castor Oil, Sulfated, Sodium Salt, 75% in the lymphocyte cultures was determined

using the mitotic index.

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 determined by the solubility (3 h

exposure time) or had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of

the lowest dose level wa s approximately the same as the mitotic index of the solvent control.

6.6.2. Cytogenetic assay

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

Castor Oil, Sulfated, Sodium Salt, 75% 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

Castor Oil, Sulfated, Sodium Salt, 75% 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 cytogenetic assay.

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

solubility of Castor Oil, Sulfated, Sodium Salt, 75% in the culture medium appropriate dose levels wereCastor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 12

- 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

Castor Oil, Sulfated, Sodium Salt, 75% 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 Castor Oil, Sulfated, Sodium Salt, 75% 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 included 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 cell 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 were 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 prepared

per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa

(Merck) solution in 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 (Klinipath) 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

1000 cells (with a maximum deviation of 5%). At least three analysable concentrations were used for

scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those

cultures with an inhibition of the mitotic index of about 50% or above whereas the mitotic index of the

lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures

treated with an intermediate dose were examined for chromosome aberrations. In case Castor Oil,

Sulfated, Sodium Salt, 75% was not cytotoxic and/or difficult to dissolve in aqueous solutions, the

highest concentration analysed at the 3 h exposure time was determined by the solubility in the culture

medium.

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 hundred 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 aberrationsCastor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 13

- and the number of aberrations were calculated.

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, onesided, 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 was considered positive (clastogenic) in the chromosome aberration test if:

a) It induced a dose-related 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 test, one-sided, p < 0.05)

increase in the number of cells with chromosome aberrations.

The preceding criteria are not absolute and other modifying factors might enter into the final evaluation

decision.

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 compared

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< 0.05) the hypothesis that the

(a+b) (c+d) (a+c) (b+d)Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 14

- 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 dose range finding study the humidity w as several times below the range as mentioned

in the protocol with a minimum of 48 %, for a maximum of 2.5 hours.

Evaluation: These deviations were only for a short time and were caused by opening and

closing of the incubator door during handling of the cell cultures. The dose range finding study

was only used to set the doses for the cytogenetic assays. Therefore these deviations of the

humidity have 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

In the dose range finding test blood cultures were treated with 100, 333, 1000, 3330 and 5000 μ g

Castor Oil, Sulfated, Sodium Salt, 75%/ml culture medium with and without S9-mix.

At a concentration of 1000 μg/ml Castor Oil, Sulfated, Sodium Salt, 75% precipitated in the culture

medium, the precipitate did not disappear within 48 h.

Table 1 (APPENDIX 1 ) shows the mitotic index of cultures treated with various Castor Oil, Sulfated,

Sodium Salt, 75% concentrations or with the negative control substance.

7.2. First cytogenetic assay

Further investigation showed that a concentration of 666 μg/ml already precipitated in the culture

medium. Therefore, a concentration of 666 μg/ml was used as the highest concentration.

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

cytogenetic assay:

Without and with S9 -mix: 100, 333 and 666 μg/ml culture medium

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

Table 2 (APPENDIX 1 ) shows the mitotic index of cultures treated with various Castor Oil, Sulfated,

Sodium Salt, 75% concentrations or with the positive or negative control substances.

All dose levels were selected for scoring of chromosome aberrations

. Both in the absence and

presence of S9-mix, Castor Oil, Sulfated, Sodium Salt, 75% 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, Castor Oil, Sulfated, Sodium Salt, 75% did not increase

the number of polyploid cells and cells with endoreduplicated chromosomes.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 15

- 7.3. Second cytogenetic assay

To obtain more information about the possible clastogenicity of Castor Oil, Sulfated, Sodium Salt,

75% , a second cytogenetic assay was performed in which human lymphocytes were continuously

exposed to Castor Oil, Sulfated, Sodium Salt, 75% 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 Castor Oil, Sulfated,

Sodium Salt, 75%. The following dose levels were selected for the second cytogenetic assay:

Without S9-mix : 10, 30, 100, 200

, 300, 400 and 500 μg/ml culture medium

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

With S9-mix : 100, 300 and 500 μg/ml culture medium

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

At a concentration of 500 μg/ml Castor Oil, Sulfated, Sodium Salt, 75% precipitated in the culture

medium.

Table 5 (APPENDIX 1 ) shows the mitotic index of cultures treated with various Castor Oil, Sulfated,

Sodium Salt, 75% concentrations or with the positive or negative control substances.

Based on these observations the following doses were selected for scoring of chromosome

aberrations:

Without S9-mix : 10, 200 and 300 μg/ml culture medium

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

10, 100 and 200 μg/ml culture medium

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

With S9-mix : 100, 300 and 500 μg/ml culture medium

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

Both in the absence and presence of S9-mix, Castor Oil, Sulfated, Sodium Salt, 75% 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, Castor Oil, Sulfated, Sodium Salt, 75% did not increase

the number of polyploid cells and cells with endoreduplicated chromosomes.

7.4. Evaluation of the results

The ability of Castor Oil, Sulfated, Sodium Salt, 75% to induce chromosome aberrations in human

peripheral lymphocytes was investigated in two independent experiments. The highest concentration

analysed was selected based on the solubility of the test substance in the culture medium (3 h

exposure time) or on toxicity, inhibition of the mitotic index of about 50% or greater (24 h and 48 h

continuous exposure times).

The mitotic indices of cultures treated with various Castor Oil, Sulfated, Sodium Salt, 75%

concentrations or with the negative control 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 Castor Oil,

Sulfated, Sodium Salt, 75% are presented in Table 3

, Table 4 and Table 6 – 8 (APPENDIX 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 statistical 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 historical

control data range (see APPENDIX 5) . The positive control chemicals (MMC-C and CP) bothCastor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 16

- 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 Castor Oil, Sulfated, Sodium Salt, 75% 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 Castor Oil, Sulfated, Sodium Salt, 75% 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 Castor Oil, Sulfated, Sodium Salt, 75% 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 Castor Oil, Sulfated, Sodium Salt, 75% 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 Gene-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.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 17

- APPENDIX 1 TABLES

Table 1 Mitotic index of human lymphocyte cultures treated with Castor Oil, Sulfated,

Sodium Salt, 75% in the dose range finding test

Castor Oil, Sulfated, Sodium Salt, 75% Number of metaphases

concentration (μg/ml) Absolute Number Percentage

of cells scored of control

Without metabolic activation (- S9 -mix)

3 h exposure time, 24 h fixation time

Control a) 89 1009 100

Control b) 70 1008 100

100 63 1043 71

333 63 1017 71

1000 c) 39 1012 44

3330 c) 0 1000 0

5000 c ) 0 1000 0

24 h exposure time, 24 h fixation time

Control a) 50 1000 100

Control b) 34 1001 100

100 40 1040 80

333 24 1006 48

1000 c) 0 1000 0

3330 c) 0 1000 0

5000 c) 0 1000 0

48 h exposure time, 48 h fixation time

Control a) 48 1002 100

Control b) 32 1012 100

100 27 1017 56

333 20 1020 42

1000 c) 0 1000 0

3330 c) 0 1000 0

5000 c) 0 1000 0

a) Ethanol (0.5%)

b) Ethanol (1.0%)

c) Castor Oil, Sulfated, Sodium Salt, 75% precipitated in the culture medium.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 18

- Table 1

– continued

–

Castor Oil, Sulfated, Sodium Salt, 75% Number of metaphases

concentration (μg/ml) Absolute Number Percentage

of cells scored of control

With metabolic activation (+S9-mix)

3 h exposure time, 24 h fixation time

Control a) 68 1004 100

Control b) 50 1009 100

100 54 1031 79

33 3 54 1016 79

1000 c) 40 1025 59

3330 c) 0 1001 0

5000 c) 0 1000 0

a) Ethanol (0.5%)

b) Ethanol (1.0%)

c) Castor Oil, Sulfated, Sodium Salt, 75% precipitated in the culture medium.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 19

- Table 2 Mitotic index of human lymphocyte cultures treated with Castor Oil, Sulfated,

Sodium Salt, 75% in the first cytogenetic assay

Castor Oil, Sulfated, Sodium Salt, 75% Number of metaphases a)

concentration (μg/ml) Absolute Number Percentage

of cells scored of control

Without metabolic activation (- S9 -mix)

3 h exposure time, 24 h fixation time

Control b) 65

- 62 1005

- 1033 100

100 57

- 59 1007

- 1015 91

333 56

- 65 1046

- 1007 95

666 c) 62

- 62 1007

- 1001 98

MMC

- C; 0.5 μg/ml 39

- 55 1001

- 1008 74

MMC

- C; 0. 7 5 μg/ml 38

- 42 1018

- 1011 63

With metabolic activation (+S9-mix)

3 h exposure time, 24 h fixation time

Control b) 76

- 78 1011

- 1014 100

100 70

- 70 1003

- 1024 91

333 60

- 69 1014

- 1012 84

666 c) 57

- 58 1012

- 1010 75

CP; 1 0 μg/ml 23

- 34 1031

- 1010 37

a) Duplicate cultures

b) Ethanol

c) Castor Oil, Sulfated, Sodium Salt, 75% precipitated in the culture medium.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 20

- Table 3 Chromosome aberrations in human lymphocyte cultures treated with Castor Oil,

Sulfated, Sodium Salt, 75% in the absence of S9-mix in the first cytogenetic

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

Conc Ethanol

( 0

. 5 % v/v) 100

μ g/ml 333

μ g/ml 666

μ g/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 91 95 97 74

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 1 1 0 1 1 3 1 4 0 2 2 14 11 *** )

25

No. of

Cells with

aberrations

(

- gaps) 0 1 1 0 1 1 2 0 2 0 2 2 14 11 *** )

25

g’ 1

g” 1

b’ 1 1 2 2 12 9

b” 2

m’

m”

exch. 1 2

dic

d’

misc. poly poly

total aberr

(+ gaps) 0 1 0 1 3 1 0 2 15 11

total aberr

(

- gaps) 0 1 0 1 2 0 0 2 15 11

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

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

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.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 21

- Table 4 Chromosome aberrations in human lymphocyte cultures treated with Castor Oil,

Sulfated, Sodium Salt, 75% in the presence of S9-mix in the first cytogenetic

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

Conc Conc

Ethanol

(0. 5 % v/v) 100

μ g/ml 333

μ g/ml 666

μ g/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 91 84 75 37

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 1 0 1 1 1 2 1 2 3 24 35 *** )

59

No. of

Cell s with

aberrations

(

- gaps) 0 1 1 1 0 1 1 1 2 1 2 3 22 32 *** )

54

g’ 2 2 4

g” 1

b’ 1 1 1 23 37

b” 1 1 2 1

m’

m”

exch. 2

dic 1

d’

misc. poly endo

total aberr

(+ gaps) 0 3 1 0 1 1 1 2 27 44

total aberr

(

- gaps) 0 1 1 0 1 1 1 2 25 39

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) were not counted as an

aberration.

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 22

- Table 5 Mitotic index of human lymphocyte cultures treated with Castor Oil, Sulfated,

Sodium Salt, 75% in the second cytogenetic assay

Castor Oil, Sulfated, Sodium Salt, 75% Number of metaphases a)

concentration (μg/ml) Absolute Number Percentage

of cells scored of control

Without metabolic activation (- S9 -mix)

24 h exposure time, 24 h fixation time

Control b) 52

- 58 1004

- 1005 100

10 55

- 55 1019

- 1033 100

30 49

- 53 1003

- 1018 93

10 0 51

- 53 1013

- 1005 95

200 48

- 50 1009

- 1020 89

300 24

- 21 1024

- 1004 41

400 15

- 14 1015

- 1019 26

500 c) 0

- 0 1000

- 1000 0

MMC

- C; 0.2 μg/ml 35

- 37 1004

- 1005 65

MMC

- C; 0.3 μg/ml 27

- 26 1009

- 1022 48

48 h exposure time, 48 h fixation time

Control b) 64

- 58 1035

- 1015 100

10 47

- 54 1009

- 1016 83

30 45

- 37 100 3

- 1027 67

100 41

- 45 1018

- 1002 70

200 35

- 31 1021

- 1005 54

300 8

- 11 1009

- 1017 16

400 0

- 0 1000

- 1000 0

500 c) 0

- 0 1 002

- 1000 0

MMC

- C; 0.1 μg/ml 39

- 42 1010

- 1031 66

MMC

- C; 0. 1 5 μg/ml 38

- 33 1012

- 1001 58

With metabolic activation (+S9-mix)

3 h exposure time, 48 h fixation time

Control b) 63

- 60 1010

- 1008 100

100 36

- 45 1014

- 1006 66

300 34

- 36 1009

- 1027 57

500 c) 40

- 44 1013

- 1017 68

CP; 10 μg/ml 36

- 33 1006

- 1003

– d)

a) Duplicate cultures

b) Ethanol

c) Castor Oil, Sulfated, Sodium Salt, 75% precipitated in the culture medium

d) CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of

control.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 23

- Table 6 Chromosome aberrations in human lymphocyte cultures treated with Castor Oil,

Sulfated, Sodium Salt, 75% in the absence of S9-mix in the second cytogenetic

assay (24 h exposure time, 24 h fixation time)

Conc Ethanol

(0. 5 % v/v) 10

μ g/ml 200

μ g/ml 300

μ g/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

Index (%) 100 100 89 41 65

No. of

Cells

scored 100 100 200 100 100 20 0 100 100 200 100 100 200 100 100 200

No. of

Cells with

aberrations

(+ gaps) a) 2 0 2 1 1 2 0 1 1 1 0 1 13 11 *** )

24

No. of

Cells with

aberrations

(

- gaps) 1 0 1 1 0 1 0 1 1 1 0 1 11 8 *** )

19

g’ 1 1 2

g” 2 3

b’ 1 1 4 4

b” 1 6 4

m’ 1 1

m” 7 1

exch. 1

dic

d’

misc. poly poly poly

total aberr

(+ gaps) 2 0 1 1 0 1 7 0 15 14

total aberr

(

- gaps) 1 0 1 0 0 1 7 0 13 9

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.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 24

- Table 7 Chromosome aberrations in human lymphocyte cultures treated with Castor Oil,

Sulfated, Sodium Salt, 75% in the absence of S9-mix in the second cytogenetic

assay (48 h exposure time, 48 h fixation time)

Conc Ethanol

(0. 5 % v/v) 10

μ g/ml 100

μ g/ml 200

μ g/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 70 54 66

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 2 2 0 2 2 0 1 1 0 2 2 8 18 *** )

26

No. of

Cells with

aberrations

(

- gaps) 0 1 1 0 0 0 0 1 1 0 1 1 8 15 *** )

23

g ’ 2

g” 1 3 1 2 2

b’ 2 7

b” 1 1 1 5 6

m’

m” 2

exch.

dic

d’

misc. p 3p

total aberr

(+ gaps) 0 2 0 3 0 1 0 2 10 22

total aberr

(

- gaps) 0 1 0 0 0 1 0 1 8 18

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.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 25

- Table 8 Chromosome aberrations in human lymphocyte cultures treated with Castor Oil,

Sulfated, Sodium Salt, 75% in the presence of S9-mix in the second cytogenetic

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

Conc Ethanol

(0. 5 % v/v) 100

μ g/ml 300

μ g/ml 500

μ g/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 66 57 68

- b)

No. of

Cells

scor ed 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 0 0 0 1 1 1 0 1 28 25 *** )

53

No. of

Cells with

aberrations

(

- gaps) 0 0 0 0 0 0 0 0 0 1 0 1 24 22 *** )

46

g’ 1 5 2

g” 3

b’ 1 15 12

b” 9 9

m’ 2

m” 2

exch. 3

dic

d’

misc.

total aberr

(+ gaps) 0 0 0 0 0 1 1 0 31 31

total aberr

(

- gaps) 0 0 0 0 0 0 1 0 26 26

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

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

b) CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 26

- APPENDIX 2 DEFINITIONS OF CHROMOSOME 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.

Double 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.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 27

- 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 trior 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

(chromatid 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.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 28

- APPENDIX 3 STATISTICAL EVALUATION OF THE TEST RESULTS

Chi-square Test

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

(INCLUSIVE/EXCLUSIVE GAPS) 1)

.

EXPOSURE S9 -MIX GAPS P-VALUE DECISION AT 95%

DOSE (μg/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.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 29

- APPENDIX 4 HISTORICAL 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 0

- 5 0

- 5 0

- 5 0

- 4

Mean 0.8 0.7 0.7 0. 6

SD 1.0 1. 0 0.9 1.0

n 378 558 382 541

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between September 2010 and September 2013

.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 30

- APPENDIX 5 HISTORICAL CONTROL DATA FOR NUMERICAL 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

- 2 0

- 1

Mean 0.0 9 0. 10 0.02 0.03

SD 0. 31 0.3 6 0.1 5 0.1 6

n 329 470 335 475

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between September 2010 and September 2013.Castor Oil, Sulfated, Sodium Salt, 75% Project 503341

- Page 31

- APPENDIX 6 CERTIFICATE OF ANALYSIS