### FINAL REPORT

Test Facility Study No. 787723, Report No. 30917



### Alcohols, C18-22 Distn. Residues (Cas No. 1160164-88-4) Chromosomal Aberrations Assay wi th Chinese Hamster Ovary Cell Cultures In Vitro

TEST FACILITY: Charles River Tranent Edinburgh EH33 2NE UK

SPONSOR: SASOL Germany GmbH Anckelmannsplatz 1 D-20537 Hamburg Germany

Page 1 of 37

### Page 2 Test Facility Study No. 787723

TABLE OF CONTENTS

1 COMPLIANCE STATEMENT................................................................................ 6

2 QUALITY ASSURANCE STATEMENT ............................................................... 7

3 RESPONSIBLE PERSONNEL ................................................................................ 8

4 SUMMARY ................................................................................................................ 9

5 INTRODUCTION.................................................................................................... 10

5.1 Justification of Test System..................................................................................... 10

5.2 Regulatory Guidelines ............................................................................................. 10

5.3 Location of Study ..................................................................................................... 11

5.4 Study Dates and Archives........................................................................................ 11

6 MATERIALS AND METHODS ............................................................................ 11

6.1 Test Item ................................................................................................................... 11

6.2 Dose Formulation and Analysis .............................................................................. 12

6.3 Cells and Cell Culture.............................................................................................. 12

6.4 Justification of Dose Levels ..................................................................................... 12

6.5 Administration of Test/Control Items.................................................................... 13

7 EXPERIMENTAL PROCEDURES ...................................................................... 13

7.1 S9 Mix (McGregor et al (1988)) .............................................................................. 13

7.2 Accessory Testing..................................................................................................... 14

7.3 Treatment ................................................................................................................. 14

7.4 Harvesting of Cultures and Slide Preparation ...................................................... 15

7.5 Slide Assessment....................................................................................................... 15

8 CALCULATIONS AND DATA ACCEPTANCE................................................. 16

8.1 Quantitative Measure of Toxicity........................................................................... 16

### Page 3 Test Facility Study No. 787723

8.2 Structural and Numerical Chromosomal Aberrations......................................... 16

8.3 Historical Control Data ........................................................................................... 17

8.4 Data Acceptance....................................................................................................... 17

9 DATA EVALUATION ............................................................................................ 17

9.1 Interpretation of Toxicity........................................................................................ 17

9.2 Interpretation of Clastogenicity.............................................................................. 17

10 RESULTS ................................................................................................................. 18

10.1 Toxicity Data (Appendices 1-5)............................................................................... 18

10.2 Chromosomal Aberrations Results (Tables 1-6) ................................................... 18

10.3 Vehicle Control Cultures......................................................................................... 18

10.4 Positive Control Treated Cultures.......................................................................... 18

10.5 Alcohols, C18-22 Distn. Residues Treated Cultures ............................................. 18

11 CONCLUSION......................................................................................................... 20

12 REFERENCES......................................................................................................... 21

13 TABLES.................................................................................................................... 22

14 APPENDICES .......................................................................................................... 29

Final Page of Report ........................................................................................................... ... 37

### Page 4 Test Facility Study No. 787723

LIST OF TABLES

Table 1 Aberration Data: Test 1, With S9 Mix, 6 h Treatment, 24 h Harvest.............. 23 Table 2 Aberration Data: Test 1, Without S9 Mix, 6 h Treatment, 24 h Harvest.............................................................................................................. 24 Table 3 Aberration Data: Test 2, With S9 Mix, 6 h Treatment, 24 h Harvest.............. 25 Table 4 Aberration Data: Test 2, Without S9 Mix, 22 h Treatment, 24 h Harvest.............................................................................................................. 26 Table 5 Aberration Data: Test 2, Without S9 Mix, 22 h Treatment, 48 h Harvest.............................................................................................................. 27 Table 6 Polyploid Data: Test 2, Without S9 Mix, 22 h Treatment, 48 h Harvest ......... 28

### Page 5 Test Facility Study No. 787723

LIST OF APPENDICES
Appendix 1 Toxicity Data: Test 1, With S9 Mix, 6 h Treatment, 24 h Harvest .................. 29 Appendix 2 Toxicity Data: Test 1, Without S9 Mix, 6 h Treatment, 24 h Harvest ............. 30 Appendix 3 Toxicity Data: Test 2, With S9 Mix, 6 h Treatment, 24 h Harvest .................. 31 Appendix 4 Toxicity Data: Test 2, Without S9 Mix, 22 h Treatment, 24 h Harvest ........... 32 Appendix 5 Toxicity Data: Test 2, Without S9 Mix, 22 h Treatment, 48 h Harvest ........... 33 Appendix 6 Certificate of Analysis ...................................................................................... 34 Appendix 7 S9 Data ............................................................................................................. 35 Appendix 8 Osmolality a nd Precipitation Data:................................................................... 36 Appendix 9 Historical Negative and Positive Control Data................................................. 37

### 1 COMPLIANCE STATEMENT Page 6 Test Facility Study No. 787723

I, the undersigned, hereby declare that the study was performed in accordance with the OECD Principles of Good Laboratory Practice as incorporated into the United Kingdom Statutory Instrument for GLP and as accepted by Regulatory Authorities throughout the European Community, United States of America (FDA and EPA) and Japan (MHLW, MAFF and METI).

The study was conducted according to the procedures herein described and this report represents a true and accurate record of the results obtained.

,r

# Elaine~' 27]';14 2010

Study Director Date2 QUALITY ASSURANCE STATEMENT Page 7 Test Facility Study No. 787723

Study Title: Alcohols, C18-22 Distn. Residues (Cas No. 1160164-88-4): Chromosomal Aberrations Assay with Chinese Hamster Ovary Cell Cultures In Vitro

The Charles River Quality Assurance Unit conducted a protocol review, process-based inspections and report audits relevant to this short-term study, as detailed below.

Date(s) of OA Activity Activitv Date of Report to Management

05 October 2009 05 October 2009 06 October 2009 06 October 2009 07 October 2009 07 October 2009 06-07 January 2010 27 January 2010 Protocol Review Establishing Cultures Dose Preparation/Dosing/Protocol Compliance Osmotic Pressure Harvesting Cultures

Slide Preparation Draft Report Audit Final Report Audit 05 October 2009 05 October 2009 06 October 2009 07 October 2009 07 October 2009 07 October 2009 07 January 2010 27 January 2010

The protocol review and report audits were reported to the Study Director on the same date as reported to Management. The outcome of each process-based inspection is also reported to the Study Director, where relevant.

Facilities relevant to this study are included in Charles River's annual facility inspection
programme. The outcome of each inspection is reported to Management.

This report is considered to describe accurately and completely the procedures used in the study and the results obtained.

AlironMcNaughton 

# RN Quality Assurance

### Page 8 Test Facility Study No. 787723

3 RESPONSIBLE PERSONNEL
Study Director: Elaine Murie BSc

Project Leader: Craig A McMillan BSc

Aberration Tests: Elaine Murie BSc Craig A McMillan BSc Dawn Innes BSc

Assessment: Elaine Murie BSc Craig A McMillan BSc Cameron Bain BSc

Quality Assurance: Alison McNaughton HNC

### Page 9 Test Facility Study No. 787723

4 SUMMARY

Alcohols, C18-22 Distn. Residues were submitte d for testing and evaluation of clastogenic potential. Chromosomal aberrations assays we re performed with duplicate, Chinese hamster ovary cell cultures. This study was conducte d incorporating 2 independent tests. Dimethylsulphoxide was the vehicle and cycl ophosphamide and methyl methanesulphonate were the positive controls used in both tests.

Both tests were conducted in the presence and absence of a post-mito chondrial supernatant fraction obtained from the livers of adult, ma le rats treated with Aroclor 1254 (S9) and a NADPH-generating system.

Cultures, established approximately 20 h before testing, were treated for 6 h in the presence and 6 h or 22 h in the absence of S9 mix. Cu ltures were harvested at 24 h (Test 1 and 2) or 48 h (Test 2) post treatment.

Alcohols, C18-22 Distn. Residues were non toxic to Chinese hamster ovary cells in vitro in both the presence and absence of S9 mix wh en tested up to the limit of solubility (200 μg/mL), (Appendices 1-5).

There was no evidence that Alcohols, C 18-22 Distn. Residues induced structural chromosomal aberrations in ei ther the presence or absence of S9 mix (Tables 1-5).

Alcohols, C18-22 Distn. Residues did not induc e an increase in polyploidy in either the presence or absence of S9 mix in cultur es harvested 48 h post treatment (Table 6).

It was concluded that Alcohols, C18-22 Distn. Residues were not clas togenic when tested with Chinese hamster ovary cells in vitro , when tested up to a c oncentration of 200 μg/mL.

### Page 10 Test Facility Study No. 787723

5 INTRODUCTION

The test item was Alcohols, C18-22 Distn. Residues.

The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in culture d mammalian cells. Structural aberrations may be of 2 types, chromosome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but ch romosome-type aberrations also occur. An increase in polyploidy may indi cate that a chemical has the potential to induce numerical aberrations. Chromosome mutations and relate d events are the cause of many human genetic diseases and there is substantial evidence th at chromosome mutations and related events causing alterations in oncogenes and tumor suppre ssor genes of somatic cells are involved in cancer induction in humans and experimental animals.

5.1 Justification of Test System
Many mutagens are clastogeni c in higher organisms both in vivo and in vitro . Cytological test systems are useful in the pre-scr eening of mutagens and carcinogens.

Chinese hamster ovary cells possess characterist ics which make them suitable for cytogenetic testing of clastogens. They can be subcu ltured indefinitely w ithout ageing, generally maintaining a stable pseudo-dipl oid karyotype with a low and easily analysable chromosome number. The short cell cycle of Chinese hamste r ovary cells also allows easy propagation and a high mitotic rate (Hsu et al (1977)). Furthermore, Chinese hamster ovary cells have been comprehensively validated in various cytogenetic tr ials and their sensitivity to a wide range of genotoxins documented (Galloway et al (1987), Ishidate et al (1988), Tezuka et al (1980)).

S9 mix (prepared from rat liver) provided an exogenous metabolic activation system, increasing the detection capability of the te st system for pre-mutagens (Natarajan et al (1976)).

5.2 Regulatory Guidelines
This study complies with OECD guideline 473, ICH Guidelines and with the European Commissions Annex V, Test Method B10.

### Page 11 Test Facility Study No. 787723

5.3 Location of Study
The experimental work of these tests was conducted at the laboratories of:

Charles River Tranent Edinburgh EH33 2NE UK

5.4 Study Dates and Archives
The dates for key stages in th e conduct of this study were:

Stage Date

Study initiation 02 October 2009 Experimental start date 12 October 2009 Experimental completion date 14 December 2009

Study completion date See Compliance statement for date of Study Director's signature



### All raw data generated and record ed during this study, will be stor ed in the Scientific Archive of Charles River, Preclinical Se rvices Edinburgh for 2 years after the issue of the final report. After the 2 year period the Sponsor will be c onsulted regarding the disposal, transfer or continued storage of raw data.

The original signed copy of th e final report will be stored i ndefinitely in the Scientific Archives of Charles River, Preclinical Serv ices Edinburgh.

6 MATERIALS AND METHODS

6.1 Test Item
Active Ingredient: Alcohols, C18-22 Distn. Residues (Cas No. 1160164-88-4) Lot No.: 03585/MA (product code: 5901RN2) Description: Beige waxy solid Purity: 100% by definition Storage Conditions: Stored in the da rk at ambient room temperature Supplier: Sasol Germany GmbH Expiry date: 31 August 2013

The Certificate of Analysis is shown in Appendix 6.

### Page 12 Test Facility Study No. 787723

6.2 Dose Formulation and Analysis

6.2.1 Preparation of Dose Formulations
The test item formed a non sedimentary suspensi on in dimethylsulphoxide at 20 mg/mL. This was with vortex mixing and warm ing at 37oC for 30 minutes.

6.2.2 Analysis of Dose Formulations
Chemical analysis was not performed.

6.3 Cells and Cell Culture

6.3.1 Cells
The cell line used was Chinese hamster ovary (CHO 10 B 4 

### ) cells, obtained from the University of Leiden in 1987. The cell line is mycoplasma tested (in house) on a regular basis. The cells were grown as monolayers , and have a generation time of approximately 12-14 h. The modal chromosome number has been determined for these cells to be 21. The cells were incubated at ca 37°C.

6.3.2 Culture Medium
The basic medium (Ham's F-10) containing HEPES buffer, was supplemented with the antibiotic minocycline. For cell growth and treatment in the absence of S9 mix, foetal bovine serum (10% v/v) was added. The medium used for treatment in the presence of S9 mix and for washing cultures before or after treatment, was serum free.

6.3.3 Cell Cultures
Cells were trypsinised from stock flasks at pa ssage numbers 8 (Test 1) and 16 (Test 2), and resuspended in fresh culture medium at densities of 0.1 x 10 6 

### or 0.05 x 10 6 

### cells/mL. These cells, in 5 mL volumes, were dispensed into 25 cm 2 

### tissue culture flasks. The high and low cell densities were for cultures harvested at 24 or 48 h post treatment respectively. Test cultures were established from the st ock flask about 20 h before testing.

6.4 Justification of Dose Levels
For the first experiment, 9 dose le vels, covering a wide concentra tion range, were tested. The highest dose was 200 μg/mL (limit of solubility), and subsequent dose levels were halving dilutions.

### Page 13 Test Facility Study No. 787723

In the second experiment, th e dose levels selected were:

25, 50, 100 and 200 μg/mL

6.5 Administration of Test/Control Items

6.5.1 Vehicle Controls
All experiments included vehicl e control cultures and these cultures were subjected to the same experimental manipulations as the treated cultures, both in the presence and absence of S9 mix.

6.5.2 Positive Controls
The following positive control materials were used:

With S9 mix:

Cyclophosphamide was used within the concentration range of 20-50 μg/mL.

Without S9 mix:

Methyl methanesulphonate was used within the concentration range of 10-40 μg/mL

7 EXPERIMENTAL PROCEDURES
All experimental procedures, up to harvesti ng, were conducted using aseptic technique and under amber light.

7.1 S9 Mix (McGregor et al (1988))
Aroclor 1254-induced S9 enzymes (supernatan t post-mitochondrial fraction obtained after centrifugation at 9000 g ) were prepared from the livers of adult, male Fischer rats, as described by Ames et al (1975). S9 was stored in sterile plastic tubes immersed in liquid nitrogen ( ca -196°C). Enzymic activity of each batc h of S9 was characterised by testing selected pre-mutagens in an Ames test with S. typhimurium TA 1538 (Appendix 7). S9 batches used demonstrated, within each test , a satisfactory clastogenic response in cells treated with cyclophosphamide.

To prepare S9 mix, Ham's F-10 was added to pre-weighed cofactors: nicotinamide adenine dinucleotide phosphate (NADP) disodium salt and glucose-6-phosphate (G-6-P) disodium salt, giving final concentrat ions in the ‘S9 mix’ of:

### Page 14 Test Facility Study No. 787723

NADP Na 2 

### 4 mM (= 3.150 mg/mL) Glucose-6-phosphate Na 2 

### 25 mM (= 7.605 mg/mL)

This solution was immediately filter-sterilised by passage th rough a 0.2 μm disposable filter assembly and mixed 9:1 (v/v) with the S9.

7.2 Accessory Testing
During the first test, the osmotic pressure of selected concentrations of the test item was measured (Appendix 8). Alcohols, C18-22 Dist n. Residues did not affect the osmolality of the culture medium, therefore no further measurements were made.

Observations of precipitation were made before cultures were washed out at the end of the treatment period (Appendix 8). Precipitati on was noted in culture s treated with 100 and 200 μg/mL, in both the presence and absence of S9 mix in Test 1 and in cultures treated with 200 μg/mL in both the presence and absence of S9 mix in Test 2.

Alcohols, C18-22 Distn. Residues did not change the colour of the culture medium, therefore no pH measurements were made.

7.3 Treatment
The following table shows the treatment schedules for this study.

S9 Mix Cultures Established Test Treatment Period Recovery Period Colcemid Harvest

Presence of S9 mix Tests 1 and 2

Test 1 only 0-6 h 6-22 h 22-24 h 24 h

none 22-24 h 24 h Absence of S9 mix ca 20 h before
exposure Test 2 only 0-22 h 22-46 h 46-48 h 48 h



### Tests were conducted both in the presence and ab sence of S9 mix. Treatments with test item or vehicle control substances were perf ormed on duplicate cell cultures. Several concentrations of the positive controls were tested using single cultures.

Cultures to be treated in the presence of S9 mix were washed before treatment with serum free medium.

Exposure medium was prepared, immediately before dosing, in sterile containers.

### Page 15 Test Facility Study No. 787723

Treatment with the test item was as follows:

S9 Mix Growth Medium S9 mix Dosing Solution Final volume (approx) Presence of S9 mix 4.5 mL 0.5 mL 50 μL 5 mL

Absence of S9 mix 5 mL - 50 μL 5 mL



### After treatment, cells were washed twice with serum free medium, then full growth medium added, for the recovery period and colcemid tr eatment. The volume of medium for the recovery period was 5 mL.

Living cultures were examined for evidence of changes to cell morphol ogy, once at the end of the treatment period and again be fore harvesting of cultures.

7.4 Harvesting of Cultures and Slide Preparation

Colcemid was added to all cultures at a final concentration of 0.1 μg/m L. Culturing the cells in medium containing colcemid for 2 h accumulated cells in metaphase; the stage of cell division at which chromosomes can be examined using light microscopy. Mitotic cells were harvested by gently tapping flasks to release these cells from the monolayer. Cells were sedimented by centrifugation (approximately 190 g ), and treated with hypotonic solution (1% trisodium citrate) for 15 min at room temper ature. The cells were then fixed (after sedimentation as before) using 4 mL of freshly pr epared fixative (methano l:glacial acetic acid, 3:1). Two further changes (after sediment ation as before) of fixative were made.

Monolayer cells were trypsini sed, counted and discarded. This provided a quantitative measure of toxicity.

For both experiments, 3 slides per culture were made. All slides were marked with the study number and assigned a unique, c oded number from a computer generated sequence. Slides were prepared by dropping the cell suspension on to clean, grease-free slides. The slides were stained with 5% Giemsa, then made permanen t by mounting coverslips with DPX mountant.

7.5 Slide Assessment

7.5.1 Screen for Toxicity
Slides were examined for evidence of metapha se cells and signs of cellular necrosis.

7.5.2 Chromosomal Aberrations

Three concentration levels were selected for assessment of chromosomal aberrations.

### Page 16 Test Facility Study No. 787723

From 2-3 slides per culture, up to 50 metaphase cells per slide, a tota l of 100 metaphase cells per culture, were examined. Slides we re scored in order of coded number.

A microscope was used for this assessment, the magnification used being x 1000 or x 1250, achieved with x 10 or x 12.5 eyepieces and x 100 objective.

The number of chromosomes in each metapha se cell and all abnormalities, using the nomenclature of Gebhart (1970) was recorde d. The types of structural and numerical aberrations recorded are listed in abbreviations page. The positions on the slides of any structurally aberrant cells were recorded us ing the Vernier scale on the microscope stage.

7.5.3 Polyploidy Assessment

As cultures harvested at both culture times were negative with regards to structural aberrations, a further assessment of polyploidy was made. The Study Director selected the later harvest time as being more appropriate for this analysis. This decision was based on results from the previous assessments. For this assessment, approximately 300 metaphase cells were cursorily examined at a magnificati on of x 400 or x 500 and deemed to be either diploid, polyploid or endoredupli cated. In this assessment no metaphase cell in a field was rejected.

This assessment was considered more objective, a voiding scorer selection of either normal or polyploid cells. In addition, the larger sample size gave a more accurate frequency of such cells in the population.

8 CALCULATIONS AND DATA ACCEPTANCE

8.1 Quantitative Measu re of Toxicity

From the cell counts, the number of cells recovered per culture, was calculated. This was then compared with the number of cells (mean of 2 cultures) recovered from the vehicle control cultures.

8.2 Structural and Numerical Chromosomal Aberrations
From the results, 5 parameters were calculated, a nd judged as negative, suspicious or positive. These parameters were:

1. Lesions per cell 2. Percentage of aberrant cells including cells with gaps only 3. Percentage of aberrant cells excluding cells with gaps only 4. Percentage of aneuploid cells

### Page 17 Test Facility Study No. 787723

5 Percentage of polyploid cells (normal and endoreduplicated) from additional assessment of polyploidy

The third parameter is considered the most impo rtant in judging the true clastogenicity of a test item.

8.3 Historical Control Data
The results obtained were compared with the histor ical control data. Hist orical control data is given in Appendix 9.

8.4 Data Acceptance
The experiments in this study were deemed to be valid because they fulfilled the following criteria:

There was no evidence of contamination Cells in vehicle control cultures had normal growth The results of vehicle and positive control cultures were typical The test item had 3 acceptable dose levels for assessment

9 DATA EVALUATION

9.1 Interpretation of Toxicity

A dose level was considered to be toxic if the ce ll count was reduced to less than 50% of the mean vehicle control culture values or if c onsistent evidence of ch anges to cell morphology was observed.

9.2 Interpretation of Clastogenicity
The results for test item and positive contro l treated cultures are evaluated by comparison with the concurrent vehicle control cultures a nd with historical nega tive control data. A negative response was recorded if responses from the test item treated cultures are within the 95% confidence limits for the historical negative control data.

The response at a single dose was classified as si gnificant if the percent of aberrant cells is consistently greater than the 99% confidence limit s for the historical negative control data or greater than double the frequency of an elevated vehicle or untreated control culture if appropriate.

A test was positive if the response in at leas t one acceptable dose level was significant by the criterion described above.

### Page 18 Test Facility Study No. 787723

A test item was positive if Test 1 was positive, as described above or if one of the tests was positive and the other test gave indications of activity. These indications may be suspicious levels of aberrant cells (betw een 95% and 99% confidence limits).

Experiments that met in part the criteria for a positive response, or marginally met all the criteria, were classe d as inconclusive.

No statistical analysis was performed.

10 RESULTS

10.1 Toxicity Data (Appendices 1-5)
No toxicity was noted in any of the cultures tr eated with the test item, when tested to a concentration of 200 μg/mL.

10.2 Chromosomal Aberrations Results (Tables 1-6)

Cultures treated with the follo wing dose levels of Alcohols, C18-22 Distn. Residues were selected for assessment of chromosomal aberrations:

Test 1: 50, 100 and 200 μg/mL Test 2: 50, 100 and 200 μg/mL

10.3 Vehicle Control Cultures

The vehicle control cultures had levels of stru ctural and numerical abe rrations within the 95% confidence limits of the hist orical negative control data.

10.4 Positive Control Treated Cultures
The positive control substances, cyclophos phamide in the presence and methyl methanesulphonate in the absence of S9 mix, induced positive frequencies of structural aberrations in at least one concentration leve l per section. These results demonstrated the sensitivity of the test system.

10.5 Alcohols, C18-22 Distn. Residues Treated Cultures

All cultures treated with Alcohol s, C18-22 Distn. Residues had leve ls of structural aberrations within the 95% confidence limits for a negative response.

### Page 19 Test Facility Study No. 787723

An extra assessment of polyploidy was carried out on the cultures treated in the absence of S9 mix and harvested at 48 h. All the cultures tr eated with Alcohols, C18-22 Distn. Residues had levels of polyploidy within the 95% conf idence limits for a negative response.

### Page 20 Test Facility Study No. 787723

11 CONCLUSION

It was concluded that Alcohols, C18-22 Distn. Residues were not clas togenic when tested with Chinese hamster ovary cells in vitro , when tested up to a c oncentration of 200 μg/mL.

### Page 21 Test Facility Study No. 787723

12 REFERENCES

Ames B N, McCann J and Yamasaki E (1975). Methods for detec ting carcinogens and mutagens with the Salmonella /mammalian microsome mutagenicity test. Mutat Res, 31 , 347- 364.

Galloway S M, Armstrong M A, Reuben C, Colm an S, Brown C, Cannon A D, Bloom A D, Nakamura F, Ahmed M, Duk S, Rimpo J, Ma rgolin B H, Resnick M A, Anderson B and Zeiger E (1987). Chromosome aberrations and si ster chromatid exchanges in Chinese hamster ovary cells: Evaluations of 108 ch emicals. Environ Mol Mutagen, 10 (Suppl 10), 1-175.

Gebhart E, (1970). The Treat ment of Human Chromosomes in vitro , in Vogel F and Rohrborn G, (Eds), Chemical Mutagenesis in Mammals and Man, Springer-Verlag, Berlin.

Hsu T L, Collie C J, Lusley A F and Johnston D A (1977). Cytogenetic assays of chemical clastogens using mammalian cells in culture. Mutat Res, 45 , 233-247.

Ishidate M Jr, Harnois M C and Sofuni T (1988) . A comparative analysis of data on the clastogenicity of 951 chemical substances test ed in mammalian cell cultures. Mutat Res, 195

, 151-213.

McGregor D B, Edwards I, Riach C G, Cattanach P, Martin R, Mitchell A and Caspary W J (1988). Studies of an S9-based metabolic act ivation system used in the mouse lymphoma L5178Y cell mutation assay. Mutagenesis, 3 , 485-490.

Natarajan A T, Tates A D, van Buul P P W, Meijers M and De Vogel N (1976). Cytogenetic effects of mutagens/carci nogens after activation in a microsomal system in vitro , I. Induction of chromosome aberrations and sister chromatid exchanges by diethyln itrosamine (DEN) and dimethylnitrosamine (DMN) in CHO cells in th e presence of rat-liver microsomes. Mutat Res, 37 , 83-90.

Tezuka H, Ando N, Suzuki R, Terahata M, Mori ya M and Shirasu Y (1980). Sister-chromatid exchanges and chromosomal aberrations in cu ltured Chinese hamster cells treated with pesticides positive in microbial reversion assays. Mutat Res, 78 , 177-191.

### Page 22 Test Facility Study No. 787723

13 TABLES

Abbreviations and Footnotes for Tables and Appendices + = Positive aberration frequency +- = Suspicious aberration frequency - = Negative aberration frequency e = Elevated aberration frequency t = Toxicity evident from morphological changes tt = Toxicity evident from reduced cell count (<50% of vehicle) ttt = Too toxic for metaphase assessment r = Reduced number of cells scored du e to extensive chromosome damage Index = Expressed as an index of the vehicle control Conc. = Concentration
Type Description Abbreviation in Tables Number of Lesions Gap Achromatic region G 1

Break Discontinuation with dislocation of chromatid section or clearly reduced chromatid arm length B 1 Chromatid: Affecting one chromatid Fragment Chromatid fragment unassociated with chromosome F 1

Gap Achromatic region G 1

Break Discontinuation with dislocation of chromosome section or clearly reduced chromosome arm length B 1 Chromosome: Affecting both chromatids at identical sites Fragment Acentric chromosome fragment unassociated with chromosome F 1

Exchange Two or more chromosomes clearly joined in a complex figure E 2

Dicentric Chromosome with 2 centromeres D 2 Complex

Ring Circular chromosome; with or without a
centromere R 2

Double minutes Very small pairs of chromatin, appear as dots DM None

Uncondensed chromosome Extended chromosome region often with a banded appearance UC None

Reciprocal translocation Complete exchange of material between chromosomes TR 2

Robertsonian translocation Fusion of 2 telocentric chromosomes RTR 1

Pericentric inversion Change in position of centromere within chromosome INV 2

Pulverised chromosome Several lesions usually breaks and gaps in one chromosome PC 5

Multiple aberration A metaphase cell with more than 10 lesions MDC 10 Aneuploid Metaphase cell with 23-25 chromosomes AE Endoreduplicated Characterist ic paired chromosomes ER Polyploidy 26 and more chromosomes PP No lesionsPage 23Test Facility Study No. 787723Table 1Aberration Data: Test 1, With S9 Mix, 6 h Treatment, 24 h HarvestStructural AberrationsAberrationFrequencyAberrant Cell FrequencyNumericalAberrationsChromatid Chromosome Complex Lesions/CellIncludingGapsExcludingGaps% of Cells WithTreatmentGroupConc.(μg/mL)DecodedCultureNo.No. ofCellsScoredG B F G B F E D RMultipleOtherJudge % Judge % Judge AE ER PP1 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 1 0Dimethylsulphoxide1%2 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 015 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 05016 100 1 1 0 0 0 0 0 0 0 0 0 0.02 - 2 - 1 - 0 0 017 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 010018 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 019 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 1Alcohols,C18-22Distn.Residues20020 100 1 0 0 0 0 0 0 0 0 0 0 0.01 - 1 - 0 - 0 0 040 23 100 1 4 4 0 1 0 4 0 0 0 0 0.18 + 14 + 13 + 1 0 1Cyclophosphamide50 24 100 4 4 3 0 0 0 1 0 0 0 0 0.13 + 11 + 8 + 1 0 0Page 24Test Facility Study No. 787723Table 2Aberration Data: Test 1, WithoutS9 Mix, 6 h Treatment, 24 h HarvestStructural AberrationsAberrationFrequencyAberrant Cell FrequencyNumericalAberrationsChromatid Chromosome Complex Lesions/CellIncludingGapsExcludingGaps% of Cells WithTreatmentGroupConc.(μg/mL)DecodedCultureNo.No. ofCellsScoredG B F G B F E D RMultipleOtherJudge % Judge % Judge AE ER PP25 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 0Dimethylsulphoxide1%26 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 039 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 1 0 05040 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 041 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 010042 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 043 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 0Alcohols,C18-22Distn.Residues20044 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 030 47 100 0 4 0 0 0 0 0 0 0 0 0 0.04 - 3 - 3 +- 1 0 0Methylmethanesulphonate40 48 100 3 11 1 0 0 0 7 0 0 0 0 0.29 + 16 + 14 + 1 0 0Page 25Test Facility Study No. 787723Table 3Aberration Data: Test 2, With S9 Mix, 6 h Treatment, 24 h HarvestStructural AberrationsAberrationFrequencyAberrant Cell FrequencyNumericalAberrationsChromatid Chromosome Complex Lesions/CellIncludingGapsExcludingGaps% of Cells WithTreatmentGroupConc.(μg/mL)DecodedCultureNo.No. ofCellsScoredG B F G B F E D RMultipleOtherJudge % Judge % Judge AE ER PP49 100 1 0 0 0 0 0 0 0 0 0 0 0.01 - 1 - 0 - 0 0 0Dimethylsulphoxide1%50 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 1 053 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 1 05054 100 1 0 0 0 0 0 0 0 0 0 0 0.01 - 1 - 0 - 0 1 055 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 1 010056 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 1 0 057 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 1 0 1Alcohols,C18-22Distn.Residues20058 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 020 59 100 0 2 0 0 0 0 0 0 0 0 0 0.02 - 1 - 1 - 1 0 0Cyclophosphamide30 60 100 0 7 1 0 0 0 1 0 0 0 0 0.10 + 8 + 8 + 1 0 0Page 26Test Facility Study No. 787723Table 4Aberration Data: Test 2, WithoutS9 Mix, 22 h Treatment, 24 h HarvestStructural AberrationsAberrationFrequencyAberrant Cell FrequencyNumericalAberrationsChromatid Chromosome Complex Lesions/CellIncludingGapsExcludingGaps% of Cells WithTreatmentGroupConc.(μg/mL)DecodedCultureNo.No. ofCellsScoredG B F G B F E D RMultipleOtherJudge % Judge % Judge AE ER PP63 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 0Dimethylsulphoxide1%64 100 1 0 0 0 0 0 0 0 0 0 0 0.01 - 1 - 0 - 0 0 067 100 1 0 0 0 0 0 0 0 0 0 0 0.01 - 1 - 0 - 0 0 15068 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 069 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 010070 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 071 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 0Alcohols,C18-22Distn.Residues20072 100 0 1 0 0 0 0 0 0 0 0 0 0.01 - 1 - 1 - 0 0 020 74 100 2 2 0 0 0 0 1 0 0 0 0 0.06 +- 5 +- 3 +- 1 0 1Methylmethanesulphonate30 75 100 3 11 1 1 0 1 6 0 0 0 0 0.29 + 11 + 10 + 1 0 2Page 27Test Facility Study No. 787723Table 5Aberration Data: Test 2, WithoutS9 Mix, 22 h Treatment, 48 h HarvestStructural AberrationsAberrationFrequencyAberrant Cell FrequencyNumericalAberrationsChromatid Chromosome Complex Lesions/CellIncludingGapsExcludingGaps% of Cells WithTreatmentGroupConc.(μg/mL)DecodedCultureNo.No. ofCellsScoredG B F G B F E D RMultipleOtherJudge % Judge % Judge AE ER PP77 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 1Dimethylsulphoxide1%78 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 081 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 05082 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 083 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 110084 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 185 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 0Alcohols,C18-22Distn.Residues20086 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 - 0 - 0 0 020 88 100 1 1 0 0 0 0 2 0 0 0 0 0.06 +- 3 - 2 - 2 0 0Methylmethanesulphonate30 89 100 6 10 3 0 0 2 2 0 0 0 0 0.25 + 13 + 12 + 7 0 1

### Page 28 Test Facility Study No. 787723

Table 6 Polyploid Data: Test 2, Without S9 Mix, 22 h Treatment, 48 h Harvest

No. of Polyploid Cells Treatment Group Conc. (μg/mL) Decoded No. No. Of Diploid

Cells Normal Endoploid Frequency of Polyploid

Cells Judge

77 300 0 0 0.00 -Dimethylsulphoxide 1% 78 300 1 0 0.33 -

81 300 0 0 0.00 - 50 82 300 1 0 0.33 -

83 300 0 0 0.00 - 100 84 300 1 0 0.33 -

85 300 2 0 0.66 - Alcohols, C18-22 Distn. Residues

200 86 300 0 0 0.00 -

### Page 29 Test Facility Study No. 787723

14 APPENDICES
Appendix 1 Toxicity Data: Test 1, With S9 Mix, 6 h Treatment, 24 h Harvest

Cell Count Data Observations Treatment

Group Conc. (μg/mL) Decoded

No. No. of Cells (x10 6 ) Index Culture Slide Toxic Judge

1 1.33 1.00 -Dimethylsulphoxide 1% 2 1.33 1.00 -

3 1.28 0.96 - 0.78 4 1.35 1.02 -

5 1.23 0.92 - 1.56 6 1.38 1.04 -

7 1.30 0.98 - 3.13 8 1.38 1.04 -

9 1.30 0.98 - 6.25 10 1.38 1.04 -

11 1.35 1.02 - 12.5 12 1.25 0.94 -

13 1.25 0.94 - 25 14 1.28 0.96 -

15 1.28 0.96 - 50 16 1.30 0.98 -

17 1.25 0.94 - 100 18 1.20 0.90 -

19 1.33 1.00 - Alcohols, C18-22 Distn. Residues

200 20 1.25 0.94 Nil toxicity Nil toxicity

-

### Page 30 Test Facility Study No. 787723

Appendix 2 Toxicity Data: Test 1, Without S9 Mix, 6 h Treatment, 24 h Harvest

Cell Count Data Observations Treatment

Group Conc. (μg/mL) Decoded

No. No. of Cells (x10 6 ) Index Culture Slide Toxic Judge

25 2.25 1.03 -Dimethylsulphoxide 1% 26 2.10 0.97 -

27 2.23 1.03 - 0.78 28 2.18 1.00 -

29 2.13 0.98 - 1.56 30 2.40 1.10 -

31 2.13 0.98 - 3.13 32 2.13 0.98 -

33 2.13 0.98 - 6.25 34 2.05 0.94 -

35 2.00 0.92 - 12.5 36 2.05 0.94 -

37 2.13 0.98 - 25 38 2.03 0.93 -

39 2.15 0.99 - 50 40 2.05 0.94 -

41 2.13 0.98 - 100 42 1.98 0.91 -

43 1.93 0.89 - Alcohols, C18-22 Distn. Residues

200 44 2.25 1.03 Nil toxicity Nil toxicity

-

### Page 31 Test Facility Study No. 787723

Appendix 3 Toxicity Data: Test 2, With S9 Mix, 6 h Treatment, 24 h Harvest

Cell Count Data Observations Treatment

Group Conc. (μg/mL) Decoded

No. No. of Cells (x10 6 ) Index Culture Slide Toxic Judge

49 1.15 0.95 -Dimethylsulphoxide 1% 50 1.28 1.05 -

51 1.23 1.01 - 25 52 1.20 0.99 -

53 1.25 1.03 - 50 54 1.23 1.01 -

55 1.10 0.91 - 100 56 1.30 1.07 -

57 1.23 1.01 - Alcohols, C18-22 Distn. Residues

200 58 1.18 0.97 Nil toxicity Nil toxicity

-

### Page 32 Test Facility Study No. 787723

Appendix 4 Toxicity Data: Test 2, Without S9 Mix, 22 h Treatment, 24 h Harvest

Cell Count Data Observations Treatment

Group Conc. (μg/mL) Decoded

No. No. of Cells (x10 6 ) Index Culture Slide Toxic Judge

63 2.20 0.97 -Dimethylsulphoxide 1% 64 2.33 1.03 -

65 2.30 1.02 - 25 66 2.30 1.02 -

67 2.35 1.04 - 50 68 2.30 1.02 -

69 2.25 0.99 - 100 70 2.13 0.94 -

71 1.90 0.84 - Alcohols, C18-22 Distn. Residues

200 72 2.00 0.88 Nil toxicity Nil toxicity

-

### Page 33 Test Facility Study No. 787723

Appendix 5 Toxicity Data: Test 2, Without S9 Mix, 22 h Treatment, 48 h Harvest

Cell Count Data Observations Treatment

Group Conc. (μg/mL) Decoded

No. No. of Cells (x10 6 ) Index Culture Slide Toxic Judge

77 3.03 0.95 -Dimethylsulphoxide 1% 78 3.33 1.05 -

79 3.18 1.00 - 25 80 2.93 0.92 -

81 3.20 1.01 - 50 82 3.15 0.99 -

83 3.00 0.94 - 100 84 2.98 0.94 -

85 3.25 1.02 - Alcohols, C18-22 Distn. Residues

200 86 3.40 1.07 Nil toxicity Nil toxicity

-

### Page 34 Test Facility Study No. 787723

Appendix 6 Certificate of Analysis



## saS O L

(('cel/Mq lIew /rOIl(/(,0.

Brunsbuettel, 9/09/2009

C E R T I FIe ATE o F ANALYSIS

Product: Lot No.: Alcohols, C1S-22, distn. Residues 03585/MA (59B1RN2)
Manufacturing date: Expiry date:

Testa

Hydroxyl

- Number Bster No. Acid NO.
water Iodine N o .

Sasol Germany GmbH P.D. Box 1160 0-25534 Brunsbuettel 25/08/2009 08/2013

Unit Result

[mg ItOH/g] SO, [mg ItOH/gJ 41,17 [mg ItOH/g] 0,09 [wt.%l 0,02 [mg I/IOOmg] 5,34

Phone, 049 (0)4852/392-0 Fax, 049 (0)4852/3285 Best regards Works inspector M. Sprung

I h"""·,.,,·f.,,,'-!**Ul<'~l"' r'IIJly~("",,1it** **~l1l~** IOliO:Ul ie eJiC1 ti rr, ,LO ' ",-,J:iv, L,r ld 1:.1("111 f., it ,C**I~E ,.** **r:~,:,'r('nt1,-,il~_** ul1d**aIUw rf;** **tlIl~'** "'-1 Cl;!' D;, 1. l l'i"

· ' ',(I**'Il~** "nt[Ji:',(kl P"II**rm~,:,~rl!: l&'-**  ni c llt \ ' ('l

> , I:;' ,\: ':" "',., -; ", dl-"'19,

· I.I'·n**'~. ';'l-"**  WW,'I**,;.mp;;n9~-r':ij' I"** **~** :,r \" -F' l ;r "i ,flf i.,(pl in,j,::,: I,lli'lf i.. I, >, p! L.id > c Urih(;1 <:1', '_ ' ':h**I,-i.~,'** v '.1. 1,. ,H-'I' IL" ",,":"-1, u W

, ("t-II I : [jf ' 1w ,n ,' r , ,--J1H"'. i i l l ":" In",,. 'I, c!p, I" ", H i

, !

, ;, !<'lL ' {' 1 1 ('11**hEf~ulIlJ lll;~'** **(.;l "~** **ifi (-<tll'-**  o f**~. I.,!-,** **~i~** i b inl i"! o rlll m i D;"j 0<1 1 \' arid**dot'~** 110! till",

r :;:nto;c**~I1')' PO'l** **clll~'** r'f LldllCt**propp.,ti,,~.** 1 1 d lXS not i"'f il L\' lto e> p l

. e rt of 1I1ci**Obl ,~** **~tI{1' \**  tr. r "'"1)'**~,u l**  i'llJrodur;l !8ce i ":llg**r~ <,p~.;t'(** **)"** I. 

## l'r,i'**:~:'~ '~** **:'.I:,~ ~~:.'** **:~~ i~.~~:** **:~t~~ ~;** **~~,8t~u~;~:~ ;~** **:t~ ~;;;:~** **:tl;~ :,~** **::~;l~ '** **(' I,** **I;l;I"'i~;;~** . "1 1,.-· ".1 ",," II 1**~O** 11-,, ' timc " C Ot I C1l1 " h ;

. S;Js ol GPrmnny Gm bH

### Page 35 Test Facility Study No. 787723

Appendix 7 S9 Data in vitro Activation Preparation Form Activation Batch: FLI 112

### Page 36 Test Facility Study No. 787723

Appendix 8 Osmolality and Precipitation Data:

Treatment Group Conc. (μg/mL) Osmotic Pressure (mmol/kg) Test 1, With S9 Mix Precipitation Test 1, With and Without S9 Mix Precipitation Test 2, With and Without S9 Mix Dimethylsulphoxide 1% 435 Nil precipitation

0.78 -

1.56 -

3.13 454

6.25 -

12.5 - Not tested

25 426

50 - Nil precipitation

100 - Nil precipitation Alcohols, C18-22 Distn. Residues

200 471 Very fine precipitation Slight precipitation

### Page 37 Test Facility Study No. 787723

Appendix 9 Historical Negative and Positive Control Data

Negative Control Data

Confidence Levels for Negative Results Mean and SD for each parameter Number of Records Parameter

0-95% >95- ≤ 99% >99% Mean SD

1833 Lesions/cell 0.00-0.04 >0.04 - ≤ 0.07 >0.07 0.01 0.02
Aberrant cell frequency

including gaps (%) 0-4 >4 - ≤ 6 >6 1.0 1.4 Structural Aberrations 2157 Aberrant cell frequency excluding gaps (%) 0-2 >2- ≤ 3 >3 0.3 0.7

1805 AE (%) 0-3 >3- ≤ 8 >8 0.6 1.4 Numerical Aberrations 1 388 PP + ER (%) 0-1.96 >1.96- ≤ 2.6 >2.6 0.77 0.65

Judgement of test item or positive control culture aberration values Negative (-) Suspicious (+-) Positive (+) NA

QUALITY ASSURANCE Last date data added to database: 07 May 2009 Auditor: Alison McNaughton Date Audited: 06 Jul y 2009

1 = Aneuploidy (AE) measured in 100 cells assessed for structural aberrations Polyploidy (PP + ER) measured in approx imately 300 cells assessed for polyploidy only. 95% confidence limits : 95% of values from negative controls fall on or within the values given. 99% confidence limits : 99% of values from negative controls fall on or within the values given.



### Positive Control Data

Number of Records Parameter Range

1642 Lesions/cell 0.00-6.72 Aberrant cell frequency including gaps (%) 0-100 Structural Aberrations 1933 Aberrant cell frequency excluding gaps (%) 0-100

Numerical Aberrations 1 1610 AE (%) 0-52

QUALITY ASSURANCE Last date data added to database: 07 May 2009 Auditor: Alison McNaughton Date audited: 06 July 2009 1 = Aneuploidy (AE) measured in 100 ce lls assessed for structural aberrations