FINAL REPORT 

Test Facility Study No. 787723, Report No. 30917 

 

Alcohols, C18-22 Distn. Residues (Cas No. 1160164-88-4) 

Chromosomal Aberrations Assay with 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 
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 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
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 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 
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 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 
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 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 and Precipitation Data:................................................................... 36 

Appendix 9 Historical Negative and Positive Control Data................................................. 37 
1 COMPLIANCE STATEMENT 

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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 

Date 
2 QUALITY ASSURANCE STATEMENT 

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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 
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 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 
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 Test Facility Study No. 787723 

4 SUMMARY 

Alcohols, C18-22 Distn. Residues were submitted for testing and evaluation of clastogenic 

potential. Chromosomal aberrations assays were performed with duplicate, Chinese hamster 

ovary cell cultures. This study was conducted incorporating 2 independent tests. 

Dimethylsulphoxide was the vehicle and cyclophosphamide and methyl methanesulphonate 

were the positive controls used in both tests. 

Both tests were conducted in the presence and absence of a post-mitochondrial supernatant 

fraction obtained from the livers of adult, male 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. Cultures 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 when tested up to the limit of solubility 

(200 μg/mL), (Appendices 1-5). 

There was no evidence that Alcohols, C18-22 Distn. Residues induced structural 

chromosomal aberrations in either the presence or absence of S9 mix (Tables 1-5). 

Alcohols, C18-22 Distn. Residues did not induce an increase in polyploidy in either the 

presence or absence of S9 mix in cultures harvested 48 h post treatment (Table 6). 

It was concluded that Alcohols, C18-22 Distn. Residues were not clastogenic when tested 

with Chinese hamster ovary cells in vitro, when tested up to a concentration of 200 μg/mL. 
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 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 cultured 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 chromosome-type aberrations also occur. An 

increase in polyploidy may indicate that a chemical has the potential to induce numerical 

aberrations. Chromosome mutations and related events are the cause of many human genetic 

diseases and there is substantial evidence that chromosome mutations and related events 

causing alterations in oncogenes and tumor suppressor genes of somatic cells are involved in 

cancer induction in humans and experimental animals. 

5.1 Justification of Test System 

Many mutagens are clastogenic in higher organisms both in vivo and in vitro. Cytological test 

systems are useful in the pre-screening of mutagens and carcinogens. 

Chinese hamster ovary cells possess characteristics which make them suitable for cytogenetic 

testing of clastogens. They can be subcultured indefinitely without ageing, generally 

maintaining a stable pseudo-diploid karyotype with a low and easily analysable chromosome 

number. The short cell cycle of Chinese hamster 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 trials 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 test 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. 
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 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 the 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 recorded during this study, will be stored in the Scientific Archive 

of Charles River, Preclinical Services Edinburgh for 2 years after the issue of the final report. 

After the 2 year period the Sponsor will be consulted regarding the disposal, transfer or 

continued storage of raw data. 

The original signed copy of the final report will be stored indefinitely in the Scientific 

Archives of Charles River, Preclinical Services 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 dark at ambient room temperature 

Supplier: Sasol Germany GmbH 

Expiry date: 31 August 2013 

The Certificate of Analysis is shown in Appendix 6. 
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 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 suspension in dimethylsulphoxide at 20 mg/mL. This 

was with vortex mixing and warming 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 B4) 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 passage numbers 8 (Test 1) and 16 (Test 2), and 

resuspended in fresh culture medium at densities of 0.1 x 106

 or 0.05 x 106 cells/mL. These 

cells, in 5 mL volumes, were dispensed into 25 cm2

 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 stock flask about 20 h before testing. 

6.4 Justification of Dose Levels 

For the first experiment, 9 dose levels, covering a wide concentration range, were tested. The 

highest dose was 200 μg/mL (limit of solubility), and subsequent dose levels were halving 

dilutions. 
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 Test Facility Study No. 787723 

In the second experiment, the 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 vehicle 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 harvesting, were conducted using aseptic technique and 

under amber light. 

7.1 S9 Mix (McGregor et al (1988)) 

Aroclor 1254-induced S9 enzymes (supernatant 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 batch 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 concentrations in the ‘S9 mix’ of: 
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 Test Facility Study No. 787723 

NADP Na2 4 mM (= 3.150 mg/mL) 

Glucose-6-phosphate Na2 25 mM (= 7.605 mg/mL) 

This solution was immediately filter-sterilised by passage through 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 Distn. 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). Precipitation was noted in cultures 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 absence of S9 mix. Treatments with test item 

or vehicle control substances were performed 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. 
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 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 treatment. The volume of medium for the 

recovery period was 5 mL. 

Living cultures were examined for evidence of changes to cell morphology, once at the end of 

the treatment period and again before 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/mL. 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 temperature. The cells were then fixed (after 

sedimentation as before) using 4 mL of freshly prepared fixative (methanol:glacial acetic acid, 

3:1). Two further changes (after sedimentation as before) of fixative were made. 

Monolayer cells were trypsinised, 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, coded 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 permanent by mounting coverslips with DPX mountant. 

7.5 Slide Assessment 

7.5.1 Screen for Toxicity 

Slides were examined for evidence of metaphase cells and signs of cellular necrosis. 

7.5.2 Chromosomal Aberrations 

Three concentration levels were selected for assessment of chromosomal aberrations. 
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 Test Facility Study No. 787723 

From 2-3 slides per culture, up to 50 metaphase cells per slide, a total of 100 metaphase cells 

per culture, were examined. Slides were 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 metaphase cell and all abnormalities, using the 

nomenclature of Gebhart (1970) was recorded. 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 using 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 magnification of x 400 or x 500 and deemed to be either 

diploid, polyploid or endoreduplicated. In this assessment no metaphase cell in a field was 

rejected. 

This assessment was considered more objective, avoiding 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 Measure 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, and 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 
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 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 important in judging the true clastogenicity of a 

test item. 

8.3 Historical Control Data 

The results obtained were compared with the historical control data. Historical 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 cell count was reduced to less than 50% of the 

mean vehicle control culture values or if consistent evidence of changes to cell morphology 

was observed. 

9.2 Interpretation of Clastogenicity 

The results for test item and positive control treated cultures are evaluated by comparison 

with the concurrent vehicle control cultures and with historical negative 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 significant if the percent of aberrant cells is 

consistently greater than the 99% confidence limits 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 least one acceptable dose level was significant by the 

criterion described above. 
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 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 (between 95% and 99% confidence limits). 

Experiments that met in part the criteria for a positive response, or marginally met all the 

criteria, were classed 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 treated 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 following 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 structural and numerical aberrations within the 95% 

confidence limits of the historical negative control data. 

10.4 Positive Control Treated Cultures 

The positive control substances, cyclophosphamide in the presence and methyl 

methanesulphonate in the absence of S9 mix, induced positive frequencies of structural 

aberrations in at least one concentration level per section. These results demonstrated the 

sensitivity of the test system. 

10.5 Alcohols, C18-22 Distn. Residues Treated Cultures 

All cultures treated with Alcohols, C18-22 Distn. Residues had levels of structural aberrations 

within the 95% confidence limits for a negative response. 
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 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 treated with Alcohols, C18-22 Distn. Residues had 

levels of polyploidy within the 95% confidence limits for a negative response. 
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 Test Facility Study No. 787723 

11 CONCLUSION 

It was concluded that Alcohols, C18-22 Distn. Residues were not clastogenic when tested 

with Chinese hamster ovary cells in vitro, when tested up to a concentration of 200 μg/mL. 
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 Test Facility Study No. 787723 

12 REFERENCES 

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. 

Galloway S M, Armstrong M A, Reuben C, Colman S, Brown C, Cannon A D, Bloom A D, 

Nakamura F, Ahmed M, Duk S, Rimpo J, Margolin B H, Resnick M A, Anderson B and 

Zeiger E (1987). Chromosome aberrations and sister chromatid exchanges in Chinese hamster 

ovary cells: Evaluations of 108 chemicals. Environ Mol Mutagen, 10 (Suppl 10), 1-175. 

Gebhart E, (1970). The Treatment 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 tested 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 activation 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/carcinogens after activation in a microsomal system in vitro, I. Induction 

of chromosome aberrations and sister chromatid exchanges by diethylnitrosamine (DEN) and 

dimethylnitrosamine (DMN) in CHO cells in the presence of rat-liver microsomes. Mutat 

Res, 37, 83-90. 

Tezuka H, Ando N, Suzuki R, Terahata M, Moriya M and Shirasu Y (1980). Sister-chromatid 

exchanges and chromosomal aberrations in cultured Chinese hamster cells treated with 

pesticides positive in microbial reversion assays. Mutat Res, 78, 177-191. 
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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 due 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 

Complex Dicentric Chromosome with 2 centromeres D 2 

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 Characteristic paired chromosomes ER 

Polyploidy 26 and more chromosomes PP 

No lesions
Table 1 Aberration Data: Test 1, With S9 Mix, 6 h Treatment, 24 h Harvest 

Structural Aberrations Aberration Frequency Aber

Chromatid Chromosome Complex Lesions/Cell Inclu

Ga

Treatment Group Conc. (μg/mL) Decoded Culture No. No. of Cells Scored

G B F G B F E D R 

Multiple Other Judge % 

1 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

Dimethylsulphoxide 1% 2 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

15 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

50 

16 100 1 1 0 0 0 0 0 0 0 0 0 0.02 - 2 

17 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

100 

18 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

19 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

Alcohols, C18-22 Distn. Residues 

200 

20 100 1 0 0 0 0 0 0 0 0 0 0 0.01 - 1 

40 23 100 1 4 4 0 1 0 4 0 0 0 0 0.18 + 14 

Cyclophosphamide 50 24 100 4 4 3 0 0 0 1 0 0 0 0 0.13 + 11 
Table 2 Aberration Data: Test 1, Without S9 Mix, 6 h Treatment, 24 h Harvest 

Structural Aberrations Aberration Frequency Aber

Chromatid Chromosome Complex Lesions/Cell Inclu

Ga

Treatment Group Conc. (μg/mL) Decoded Culture No. No. of Cells Scored

G B F G B F E D R 

Multiple Other Judge % 

25 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

Dimethylsulphoxide 1% 26 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

39 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

50 

40 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

41 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

100 

42 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

43 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

Alcohols, C18-22 Distn. Residues 

200 

44 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

Methyl 30 47 100 0 4 0 0 0 0 0 0 0 0 0 0.04 - 3 methanesulphonate 40 48 100 3 11 1 0 0 0 7 0 0 0 0 0.29 + 16 
Table 3 Aberration Data: Test 2, With S9 Mix, 6 h Treatment, 24 h Harvest 

Structural Aberrations Aberration Frequency Aber

Chromatid Chromosome Complex Lesions/Cell Inclu

Ga

Treatment Group Conc. (μg/mL) Decoded Culture No. No. of Cells Scored

G B F G B F E D R 

Multiple Other Judge % 

49 100 1 0 0 0 0 0 0 0 0 0 0 0.01 - 1 

Dimethylsulphoxide 1% 50 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

53 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

50 

54 100 1 0 0 0 0 0 0 0 0 0 0 0.01 - 1 

55 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

100 

56 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

57 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

Alcohols, C18-22 Distn. Residues 

200 

58 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

20 59 100 0 2 0 0 0 0 0 0 0 0 0 0.02 - 1 

Cyclophosphamide 30 60 100 0 7 1 0 0 0 1 0 0 0 0 0.10 + 8 
Table 4 Aberration Data: Test 2, Without S9 Mix, 22 h Treatment, 24 h Harvest 

Structural Aberrations Aberration Frequency Aber

Chromatid Chromosome Complex Lesions/Cell Inclu

Ga

Treatment Group Conc. (μg/mL) Decoded Culture No. No. of Cells Scored

G B F G B F E D R 

Multiple Other Judge % 

63 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

Dimethylsulphoxide 1% 64 100 1 0 0 0 0 0 0 0 0 0 0 0.01 - 1 

67 100 1 0 0 0 0 0 0 0 0 0 0 0.01 - 1 

50 

68 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

69 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

100 

70 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

71 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

Alcohols, C18-22 Distn. Residues 

200 

72 100 0 1 0 0 0 0 0 0 0 0 0 0.01 - 1 

Methyl 20 74 100 2 2 0 0 0 0 1 0 0 0 0 0.06 +- 5 methanesulphonate 30 75 100 3 11 1 1 0 1 6 0 0 0 0 0.29 + 11 
Table 5 Aberration Data: Test 2, Without S9 Mix, 22 h Treatment, 48 h Harvest 

Structural Aberrations Aberration Frequency Aber

Chromatid Chromosome Complex Lesions/Cell Inclu

Ga

Treatment Group Conc. (μg/mL) Decoded Culture No. No. of Cells Scored

G B F G B F E D R 

Multiple Other Judge % 

77 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

Dimethylsulphoxide 1% 78 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

81 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

50 

82 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

83 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

100 

84 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

85 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

Alcohols, C18-22 Distn. Residues 

200 

86 100 0 0 0 0 0 0 0 0 0 0 0 0.00 - 0 

Methyl 20 88 100 1 1 0 0 0 0 2 0 0 0 0 0.06 +- 3 methanesulphonate 30 89 100 6 10 3 0 0 2 2 0 0 0 0 0.25 + 13 
 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 

(x106

) 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 

(x106

) 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 

(x106

) 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 

(x106

) 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 

(x106

) 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 

saSOL 

(('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 No . 

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 ieeJiC1 tirr, ,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.ll'i"·' ',(I 'Il~ "nt[Ji:',(kl P"II rm~,:,~rl!:l&'- nicllt \'('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, uW,("t-III:[jf'1w,n,'r, 

,--J1H"'. iil l":" In",,. 'I, c!p, I"", Hi,!, ;,!<'lL '{'11('11 hEf~ulIlJ 

lll;~' (.;l"~ ifi (-<tll'- o f ~. I.,!-, ~i~ ib inl i"!orlllmiD;"j 0<11\' arid dot'~ 110! till", 

r:;:nto;c ~I1')' PO'l clll~' r'fLldllCt propp.,ti,,~. 11 dlXS not i"'f il L\' ltoe>p l. 

ert of 1I1ci Obl ,~ ~tI{1' \ tr. r "'"1)' ~,u l i'llJrodur;l !8cei":llg r~<,p~.;t'()" I. l'r,i' :~:'~'~:'.I:,~ ~~:.' :~~ i~.~~: :~t~~~; ~~,8t~u~;~:~ ;~ :t~ ~;;;:~:tl;~:,~ ::~;l~ ' (' I, I;l;I"'i~;;~ ."1 1,.-· ".1",," II1 ~O 11-,, ' timc" COt IC1l1 " h ;. 

S;Jsol GPrmnny GmbH 
 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 

Aberrations1

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 July 2009

1 = Aneuploidy (AE) measured in 100 cells assessed for structural aberrations 

 Polyploidy (PP + ER) measured in approximately 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 (%) Structural 0-100 

Aberrations 1933 

Aberrant cell frequency 

excluding gaps (%) 0-100 

Numerical 

Aberrations1 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 cells assessed for structural aberrations