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Working for a better future

In Vitro Mammalian Chromosome Aberration Test 

In Human Lymphocytes 

AMENDED FINAL REPORT 

HLS study number: NBJ0048 

Version ID: Final 

Issue date: 21 June 2010 

Amended report issue date: 31 March 2011 
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Sponsor and Test Facility Details 

Sponsor 

 

Test facility Huntingdon Life Sciences 

Eye Research Centre 

 Eye 

 Suffolk 

 IP23 7PX 

 UK 
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Table of Contents 

Sponsor and Test Facility Details ........................................................................................... 2

Compliance with Good Laboratory Practice............................................................................ 5

Quality Assurance Statement ................................................................................................. 6

Contributing Scientists ............................................................................................................ 7

Summary................................................................................................................................. 8

1. Introduction .................................................................................................................... 9

2. Experimental Procedure............................................................................................... 11

2.1 Test substance................................................................................................. 11

2.2 Culture of lymphocytes..................................................................................... 11

2.3 Positive controls ............................................................................................... 11

2.3.1 In the absence of S9 mix ...................................................................... 11

2.3.2 In the presence of S9 mix..................................................................... 11

2.4 S9 metabolizing system ................................................................................... 12

2.4.1 Preparation of S9 fraction..................................................................... 12

2.4.2 Preparation of S9 mix ........................................................................... 12

2.5 Selection of solvent .......................................................................................... 12

2.6 Treatment of cells with test substance – first test............................................. 12

2.7 Harvesting and fixation..................................................................................... 13

2.8 Slide preparation .............................................................................................. 13

2.9 Microscopic examination .................................................................................. 13

2.10 Second test ...................................................................................................... 14

2.11 Stability, homogeneity and formulation analysis .............................................. 15

3. Assessment of Results................................................................................................. 16

4. Deviations from Protocol.............................................................................................. 16

5. Maintenance of Records .............................................................................................. 16

6. Results ......................................................................................................................... 17

6.1 First test............................................................................................................ 17

6.1.1 Toxicity data ......................................................................................... 17

6.1.2 Metaphase analysis.............................................................................. 17

6.2 Second test ...................................................................................................... 17

6.2.1 Toxicity data ......................................................................................... 17

6.2.2 Metaphase analysis.............................................................................. 18

6.2.3 Polyploid analysis ................................................................................. 18

7. Conclusion ................................................................................................................... 18

8. References................................................................................................................... 19
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List of Tables 

Table 1 Summary of results ....................................................................................... 20

Table 2 Mitotic index data – first test.......................................................................... 21

Table 3 Metaphase analysis data – first test .............................................................. 23

Table 4 Mitotic index data – second test .................................................................... 25

Table 5 Metaphase analysis data – second test ........................................................ 28

List of Appendices 

Appendix 1 Historical Control Data .................................................................................. 30

Appendix 2 GLP Compliance Statement.......................................................................... 33

Appendix 3 S9 Quality Control Certificates ...................................................................... 34

Appendix 4 Certificate of Analysis.................................................................................... 36

Appendix 5 Apparatus for Vapour/Gas Phase Exposure of Cultivated Mammalian Cells 37

Appendix 6 Protocol and Amendments ............................................................................ 38


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Contributing Scientists 

In Vitro Mammalian Chromosome Aberration Test In Human 

Lymphocytes 

Study management 

Lincoln Pritchard BSc (Hons) 

(Study Director) 

Department of Genetic Toxicology 

Claire Damant 

(Study Supervisor) 

Department of Genetic Toxicology 
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Summary 

A study was performed to assess the ability of (a liquefied gas) to induce 

chromosomal aberrations in human lymphocytes cultured in vitro. 

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of 

phytohaemagglutinin, and exposed to the test substance both in the absence and presence of 

S9 mix derived from rat livers. Solvent and positive control cultures were also included. 

Two hours before the end of the incubation period, cell division was arrested using 

Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be 

examined for chromosomal damage. 

In order to determine the toxicity of to cultured human lymphocytes, the mitotic 

index was assessed for all cultures treated with the test substance and the solvent control, 

ethanol. Justification for the highest selected concentration was based on the limit final 

concentration for this test system (10 mM). On the basis of these data, the following 

concentrations were selected for metaphase analysis: 

First test 

In the absence of S9 mix - 3 hour treatment, 18 hour recovery: 469.80, 783 and 1305 μg/mL. 

In the presence of S9 mix (2% v/v) - 3 hour treatment, 18 hour recovery: 469.80, 783 and 

1305 μg/mL. 

Second test 

In the absence of S9 mix - 21 hour continuous treatment: 469.80, 783 and 1305 μg/mL. 

In the presence of S9 mix (5% v/v) - 3 hour treatment, 18 hour recovery: 469.80, 783 and 

1305 μg/mL. 

In both the absence and presence of S9 mix, caused no statistically significant 

increases in the proportion of metaphase figures containing chromosomal aberrations, at any 

concentration, when compared with the solvent control, in either test. 

No statistically significant increases in the proportion of polyploid cells were observed during 

metaphase analysis, in either test. 

All positive control compounds caused statistically significant increases in the proportion of 

aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. 

It is concluded that has shown no evidence of causing an increase in the frequency of 

structural chromosome aberrations in this in vitro cytogenetic test system, under the 

experimental conditions described. 
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1. Introduction 

This report describes a study designed to assess the ability of to cause chromosomal 

aberrations in human lymphocytes cultured in vitro. 

The study was conducted in compliance with the following guidelines: 

OECD Guideline for the Testing of Chemicals. (1997) Genetic Toxicology: In Vitro 

Mammalian Chromosome Aberration Test, Guideline 473. 

US EPA (1998) Health Effects Test Guidelines. OPPTS 870.5375 In Vitro 

Mammalian Chromosome Aberration Test. EPA 712-C-98-223. 

EC Commission Regulation No. 440/2008. Method B.10: Mutagenicity – In Vitro 

Mammalian Chromosome Aberration Test. OJ L 142/225. 

ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory 

Genotoxicity Tests for Pharmaceuticals. 

ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity 

Testing of Pharmaceuticals. 

Human lymphocytes have been used in this type of study for a number of years (Evans and 

O'Riordan, 1975, Scott et al., 1990). They are cultured in vitro but do not divide unless 

stimulated to do so. This is achieved by adding phytohaemagglutinin (PHA) to the culture 

which results in a high mitotic yield (Nowell, 1960). 

In this study, blood taken from healthy male non-smoking donors was pooled and diluted 

with tissue culture medium. The cultures were incubated in the presence of PHA before 

being treated with the test substance. Following treatment the cells were arrested at 

metaphase using the mitotic inhibitor, Colcemid®. Chromosomes in these metaphase cells 

were then examined for the presence of chromosome aberrations. The best estimate of the 

aberration frequency is at the first cell division after initiation of treatment since certain types 

of damage may be lost during subsequent cell divisions. In this laboratory the cell cycle time 

for human lymphocytes in whole blood culture is approximately 13-14 hours. 

Some substances do not exert a mutagenic effect until they have been metabolised by enzyme 

systems that are not found in cultured cells. Therefore the cultures and test substance were 

incubated in both the absence and presence of a supplemented liver fraction (S9 mix) 

prepared from rats previously treated with a substance (phenobarbital and 5,6-benzoflavone) 

known to induce a high level of enzymic activity. 
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The study comprised of two independent tests. In the first test, a three hour treatment was 

used in both the absence and presence of S9 mix. In the second test, a 21 hour continuous 

treatment was used in the absence of S9 mix, and a three hour treatment using an increased 

S9 concentration (5% v/v) was used in the presence of S9 mix. 

Aberrations were scored according to the classification of the ISCN (1985). Traditionally 

gaps have been excluded from the quantitation of chromosome aberrations. Some gaps, 

however, have been shown to be real discontinuities in DNA (Heddle and Bodycote, 1970, 

Satya-Prakash et al., 1981). In this study the total number of cells containing aberrations both 

with and without gaps has been calculated. 

The protocol was approved by Huntingdon Life Sciences Management and the Study 

Director on 19 February 2010, and by the Sponsor on 21 April 2010. 

The experimental start and completion dates of the study were 2 March 2010 and 

26 May 2010, respectively. 
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2. Experimental Procedure 

2.1 Test substance 

Identity: 

Chemical name: 

Molecular weight: 

Batch number: 41024-47 

Expiry date: 29 January 2011 

Appearance: Clear Liquid 

Storage conditions: Room temperature, in the dark 

Purity/Assay: 99.98% 

Date received: 4 February 2010 

Certificate of Analysis: Appendix 4 

2.2 Culture of lymphocytes 

Human blood was collected aseptically from two healthy, non-smoking male donors, pooled 

and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf 

serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM 

glutamine. Aliquots (0.4 mL blood : 4.5 mL medium : 0.1 mL phytohaemagglutinin) of the 

cell suspension were placed in sterile universal containers and incubated at 37°C in a 5% CO2

atmosphere for approximately 48 hours. The cultures were gently shaken daily to resuspend 

the cells. 

In the second test only, the culture volume was increased from 5 mL to 10 mL for continuous 

treatment in the absence of S9 mix, to attain greater cell numbers within this test system. All 

cultures were prepared as larger 10 mL aliquots (0.8 mL blood: 9 mL medium: 0.2 mL PHA 

solution). All cultures were therefore treated at 1% v/v (100 μL) as a result of the increased 

culture volume. All cultures using the increased 5% v/v final concentration of S9 fraction in 

the presence of S9 mix were cultured as 5 mL cultures as previously detailed above. 

2.3 Positive controls 

2.3.1 In the absence of S9 mix 

Identity: Mitomycin C 

Appearance: Blue powder 

Batch number: 028K1815 

Solvent: Sterile purified water 

Final concentration: 0.2 μg/mL (3 hour treatment) 

0.1 μg/mL (21 hour continuous treatment)

2.3.2 In the presence of S9 mix 

Identity: Cyclophosphamide 

Appearance: White powder 

Batch number: 068K1131 
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Solvent: Sterile purified water 

Final concentration: 5 μg/mL (3 hour treatment) 

2.4 S9 metabolizing system 

2.4.1 Preparation of S9 fraction 

The S9 fraction was obtained from male Sprague-Dawley derived rats, dosed with 

phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver. The 

S9 fraction was purchased from a commercial source and stored at -80°C or below. A copy 

of each quality control statement relating to the batch of S9 preparation used (batch numbers 

2440 and 2472) is included in Appendix 3. 

2.4.2 Preparation of S9 mix 

S9 mix contained: S9 fraction (10% v/v : test 1 and 25% v/v : test 2), MgCl2 (8 mM), KCl 

(33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP 

(4 mM). All the cofactors were filter sterilised before use. 

2.5 Selection of solvent 

Prior to commencing testing, the solubility of was assessed. was found to be 

soluble in ethanol at 130.5 mg/mL. On dosing a 130.5 mg/mL solution at 1% v/v into 

aqueous tissue culture medium, giving a final concentration of 1305 μg/mL 

(10 mM), no observations were noted. 

Concentrations with high ionic strength and osmolality may cause chromosomal aberrations 

(Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not 

used in this test system. 

The osmolality and pH of the test substance in medium was tested at 1305 μg/mL; no 

fluctuation in osmolality of more than 50 mOsm/kg and no change in pH of more than 1.0 

unit were observed compared with the solvent control. 

In this case, the highest final concentration used for subsequent testing was 1305 μg/mL 

(10 mM), the limit guideline concentration. In this study was added to cultures at 

1% v/v (50 μL per 5 mL culture). 

All concentrations cited in this report are expressed in terms of the sample as 

received. 

2.6 Treatment of cells with test substance – first test 

Solvent, positive and treatment cultures were established approximately 48 hours after 

commencement of incubation of lymphocyte cultures. Duplicate cultures were prepared 

throughout for each 3 hour treatment in the absence and presence of S9 mix. All cultures 

were centrifuged and resuspended in fresh medium just before treatment. All cultures were 

treated in sealed glass bottles (with an internal volume of 160 mL) using a syringe and 

needle, inserted through the septum cap to prevent any loss of within the test system. 
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The glass bottles were then incubated on their sides at 37 °C in a roller apparatus (see 

Appendix 5: Apparatus for vapour/gas phase exposure of cultivated mammalian cells), which 

rotates the bottles once every eight minutes approximately. The lymphocytes coat the inside 

of the bottles and were immersed in culture medium once every revolution and exposed 

directly to for the rest of the revolution. 

In the absence of S9 mix, 50 μL aliquots of liquid were added to give final 

concentrations of 60.89, 101.48, 169.13, 281.88, 469.80, 783 and 1305 μg/mL. Ethanol was 

used as the solvent control. Mitomycin C at a final concentration of 0.2 μg/mL was the 

positive control. 

For treatments in the presence of S9 mix, 1 mL of medium was removed from each culture 

and discarded. This was replaced with 1 mL of S9 mix (2% v/v final concentration), 

followed by 50 μL of each test substance dilution (giving the same series of final 

concentrations as above). Ethanol was the solvent control. Cyclophosphamide at a final 

concentration of 5 μg/mL was the positive control. 

In both the absence and presence of S9 mix, no notable culture medium changes were 

observed either at or after 3 hour treatment. 

Three hours after dosing, the cultures were transferred to universal containers and centrifuged 

at 500g for 5 minutes. The supernatant removed and the cell pellets resuspended in fresh 

medium. They were then incubated for a further 18 hours. 

2.7 Harvesting and fixation 

Two hours before the cells were harvested, mitotic activity was arrested by addition of 

Colcemid® to each culture at a final concentration of 0.1 μg/mL. After 2 hours incubation, 

each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 

500g. The cell pellets were treated with a hypotonic solution (0.075M KCl), pre-warmed at 

37°C. After a 10 minute period of incubation at 37°C, the suspensions were centrifuged at 

500g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 

parts methanol : 1 part glacial acetic acid). The fixative was replaced until it was clear. 

2.8 Slide preparation 

The pellets were resuspended, then centrifuged at 500g for 5 minutes and finally resuspended 

in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto 

pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10% 

Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were 

left to air-dry and mounted in DPX. The remainder of the cell pellets in fixative were stored 

at 4°C until slide analysis was completed. 

2.9 Microscopic examination 

The prepared slides were examined by light microscopy using a low power objective. The 

proportion of mitotic cells per 1000 cells in each culture was recorded except for positive 

control treated cultures, or cultures where there were no signs of cytotoxicity. As no 

significant decrease in toxicity was observed, the maximum concentration tested was the 
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highest concentration selected for metaphase analysis. Lower concentrations were also 

selected. 

The selected slides were then coded. Metaphase cells were identified using a low power 

objective and examined at a magnification of x1000 using an oil immersion objective. One 

hundred metaphase figures were examined from each culture, however, this number was 

reduced in cultures showing a high level of aberrant cells, where 10 cells in 100 metaphases 

with structural aberrations (excluding gaps) were observed. Chromosome aberrations were 

scored according to the classification of the ISCN (1985). Only cells with 44 - 48 

chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen. 

The vernier readings of all aberrant metaphase figures were recorded. 

The number of aberrant metaphase cells in each test substance group was compared with the 

solvent control value using the one-tailed Fisher exact test (Fisher 1973).

A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test 

substance groups. If this is significant at the 1% level, the test is reiterated excluding the 

highest concentration group - this process continues until the trend test is no longer 

significant. 

D20s (the minimum concentration (mg/mL) at which aberrations were found in 20% of 

metaphases) were estimated using logistic regression on a log(concentration) scale, allowing 

the number of control aberrations to be non-zero (Armitage et al., 2002). The following 

model was used: 

p is the proportion of cells with aberrations, conc is the concentration of test substance. C is a 

parameter estimating the control proportion of aberrations. 

2.10 Second test 

Cultures were initiated and maintained as previously described. In this second test a 21 hour 

continuous treatment was used in the absence of S9 mix. In the presence of S9 mix, a 3 hour 

treatment was used, as in the first test. However, to modify study parameters the final 

concentration of S9 mix was increased from 2% v/v to 5% v/v. All cultures were centrifuged 

and resuspended in fresh medium just before treatment. 

Concentrations of were as follows: 

In the absence of S9 mix: 169.13, 281.88, 469.80, 783 and 1305 μg/mL. 

In the presence of S9 mix (5% v/v): 169.13, 281.88, 469.80, 783 and 1305 μg/mL. 

Duplicate cultures were used for each treatment and two cultures were treated with the 

solvent control. Positive control cultures were treated as in the first test. Mitomycin C, at a 

final concentration of 0.1 μg/mL, and Cyclophosphamide, at a final concentration of 

5 μg/mL, were added to duplicate cultures. 
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In both the absence and presence of S9 mix, no notable culture medium changes were 

observed either at or after treatment. 

Three hours after dosing, the cultures containing S9 mix were centrifuged. The cell pellets 

were rinsed and resuspended in fresh medium. They were then incubated for a further 

18 hours. Cultures treated in the absence of S9 mix were incubated continuously for 

21 hours. 

All cultures were treated with Colcemid®, at a final concentration of 0.1 μg/mL, two hours 

before the end of the incubation period. They were then harvested, fixed and the slides 

prepared as previously described. The slides were examined microscopically as previously 

described. 

In the presence of S9 mix all treatment concentrations were non-cytotoxic. However, 

in the absence of S9 mix, further testing was conducted to achieve the required toxicity under 

21 hour continuous treatment conditions. 

As low solvent control and treatment values were obtained for cultures in the absence 

of S9 following extended treatment, all cultures were prepared as larger 10 mL aliquots (0.8 

mL blood: 9 mL medium: 0.2 mL PHA solution) for the subsequent repeat test. 

There was no change in study methodology, however, all cultures were treated at 1% v/v 

(100 μL) as a result of the increased culture volume. 

Concentrations of were as follows: 

In the absence of S9 mix: 169.13, 281.88, 469.80, 783 and 1305 μg/mL. 

No notable culture medium changes were observed either at or after treatment.

2.11 Stability, homogeneity and formulation analysis 

The stability and homogeneity of the test substance and of the test substance in the solvent 

were not determined as part of this study. 
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3. Assessment of Results 

An assay is considered to be acceptable if the negative and positive control values lie within 

the current historical control range. 

The test substance is considered to cause a positive response if the following conditions are 

met: 

Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant 

chromosomes (excluding gaps) are observed at one or more test concentration. 

The increases exceed the negative control range of this laboratory, taken at the 99% 

confidence limit. 

The increases are reproducible between replicate cultures. 

The increases are not associated with large changes in pH, osmolality of the treatment 

medium or extreme toxicity. 

Evidence of a concentration-related response is considered to support the conclusion. 

A negative response is claimed if no statistically significant increases in the number of 

aberrant cells above concurrent control frequencies are observed, at any concentration. 

A further evaluation may be carried out if the above criteria for a positive or a negative 

response are not met. 

4. Deviations from Protocol 

There were no deviations from the protocol. 

5. Maintenance of Records 

All raw data, samples and specimens (if appropriate) arising from the performance of this 

study will remain the property of the Sponsor. Types of sample and specimen which are 

unsuitable, by reason of instability, for long term retention and archiving may be disposed of. 

All other samples and specimens and all raw data will be retained by Huntingdon Life 

Sciences in its archive for a period of one year from the date on which the Study Director 

signs the final report. After such time, the Sponsor will be contacted and advice sought on 

the return, disposal or further retention of the materials. If requested, Huntingdon Life 

Sciences will continue to retain the materials subject to a reasonable fee being agreed with 

the Sponsor. 

Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and 

a copy of the final report in its archive indefinitely. 
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6. Results 

6.1 First test 

6.1.1 Toxicity data 

Mitotic indices of cultured human lymphocytes treated with are shown in Table 2. 

In the absence of S9 mix following three hour treatment, caused no reduction in the 

mitotic index at 1305 μg/mL, the highest tested concentration, compared to the solvent 

control value. The concentrations selected for metaphase analysis were 469.80, 783 and 

1305 μg/mL. 

In the presence of S9 mix (2% v/v final concentration) following three hour treatment, 

 caused no reduction in the mitotic index at 1305 μg/mL, the highest tested 

concentration, compared to the solvent control value. The concentrations selected for 

metaphase analysis were 469.80, 783 and 1305 μg/mL. 

6.1.2 Metaphase analysis 

The effects of on the chromosomes of cultured human lymphocytes are shown in 

Table 3 and summarised in Table 1. 

In both the absence and the presence of S9 mix, caused no statistically significant 

increases in the proportion of cells with chromosomal aberrations at any concentration, when 

compared with the solvent control. 

All mean values for the solvent control (ethanol), and all treatment concentrations 

were within the historical control range for this laboratory, when taken at the 99% confidence 

limit. 

Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically 

significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the 

efficacy of the S9 mix and the sensitivity of the test system. 

6.2 Second test 

6.2.1 Toxicity data 

Mitotic indices of cultured human lymphocytes treated with are shown in Table 4. 

In the absence of S9 mix following 21 hour continuous treatment, caused no 

significant reduction in the mitotic index at 1305 μg/mL, the highest tested concentration, 

compared to the solvent control value. The concentrations selected for metaphase analysis 

were 469.80, 783 and 1305 μg/mL. 

In the presence of S9 mix (5% v/v final concentration) following three hour treatment, 

 caused no reduction in the mitotic index at 1305 μg/mL, the highest tested 

concentration, compared to the solvent control value. The concentrations selected for 

metaphase analysis were 469.80, 783 and 1305 μg/mL. 
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6.2.2 Metaphase analysis 

The effects of on the chromosomes of cultured human lymphocytes are shown in 

Table 5 and summarised in Table 1. 

In both the absence and the presence of S9 mix, caused no statistically significant 

increases in the proportion of cells with chromosomal aberrations at any concentration, when 

compared with the solvent control. 

In the absence of S9 mix, the mean values for the solvent control (ethanol) were on the limit 

of the laboratory historical control range (including gaps), and outside the laboratory 

historical range (excluding gaps). Although increased values were also observed at 

 treatment concentrations, as no statistical significance was observed at any treatment 

level with no associated concentration-related response, the result was considered negative. 

In the presence of S9 mix, all mean values for the solvent control (ethanol), and all 

 treatment concentrations were within the laboratory historical control range, when 

taken at the 99% confidence limit. 

Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically 

significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the 

efficacy of the S9 mix and the sensitivity of the test system. 

6.2.3 Polyploid analysis 

No statistically significant increases in polyploid metaphases were observed during 

metaphase analysis in either test. 

7. Conclusion 

It is concluded that the test substance has shown no evidence of causing an increase 

in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, 

under the experimental conditions described. 
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8. References 

ARMITAGE, P. (1955) Tests for linear trends in proportions and frequencies. Biometrics, 11, 

375-386. (Cochran-Armitage test). 

ARMITAGE, P., BERRY, G. and MATTHEWS, J.N.S. (2002) Statistical Methods in Medical 

Research, 4th edition. Blackwell Science, Oxford, UK. 

 

EVANS, H.J. and O’RIORDAN, M.L. (1975) Human peripheral blood lymphocytes for the 

analysis of chromosome aberrations in mutagen tests. Mutation Research, 31, 135. 

FISHER, R.A. (1973) The Exact Treatment of 2 x 2 Table in: Statistical Methods for 

Research Workers. Hafner Publishing Company, New York. 

GALLOWAY, S.M., DEASY, D.A., BEAN, C.L., KRAGNAK, A.R., ARMSTRONG, M.J. 

and BRADLEY, M.O. (1987) Effects of high osmotic strength on chromosome aberrations, 

sister chromatid exchanges and DNA strand breaks, and the relation to toxicity. Mutation 

Research, 189, 15. 

HEDDLE, J.A. and BODYCOTE, D.J. (1970) On the formation of chromosomal aberrations. 

Mutation Research, 9, 117. 

ISCN (1985) An International System for Human Cytogenetic Nomenclature, HARNDEN, 

D.G. and KLINGER, H. P. (Eds). S. Karger AG, Basel. 

NOWELL, P.C. (1960) Phytohaemagglutinin: an initiator of mitosis in cultures of normal 

human leukocytes. Cancer Research 20, 462. 

SAS INSTITUTE (2002) SAS OnlineDoc® Version Nine. SAS Institute Inc., Cary, NC, 

USA. 

SATYA-PRAKASH, K.L., HSU, T.C. and PATHAK, S. (1981) Chromatid lesions and 

chromatid core morphology. Cytogenetics and Cell Genetics, 30, 248. 

SCOTT, D., DEAN, B., DANFORD, N.D. and KIRKLAND, D.J. (1990) Metaphase 

chromosome aberration assays in vitro in KIRKLAND, D.J. (Ed.) Basic Mutagenicity Tests: 

UKEMS Recommended Procedures. Report of the UKEMS sub-committee on guidelines for 

mutagenicity testing. Report, Part I revised p. 62. Cambridge University Press, Cambridge. 
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Table 1 Summary of results 

First Test 

Exposure 

period 

S9 

mix 

Nominal concentration of Cells with aberrations 

excluding gaps 

Cells with aberrations 

including gaps 

(hours) (v/v) (μg/mL) Individual 

values (%) 

Mean 

(%) 

Individual 

values (%) 

Mean 

(%) 

Relative 

mitotic 

index 

(%) 

Polyploidy

mean 

incidence

% 

 3 - 0 (Ethanol) 2.0 1.0 1.5 2.0 2.0 2.0 100 0.0 

 469.8 1.0 2.0 1.5 1.0 2.0 1.5 97 0.0 

 783 3.0 3.0 3.0 3.0 3.0 3.0 106 0.0 

 1305 1.0 3.0 2.0 2.0 3.0 2.5 106 1.0 

 0.2 (Mitomycin C) 25.0 30.3 27.4*** 25.0 30.3 27.4*** - 0.0 

 

 3 + 0 (Ethanol) 0.0 2.0 1.0 0.0 2.0 1.0 100 0.0 

 (2%) 469.8 3.0 0.0 1.5 4.0 0.0 2.0 107 0.5 

 783 1.0 0.0 0.5 1.0 0.0 0.5 116 0.0 

 1305 0.0 0.0 0.0 0.0 1.0 0.5 111 0.0 

 5 (Cyclophosphamide) 62.5 66.7 64.5*** 62.5 66.7 64.5*** - 0.0 

One-tailed Fisher's exact test 

*** p<0.001 

Otherwise p>0.01 

Second Test 

Exposure 

period 

S9 

mix 

Nominal concentration of Cells with aberrations 

excluding gaps 

Cells with aberrations 

including gaps 

(hours) (v/v) (μg/mL) Individual 

values (%) 

Mean 

(%) 

Individual 

values (%) 

Mean 

(%) 

Relative 

mitotic 

index 

(%) 

Polyploidy

mean 

incidence

(%) 

21 - 0 (Ethanol) 5.0 4.0 4.5 5.0 6.0 5.5 100 0.0 

 469.8 6.0 10.0 8.0 9.0 14.0 11.5 103 0.5 

 783 11.5 5.0 8.0 11.5 8.0 9.6 68 1.6 

 1305 7.0 7.0 7.0 7.0 8.0 7.5 93 1.0 

 0.1 (Mitomycin C) 35.7 22.7 27.8*** 39.3 25.0 30.6*** - 0.0 

 

 3 + 0 (Ethanol) 1.0 0.0 0.5 3.0 1.0 2.0 100 0.5 

 (5%) 469.8 1.0 2.0 1.5 2.0 5.0 3.5 112 1.0 

 783 2.0 0.0 1.0 3.0 0.0 1.5 114 1.5 

 1305 1.0 2.0 1.5 3.0 2.0 2.5 124 0.0 

 5 (Cyclophosphamide) 27.0 23.8 25.3*** 27.0 23.8 25.3*** - 0.0 

One-tailed Fisher's exact test 

*** p<0.001 

Otherwise p>0.01 
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Table 2 Mitotic index data – first test 

In the absence of S9 mix, 3 hour treatment and 18 hour recovery 

Concentration of Mitotic Index Relative 

Mitotic Index 

(μg/mL) Incidence % Mean # (%) # 

0 110/1000 10.8 100 

(Ethanol) 106/1000 

 

60.89 a a 

 

101.48 a a 

 

169.13 a a 

 

281.88 a a 

 

469.8 95/1000 10.5 97 

 114/1000 

 

783 119/1000 11.4 106 

 109/1000 

 

1305 118/1000 11.5 106 

 111/1000 

 a Visual assessment of toxicity performed, no toxicity observed. 

# Occasional apparent errors of ±1% may occur due to rounding of values. 
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Table 2 Mitotic index data – first test (continued) 

In the presence S9 mix (2% v/v), 3 hours treatment and 18 hours recovery 

Concentration of Mitotic Index Relative 

Mitotic Index 

(μg/mL) Incidence % Mean # (%) # 

0 100/1000 10.5 100 

(Ethanol) 109/1000 

 

60.89 a a 

 

101.48 a a 

 

169.48 a a 

 

281.88 a a 

 

469.8 120/1000 11.2 107 

 104/1000 

 

783 119/1000 12.2 116 

 124/1000 

 

1305 116/1000 11.6 111 

 116/1000 

 a Visual assessment of toxicity performed, no toxicity observed. 

# Occasional apparent errors of ±1% may occur due to rounding of values. 
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Table 3 Metaphase analysis data – first test 

In the absence of S9 mix, 3 hour treatment and 18 hour recovery 

Nominal concentration No. cells Aberrations No. of aberrant cells Relative

of examined Chromatid Chromosome Others Gaps Exc. Mean Inc. Mean mitotic

 type type gaps (%) gaps (%) index 

(μg/mL) ctb cte csb cse ctg csg (%) 

0 100 2 2 1.5 2 2.0 100 

(Ethanol) 100 1 1 1 2 

 

469.8 100 1 1 1.5 1 1.5 97 

100 1 1 2 2 

783 100 2 1 3 3.0 3 3.0 106 

100 3 1 3 3 

 

1305 100 1 1 2 1 2.0 2 2.5 106 

100 2 1 3 3 

0.2 40 13 3 2 2 10 27.4 10 27.4 - 

(Mitomycin C) 33 10 3 10 *** 10 *** 

ctb Chromatid break cte Chromatid exchange 

csb Chromosome break cse Chromosome exchange 

ctg Chromatid gap csg Chromosome gap 

Others Cells with greater than eight aberrations, pulverised cells and pulverised chromosomes 

One-tailed Fisher's exact test 

*** p<0.001 

Otherwise p>0.01 
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Table 3 Metaphase analysis data – first test (continued) 

In the presence of S9 mix (2% v/v), 3 hour treatment and 18 hour recovery 

Nominal concentration No. cells Aberrations No. of aberrant cells Relative

of examined Chromatid Chromosome Others Gaps Exc. Mean Inc. Mean mitotic

 type type gaps (%) gaps (%) index 

(μg/mL) ctb cte csb cse ctg csg (%) 

0 100 0 1.0 0 1.0 100 

(Ethanol) 100 2 2 2 

 

469.8 100 4 1 3 1.5 4 2.0 107 

100 0 0 

 

783 100 1 1 0.5 1 0.5 116 

100 0 0 

 

1305 100 0 0.0 0 0.5 111 

100 1 0 1 

 

5 16 10 2 8 1 2 10 64.5 10 64.5 - 

(Cyclophosphamide) 15 17 2 1 10 *** 10 *** 

ctb Chromatid break cte Chromatid exchange 

csb Chromosome break cse Chromosome exchange 

ctg Chromatid gap csg Chromosome gap 

Others Cells with greater than eight aberrations, pulverised cells and pulverised chromosomes 

One-tailed Fisher's exact test 

*** p<0.001 

Otherwise p>0.01 
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Table 4 Mitotic index data – second test 

In the absence of S9 mix, 21 hour continuous treatment 

Concentration of Mitotic Index Relative 

Mitotic Index 

(μg/mL) Incidence % Mean # (%) # 

0 25/1000 1.6 100 

(Ethanol) 7/1000 

 

169.48 7/1000 0.8 47 

 8/1000 

 

281.88 a a 

 

469.8 a a 

 

783 5/1000 0.7 44 

 9/1000 

 

1305 7/1000 0.4 22 

 0/1000 

a Slides not analysed due to low control values. 

 # Occasional apparent errors of ±1% may occur due to rounding of values. 
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Table 4 Mitotic index data – second test (continued) 

In the presence of S9 mix (5% v/v), 3 hour treatment and 18 hour recovery 

Concentration of Mitotic Index Relative 

Mitotic Index 

(μg/mL) Incidence % Mean # (%) # 

0 71/1000 8.0 100 

(Ethanol) 89/1000 

 

169.48 a a 

 

281.88 a a 

 

469.8 80/1000 9.0 112 

 99/1000 

 

783 89/1000 9.2 114 

 94/1000 

 

1305 98/1000 9.9 124 

 100/1000 

 a Visual assessment of toxicity performed, no toxicity observed. 

# Occasional apparent errors of ±1% may occur due to rounding of values. 
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Table 4 Mitotic index data – second test 

In the absence of S9 mix, 21 hour continuous treatment (additional test) 

Concentration of Mitotic Index Relative 

Mitotic Index 

(μg/mL) Incidence % Mean # (%) # 

0 81/1000 8.1 100 

(Ethanol) 80/1000 

 

169.13 a a 

 

281.88 a a 

 

469.8 82/1000 8.3 103 

 84/1000 

 

783 63/1000 b 5.5 68 

 47/1000 b 

 

1305 78/1000 b 7.5 93 

 71/1000 b 

 a Visually assessed for toxicity performed, no toxicity observed. 

b

 Many non-viable cells. 

# Occasional apparent errors of ±1% may occur due to rounding of values. 
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Table 5 Metaphase analysis data – second test 

In the absence of S9 mix, 21 hour continuous treatment 

Nominal concentration No. cells Aberrations No. of aberrant cells Relative

of examined Chromatid Chromosome Others Gaps Exc. Mean Inc. Mean mitotic

 type type gaps % gaps % index 

(μg/mL) ctb cte csb cse ctg csg % 

0 100 5 2 5 4.5 5 5.5 100 

(Ethanol) 100 2 2 3 4 6 

 

469.8 100 5 1 5 6 8.0 9 11.5 103 

 100 10 5 10 14 

 

783 87 10 1 10 8.0 10 9.6 68 

100 2 2 1 2 1 5 8 

 

1305 100 6 1 2 7 7.0 7 7.5 93 

100 7 1 7 8 

 

0.1 28 5 1 5 4 10 27.8 11 30.6 - 

(Mitomycin C) 44 5 2 5 1 1 10 *** 11 *** 

ctb Chromatid break cte Chromatid exchange 

csb Chromosome break cse Chromosome exchange 

ctg Chromatid gap csg Chromosome gap 

Others Cells with greater than eight aberrations, pulverised cells and pulverised chromosomes 

One-tailed Fisher's exact test 

*** p<0.001 

Otherwise p>0.01 
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Table 5 Metaphase analysis data – second test (continued) 

In the presence of S9 mix (5% v/v), 3 hour treatment and 18 hour recovery 

Nominal concentration No. cells Aberrations No. of aberrant cells Relative

of examined Chromatid Chromosome Others Gaps Exc. Mean Inc. Mean mitotic

 type type gaps % gaps % index 

(μg/mL) ctb cte csb cse ctg csg % 

0 100 1 1 2 1 0.5 3 2.0 100 

(Ethanol) 100 1 0 1 

 

469.8 100 1 1 1 1.5 2 3.5 112 

100 1 1 4 2 5 

 

783 100 1 1 1 2 1.0 3 1.5 114 

100 0 0 

 

1305 100 1 2 1 1.5 3 2.5 124 

100 1 1 2 2 

 

5 37 3 1 9 2 10 25.3 10 25.3 - 

(Cyclophosphamide) 42 4 1 10 1 10 *** 10 *** 

ctb Chromatid break cte Chromatid exchange 

csb Chromosome break cse Chromosome exchange 

ctg Chromatid gap csg Chromosome gap 

Others Cells with greater than eight aberrations, pulverised cells and pulverised chromosomes 

One-tailed Fisher's exact test 

*** p<0.001 

Otherwise p>0.01 
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Appendix 1 Historical Control Data 

April 2008 – March 2010 (3 hours) 

Solvent Control Values (46 studies) 

In the Absence of S9 mix Frequency of aberrant metaphases 

Excluding gaps 

Mean (%) 

Including gaps 

Mean (%) 

Minimum 0.0 0.0 

Maximum 2.5 5.5 

Mean 0.9 1.8 

Standard Deviation 0.7 1.3 

99% Upper confidence limit 2.50 5.28 

Positive Control Values (46 studies)

In the Absence of S9 mix Frequency of aberrant metaphases 

Excluding gaps 

Mean (%) 

Including gaps 

Mean (%) 

Minimum 7.0 8.0 

Maximum 51.3 51.3 

Mean 22.2 25.2 

Standard Deviation 10.0 9.2 

99% Upper confidence limit 48.69 48.69 

99% Lower confidence limit 7.90 9.80 
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Appendix 1 Historical Control Data - continued 

April 2008 – March 2010 (3 hours) 

Solvent Control Values (85 studies) 

In the Presence of S9 mix Frequency of aberrant metaphases 

Excluding gaps 

Mean (%) 

Including gaps 

Mean (%) 

Minimum 0.0 0.0 

Maximum 4.0 5.0 

Mean 0.9 1.8 

Standard Deviation 0.9 1.1 

99% Upper confidence limit 3.58 5.00 

Positive Control Values (85 studies)

In the Presence of S9 mix Frequency of aberrant metaphases 

Excluding gaps 

Mean (%) 

Including gaps 

Mean (%) 

Minimum 7.0 8.0 

Maximum 51.3 53.8 

Mean 20.3 22.8 

Standard Deviation 9.1 9.0 

99% Upper confidence limit 49.20 49.60 

99% Lower confidence limit 7.42 9.51 
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Appendix 1 Historical Control Data - continued 

April 2008 – March 2010 (continuous) 

Solvent Control Values (36 studies) 

In the Absence of S9 mix Frequency of aberrant metaphases 

Excluding gaps 

Mean (%) 

Including gaps 

Mean (%) 

Minimum 0.0 0.0 

Maximum 3.0 5.5 

Mean 1.4 2.3 

Standard Deviation 0.8 1.2 

99% Upper confidence limit 3.00 5.33 

Positive Control Values (36 studies)

In the Absence of S9 mix Frequency of aberrant metaphases 

Excluding gaps 

Mean (%) 

Including gaps 

Mean (%) 

Minimum 7.5 12.5 

Maximum 57.5 62.5 

Mean 26.8 29.7 

Standard Deviation 13.3 12.9 

99% Upper confidence limit 57.36 61.63 

99% Lower confidence limit 8.73 13.20 
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Appendix 2 GLP Compliance Statement 
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Appendix 3 S9 Quality Control Certificates 
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Appendix 3 S9 Quality Control Certificates (continued) 
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Appendix 4 Certificate of Analysis 
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Appendix 5 Apparatus for Vapour/Gas Phase Exposure of Cultivated 

Mammalian Cells 

Culture bottle 

Rotating bottle holder

Motorised rollers

FIGURE 2 

Roller apparatus 

Septum cap 

Culture medium containing mammalian cells 

± S9 mix 

FIGURE 1 

Culture bottle (volume 160ml)
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Appendix 6 Protocol and Amendments 
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