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## 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 2011Huntingdon Life Sciences NBJ0048

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## Sponsor and Test Facility Details

Sponsor

Test facility Huntingdon Life Sciences Eye Research Centre Eye Suffolk IP23 7PX UKHuntingdon Life Sciences NBJ0048

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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................................................................................................................... 19Huntingdon Life Sciences NBJ0048

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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 ............................................................................ 38Huntingdon Life Sciences NBJ0048

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## Contributing Scientists

In Vitro Mammalian Chromosome A berration Test In Human Lymphocytes 

## Study management

Lincoln Pritchard BSc (Hons) (Study Director) Department of Genetic Toxicology

Claire Damant (Study Supervisor) Department of Genetic ToxicologyHuntingdon Life Sciences NBJ0048

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## Summary

A study was performed to assess the ability of (a liquef ied gas) to induce chromosomal aberrations in human lymphocytes cultured in vitro.
Human lymphocytes, in whole bl ood 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 concentrati on 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 μ g/mL.
In the presence of S9 mix (2% v/v) - 3 hour treatment, 18 hour recovery: 469.80, 783 and 1305 μ g/mL.

Second test

In the absence of S9 mix - 21 hour continuous treatment: 469.80, 783 and 1305 μ g/mL.
In the presence of S9 mix (5% v/v) - 3 hour treatment, 18 hour recovery: 469.80, 783 and 1305 μ g/mL.
In both the absence and presence of S9 mix, caused no statistically significant increases in the proportion of metaphase figures containing ch romosomal aberrations, at any concentration, when compared with th e 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 cau sed 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 eviden ce of causing an increas e in the frequency of structural chromosome aberra tions in this in vitro cyt ogenetic test system, under the experimental conditions described.Huntingdon Life Sciences NBJ0048

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## 1. Introduction

This report describes a study designed to asse ss 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 Chemi cals. (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 Aberra tion Test. EPA 712-C-98-223.
EC Commission Regulation No. 440/2008. Method B.10: Mutagenicity – In Vitro Mammalian Chromosome Abe rration 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 th is 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 a dding phytohaemagglutinin (PHA) to the culture which results in a high mitotic yield ( Nowell, 1960 ).
In this study, blood taken from healthy ma le non-smoking donors was pooled and diluted with tissue culture medium. The cultures we re incubated in the pr esence of PHA before being treated with the test substance. Fo llowing treatment the cells were arrested at metaphase using the mitotic inhibitor, Colcemid®. Chromosomes in these metaphase cells were then examined for the presence of chromo some aberrations. The best estimate of the aberration frequency is at the firs t cell division after initiation of treatment since certain types of damage may be lost during subsequent cell divi sions. 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 (phe nobarbital and 5,6-benzoflavone) known to induce a high leve l of enzymic activity.Huntingdon Life Sciences NBJ0048

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### The study comprised of two independent tests. In the first test, a th ree hour treatment was used in both the absence and presence of S9 mix. In the second te st, 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 us ed in the presence of S9 mix.

Aberrations were scored accordi ng to the classification of the ISCN (1985). Traditionally gaps have been excluded from the quantitat ion 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 Li fe Sciences Management and the Study Director on 19 February 2010, a nd by the Sponsor on 21 April 2010.
The experimental start and completion da tes of the study were 2 March 2010 and 26 May 2010, respectively.Huntingdon Life Sciences NBJ0048

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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 ti ssue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium hepa rin, 20 IU/mL penicillin / 20 μ g/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 incuba ted at 37°C in a 5% CO 2 

### atmosphere for approximately 48 hours. The cultu res were gently shaken daily to resuspend the cells.
In the second test only, the culture volume wa s increased from 5 mL to 10 mL for continuous treatment in the absence of S9 mix, to attain gr eater cell numbers within this test system. All cultures were prepared as larger 10 mL aliquot s (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 concentra tion 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 μ g/mL (3 hour treatment)
0.1 μ g/mL (21 hour continuous treatment)
2.3.2 In the presence of S9 mix
Identity: Cyclophosphamide Appearance: White powder Batch number: 068K1131Huntingdon Life Sciences NBJ0048

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### Solvent: Sterile purified water Final concentration: 5 μ g/mL (3 hour treatment)



## 2.4 S9 metabolizing system

2.4.1 Preparation of S9 fraction
The S9 fraction was obtained from male Sp rague-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 so urce and stored at -80° C or below. A copy of each quality control statement relating to the batch of S9 pr eparation used (batch numbers 2440 and 2472) is included in Appendix 3

. 2.4.2 Preparation of S9 mix

S9 mix contained: S9 frac tion (10% v/v : test 1 a nd 25% v/v : test 2), MgCl 2 

### (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, th e solubility of was asse ssed. 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 μ g/mL (10 mM), no observations were noted.
Concentrations with high ionic strength and osmolality may cause chromosomal aberrations ( Galloway et al. 1987 ). Therefore, concentr ations greater than 5000 μ g/mL or 10 mM are not used in this test system.
The osmolality and pH of the test su bstance in medium was tested at 1305 μ g/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 concentr ation used for subsequent testing was 1305 μ g/mL (10 mM), the limit guideline concentration. In this study was added to cultures at 1% v/v (50 μ L 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 cult ures. 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 inte rnal volume of 160 mL) using a syringe and needle, inserted through the septum cap to preven t any loss of within the test system.Huntingdon Life Sciences NBJ0048

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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 ei ght minutes approximately. Th e lymphocytes coat the inside of the bottles and were immersed in cultu re medium once every revolution and exposed directly to for the re st of the revolution.

In the absence of S9 mix, 50 μ L aliquots of liquid we re added to give final concentrations of 60.89, 101.48, 169.13, 281.88, 469.80, 783 and 1305 μ g/mL. Ethanol was used as the solvent control. Mitomycin C at a final concentration of 0.2 μ g/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 μ L of each test substance dilution (g iving the same series of final concentrations as above). Ethanol was the solvent control. Cyclophosphamide at a final concentration of 5 μ g/mL was the positive control.
In both the absence and presence of S9 mi x, no notable culture medium changes were observed either at or after 3 hour treatment.
Three hours after dosing, the cultures were transf erred to universal cont ainers 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, m itotic activity was arre sted by addition of Colcemid® to each culture at a final concentrat ion of 0.1 μg/mL. After 2 hours incubation, each cell suspension was transferred to a centrif uge tube and centrifuged for 5 minutes at 500g. The cell pellets were tr eated 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 ai r dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). Af ter rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remai nder 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 theHuntingdon Life Sciences NBJ0048

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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 ab errant 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 en doreduplicated 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 th e 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 si gnificant at the 1% level, the test is reiterated excluding the highest concentration group - this process c ontinues until the tre nd test is no longer significant.
D20s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated usi ng logistic regression on a log(c oncentration) scale, allowing the number of control abe rrations 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 contro l proportion of aberrations.



## 2.10 Second test

Cultures were initiated a nd maintained as previous ly 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. Ho wever, to modify study parameters the final concentration of S9 mix was incr eased 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 μ g/mL.
In the presence of S9 mix (5% v/v): 169.13, 281.88, 469.80, 783 and 1305 μ g/mL.
Duplicate cultures were used for each treatment and two cult ures were treated with the solvent control. Positive control cultures were tr eated as in the first test. Mitomycin C, at a final concentration of 0.1 μ g/mL, and Cyclophosphamide, at a final concentration of 5 μ g/mL, were added to duplicate cultures.Huntingdon Life Sciences NBJ0048

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### In both the absence and presence of S9 mi x, no notable culture medium changes were observed either at or after treatment.
Three hours after dosing, the cultures containi ng S9 mix were centrifuge d. 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 Colcem id®, at a final concentration of 0.1 μ g/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 c oncentrations were non-cytotoxic. However, in the absence of S9 mix, furt her testing was conducted to ac hieve 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 solutio n) for the subsequent repeat test.
There was no change in study methodology, howev er, all cultures were treated at 1% v/v (100 μL) as a result of th e increased culture volume.

Concentrations of were as follows:

In the absence of S9 mix: 169.13, 281.88, 469.80, 783 and 1305 μ g/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.Huntingdon Life Sciences NBJ0048

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## 3. Assessment of Results

An assay is considered to be acceptable if the negative and positive cont rol 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 meta phases 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 s upport the conclusion.
A negative response is claimed if no statisti cally significant increas es in the number of aberrant cells above concurrent control fre quencies are observed, at any concentration.
A further evaluation may be carried out if th e 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 appropr iate) 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 te rm retention and archiving may be disposed of.
All other samples and specimens and all ra w data will be retained by Huntingdon Life Sciences in its archive for a period of one y ear 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 material s 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.Huntingdon Life Sciences NBJ0048

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## 6. Results

6.1 First test

6.1.1 Toxicity data
Mitotic indices of cultured human lymphoc ytes 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 μ g/mL, the highest tested concentration, compared to the solvent control value. The concentra tions selected for metaphase an alysis were 469.80, 783 and 1305 μ g/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 μ g/mL, the highest tested concentration, compared to the solvent control value. The concentrations selected for metaphase analysis were 469.80, 783 and 1305 μ g/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 pr esence of S9 mix, cause d no statistically significant increases in the proportion of ce lls with chromosomal aberrations at any concentration, when compared with the solvent control.

All mean values for the solven t control (ethanol), and all treatment concentrations were within the historical control range for th is laboratory, when taken at the 99% confidence limit.
Both positive control compounds, Mitomycin C and Cycloph osphamide, 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 lymphoc ytes 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 μ g/mL, the highest tested concentration, compared to the solvent control value. The c oncentrations selected for metaphase analysis were 469.80, 783 and 1305 μ g/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 μ g/mL, the highest tested concentration, compared to the solvent control value. The concentrations selected for metaphase analysis were 469.80, 783 and 1305 μ g/mL.Huntingdon Life Sciences NBJ0048

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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 pr esence of S9 mix, cause d no statistically significant increases in the proportion of ce lls with chromosomal aberrations at any concentration, when compared with the solvent control.

In the absence of S9 mix, the mean values fo r 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 in creased values were also observed at treatment concentrations, as no statisti cal significance was obser ved 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 th e laboratory historical control range, when taken at the 99% confidence limit.
Both positive control compounds, Mitomycin C and Cycloph osphamide, 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 aberra tions in this in vitro cytogenetic test system, under the experimental conditions described.Huntingdon Life Sciences NBJ0048

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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. a nd MATTHEWS, J.N.S. (2002) Statistical Methods in Medical Research, 4 th 

### 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 mu tagen 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 os motic strength on chromosome aberrations, sister chromatid exchanges and DNA strand brea ks, 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 PA THAK, 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 KIRK LAND, 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.Huntingdon Life Sciences NBJ0048

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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 > 0.01Huntingdon Life Sciences NBJ0048

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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.Huntingdon Life Sciences NBJ0048

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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.Huntingdon Life Sciences NBJ0048

23 

### Table 3 Metaphase analysis data – first test

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

Nominal concentration No. cells Aberra tions No. of aberrant cells Relative of examined Chromatid Chromosome O thers 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 cs e Chromosome exchange ctg Chromatid gap csg Chromosome gap Others Cells with greater than eight aberratio ns, pulverised cells and pulverised chromosomes

One-tailed Fisher's exact test *** p <0.001 Otherwise p > 0.01Huntingdon Life Sciences NBJ0048

24 

### 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 Aberra tions No. of aberrant cells Relative of examined Chromatid Chromosome O thers 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 cs e Chromosome exchange ctg Chromatid gap csg Chromosome gap Others Cells with greater than eight aberratio ns, pulverised cells and pulverised chromosomes

One-tailed Fisher's exact test *** p <0.001 Otherwise p > 0.01Huntingdon Life Sciences NBJ0048

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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.Huntingdon Life Sciences NBJ0048

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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.Huntingdon Life Sciences NBJ0048

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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.Huntingdon Life Sciences NBJ0048

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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 Aberra tions No. of aberrant cells Relative of examined Chromatid Chromosome O thers 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 cs e Chromosome exchange ctg Chromatid gap csg Chromosome gap Others Cells with greater than eight aberratio ns, pulverised cells and pulverised chromosomes

One-tailed Fisher's exact test *** p <0.001 Otherwise p > 0.01Huntingdon Life Sciences NBJ0048

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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 Aberra tions No. of aberrant cells Relative of examined Chromatid Chromosome O thers 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 cs e Chromosome exchange ctg Chromatid gap csg Chromosome gap Others Cells with greater than eight aberratio ns, pulverised cells and pulverised chromosomes

One-tailed Fisher's exact test *** p <0.001 Otherwise p > 0.01Huntingdon Life Sciences NBJ0048

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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 9.80Huntingdon Life Sciences NBJ0048

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### Appendix 1 Historical C ontrol 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 9.51Huntingdon Life Sciences NBJ0048

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### Appendix 1 Historical C ontrol 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 13.20Huntingdon Life Sciences NBJ0048

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### Appendix 2 GLP Compliance StatementHuntingdon Life Sciences NBJ0048

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### Appendix 3 S9 Quality Control CertificatesHuntingdon Life Sciences NBJ0048

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### Appendix 3 S9 Quality Contro l Certificates (continued)Huntingdon Life Sciences NBJ0048

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### Appendix 4 Certifi cate of AnalysisHuntingdon Life Sciences NBJ0048

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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 a pp aratus Septum cap

Culture medium containing mammalian cells ± S9 mix 

### FIGURE 1

Culture bottle ( volume 160ml )Huntingdon Life Sciences NBJ0048

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### Appendix 6 Protocol and AmendmentsHuntingdon Life Sciences NBJ0048

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