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Title: The Micronucleus Assay

METHODOLOGY

Applies to: British American Tobacco Group

Research and Development

Effective from: 2010 (Archived methodology)

British American Tobacco Group Research & Development,

Southampton, UK.

Method – The Micronucleus assay for the determination of the

genotoxic potential of cigarette smoke.

Form No: GRD-107-COM

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Methodology : The Micronucleus Assay

Applies to : British American Tobacco Group
Research and Development

1. INTRODUCTION

The in vitro micronucleus test detects chromosome breakage and loss by measuring the formation
of micronuclei. These are small membrane bound fragments or whole chromosomes, which are
unable to attach to the spindle at mitosis and appear as small bodies within the cell.

Cells are treated with Cytochalasin B which blocks cell division but not nuclear division resulting in
cells containing two or more nuclei. The proportion of cells that have undergone cell division and
suffered chromosome breakage or loss, resulting in micronucleus formation can then be counted,
giving a representation of the genotoxicity of the test item.

The V79 (Chinese male hamster lung) cells used in this methodology have been fully screened
and characterised by the European Collection of Cell Cultures (ECACC). The cells have also been
screened and tested negative for mycoplasma contamination.

2. REAGENTS AND MATERIALS

2.1 The following reagents and materials should be made available prior to performing this
procedure:
• Complete Dulbecco’s Modified Eagles Medium (DMEM)
• V79 cells (near confluent)
• Trypsin EDTA Universals
• Phosphate Buffered Saline(PBS) pH 7.45
• Hank’s Balanced Salt Solution (HBSS)
• Cytospin and Cytofunnels
• AnalR grade Dimethyl Sulphoxide (DMSO)
• Cytochalasin B
• Methanol
• Coulter Counter and Isoton II solution
• Acridine Orange
• 5% CO2 Incubator (37°C)
• Positive controls

3. DAY 1 PROCEDURE – SETTING UP THE FLASKS

3.1 Examine flasks to determine level of confluency. This observation will reflect the
amount of media added in step 6.5.

Form No.: GRD-107-COM

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Methodology : The Micronucleus Assay

Applies to : British American Tobacco Group
Research and Development

3.2 Aspirate media from cells with the Biovac aspirator pump or disposable pipette. Wash
cells by adding approximately 10ml of warm HBSS to each flask with a sterile 10ml
dispenser pump or disposable pipette then aspirate off.

3.3 Trypsinise cells by adding 2ml of Trypsin EDTA to each flask then incubate at 37(±1)°C
for 2 minutes. After incubation, the flasks should be removed from incubator and
tapped to detach cells.

3.4 Check the flasks under the microscope to ensure cells have detached. If not, the flasks
should be returned to the incubator for another minute. NOTE - LEAVING TRYPSIN
ON FOR TOO LONG WILL DAMAGE THE CELLS.

3.5 Add 4-8 mls of complete medium to each 75cm3 flask. Then pool the contents of each
75cm3 flask into one of the flasks. If the cell suspension appears clumpy, clumps should
be broken up by repeated agitation, for example by repeatedly pipetting the suspension
up and down with a disposable pipette.

4. DAY 2 PROCEDURE – DOSING THE FLASKS

4.1 Prepare 56 x 8.9ml aliquots of serum-free media (3hrs +S9) or 56 x 9.9ml aliquots of
complete DMEM (3hrs -S9 and 20hrs -S9) in labelled plastic universals.

4.2 For 3hrs +S9 only, add 1ml of S9 cofactor mix to each universal.

4.3 Tubes can be stored in the 37(±1)°C incubator until ready for use.

4.4 Examine flasks microscopically for growth and absence of contamination (see section
11). Typical signs of contamination are changes in colour or clouding of cell medium
and changes in cell shape. Report any suspected contamination to the Project Leader
as soon as possible as this may invalidate the assay. An entry to acknowledge
microscopic examination should be made on the ‘Comments’ section of the datasheets.

4.5 Select the best 56 flasks for the assay and label with sample code. Less confluent
flasks should be allocated solvent control and low dose treatments, more confluent
flasks should be allocated higher doses and positive controls (refer to table 1 below for
details of positive and solvent controls).

Table 1: Positive and solvent controls in the micronucleus test

Treatment Solvent control Positive control
Mitomycin C (250µg/ml)
20hrs-S9 DMSO
Vinblastine (0.4 µg/ml)
3hrs-S9 DMSO Mitomycin C (250 µg/ml)
3hrs+S9 DMSO Benzo(a)pyrene (1mg/ml)

4.6 Prepare smoke condensate dilutions in sterile Eppendorfs (as detailed in the assay data
sheets).

Form No.: GRD-107-COM

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Methodology : The Micronucleus Assay

Applies to : British American Tobacco Group
Research and Development

4.7 Add 100µl of each dose level solution to the relevant universal tube. Change pipette
tips between each sample and dose. This should be done as close to the time of dosing
as possible.

4.8 Aspirate media from each flask with the Biovac aspirator pump or disposable pipette.

4.9 Wash cells by adding approximately 10ml of warm HBSS to each flask with a sterile
10ml dispenser pump or disposable pipette then aspirate off the HBSS medium.

4.10 Add 10ml of each dose level to the relevant flask with a disposable pipette. Change
pipettes between each sample and dose.

4.11 Flush each flask for approximately 5 seconds with filtered 5% CO2 and incubate for 3
hours at 37(±1)°C in a 5% CO2 incubator.

4.12 3hrs +/-S9 only

A. prepare Cytochalasin B medium and store in dark until needed.

B. After 3hrs, aspirate dosing solution from flasks with the Biovac
aspirator pump or disposable pipette into a vessel labelled toxic waste.

C. Wash cells as described in step 7.9.

D. With disposable pipettes, add 10mls of Cytochalasin B medium to each

flask (under subdued lighting as Cytochalasin B is light sensitive).

E. Flush each flask for approximately 5 seconds with filtered 5% CO2 and
incubate for 17 hours at 37(±1)°C in a 5% CO2 incubator.

4.13 20hr -S9

A. After 3hrs, add 20µl of Cytochalasin B into each flask. Pipette into the
media NOT directly onto the cell monolayer. Change pipette tips
between each sample and dose.

B. Incubate flasks for 17 hours at 37(±1)°C in a 5% CO2 incubator.

4.14 Prepare slides for harvesting; label 4 slides per dose with test item, dose level and date
using a black cryogenic marker pen or a pencil or slide etching equipment (refer to SOP
TOX-MET-030 for use of slide etcher)

4.15 Place 1 bottle of complete media and HBSS (or PBS) in the incubator 37(±1)°C to warm
overnight.

Form No.: GRD-107-COM

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Methodology : The Micronucleus Assay

Applies to : British American Tobacco Group
Research and Development

Perform a cell count using a cell counter.

4.16 Adjust the cell concentration to approximately 5 x 105 cells/ml with complete DMEM.

4.17 Add 1ml of cell suspension to 58 x 25cm2 labelled flasks containing 9ml complete
DMEM (at least 2 spare flasks should be set up). Flasks should be labelled with study
number, date and initials.

4.18 Flush each flask for approximately 5 seconds with filtered 5% CO2 and incubate
overnight at 37(±1)°C in a 5% CO2 incubator.

5. DAY 3 PROCEDURE – HARVESTING CELLS

5.1 Prepare 300ml of 90% methanol for slide fixing and place in the freezer until needed.

5.2 Check cells microscopically. Cells should appear rounded due to the action of the
Cytochalasin B. An entry to acknowledge microscopic examination should be made on
the ‘Comments’ section of the test datasheets.

5.3 Aspirate media from each flask with the Biovac aspirator pump or disposable pipette.

5.4 Wash cells as described in step 7.9.

5.5 Trypsinise cells by adding 1ml of Trypsin EDTA to each flask then incubate at 37(±1)°C
for 2 minutes. After incubation, the flasks should be removed from incubator and tapped
to detach cells.

5.6 Examine flasks microscopically to check cells have detached, then stand flasks upright.

5.7 Add 3-4mls of complete DMEM to each flask to de-activate the Trypsin. (More
confluent lower dosed flasks add 4mls; less confluent higher dosed flasks add 3mls).

5.8 Perform a cell count on one culture per test item dose

5.9 Cell counts only need to be performed on one of each pair of replicate cultures as it is
assumed the number of cells for replicate treatment flasks will be similar. The cell count
for each test item dose level should be used to adjust the cell concentrations for both
replicate flasks (for example the cell count for flask A1 should be used to calculate cell
concentration and adjustment for flasks A1 and A1’).

5.10 Adjust the concentration of the cell suspension aliquots to approximately 5x104 cells/ml
with PBS, HBSS or completed DMEM.

5.11 Load the Cytospin carousel with the 2 labelled slides and Cytofunnels per dose.

5.12 Add 250µl of each cell suspension to the relevant slide/Cytofunnel. Change pipette tips
between each sample and dose.

5.13 Spin the slides at 1000rpm for 5 minutes.

5.14 Remove slides from Cytospin and microscopically examine slides for cell density.

Form No.: GRD-107-COM

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Methodology : The Micronucleus Assay

Applies to : British American Tobacco Group
Research and Development

5.15 Rate the slides as follows and allocate a new volume of cell suspension if necessary
(150-350µl) to add to the next 2 repeat slides of each dose.

5.16 Repeat steps 8.10 – 8.13 with the duplicate slides and new cell suspension volume. A
total of 4 slides per dose should be produced; at least 2 of the 4 slides should be of
suitable quality to score. If 0 of the 4 slides are of a suitable quality to score, clean the
slides with 70% ethanol and re-dose with a new volume of cell suspension.

6. DAY 3 PROCEDURE – FIXING CELLS

6.1 Fix all slides in 90% high-grade methanol at -20(±1)°C for 9 minutes and allow to air
dry. Slides for Acridine Orange staining can be kept at room temperature prior to
staining.

7. STAINING CELLS

7.1 Add 200ml of PBS (using a 25ml disposable pipette) to a large cell culture flask and
drop in a 1ml aliquot of 25mg/ml Acridine Orange. Shake well.

7.2 Pour the Acridine Orange dye into a staining box. For each rack of slides to be stained
prepare 3 more staining boxes containing approximately 200ml of PBS

7.3 Submerge the slides in the Acridine Orange dye for 10 seconds, remove from stain
solution and transfer immediately into the first PBS wash. Briefly rinse slides.

7.4 Transfer to a second PBS wash and rinse again. Transfer slides to a third PBS wash
and leave in the dark for 10 minutes at room temperature.

7.5 Remove slides from third PBS wash and store in a dark box at room temperature until
ready to be coded and scored. Prior to coding a visual check should be carried out on a
selection of the stained slides to ensure the staining and distribution of cells is of a
suitable standard for scoring.

8. ACCEPTANCE CRITERIA

8.1 The assay will usually be considered valid if the following criteria are met:

A. The positive control chemicals induce at statistically significant increase (tested

by One-way ANOVA) in the proportion of cells with micronuclei compared to the
negative control cultures.

B. At least of 50% of cells have gone through at least one cell division (as measured
by binucleate + multinucleate cell counts) in negative control cultures.

C. At least 3 dose levels for each test item have a level of cytotoxicity below 60%.

D. For items exhibiting 60% cytotoxicity or below, at least 800 cells (per culture)
must have been scored.

Form No.: GRD-107-COM