2. INTRODUCTION
• The adverse health effects caused by the Geno toxins in vivo are of
concern. The effects of exposure to relatively low concentrations of Geno
toxic substances may not be immediately apparent, this means that there
are no warning signs so that exposure can be avoided
• The short term in vitro mammalian cell chromosome aberrations test is
used to assess the potential Genotoxic hazard of test substances
• Abnormality in Chromosome can be of following Types
1. Deletion
2. Duplication
3. Inversion
4. translocation
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3. METHODS
• White Blood Cell Culture
• Dosing Formulation: Cyclophosphamide(treat cancer), anhydrous
dimethyl sulphoxide as a vehicle, S9 fraction
• Sampling, Slide preparation and Staining:
1. KCl in water(0.56%)
2. Methanol : Glacial acetic Acid(3:1 v/v)
3. Microscopic Slides
• Conduct of Test: High quality metaphase preparation is preferred, use
of a trained and experienced Observer
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4. METHODS
• Study Design: Culture initiation
Add WBC sample to culture medium including PHA and incubate(37±1°C)
for 48 Hrs, cells mostly in G0 of cell cycle
Start of Exposure: 0hrs
Add dosing formulations dimethyl sulphoxide & incubate for 3Hrs, cells at
all stages of cell cycle
End of Exposure: 3 hrs
Remove dosing Formulation. Incubate for 15Hrs. Cells should have
undergone S phase since 0 Hr
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5. Study Design
Metaphase Arrest: 18 hrs
Add Colcemid. Incubate for 2 hrs. Cells should be at first M phase since 0 hrs
Harvest: 20 hrs
Hypotonic to swell cells, fixative cell suspension on slides, stain and cover slip
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6. White Blood Culture
• Blood is to be taken by addition of Heparin tubes to avoid clotting for Blood
collection
• The sample is been then incubated at 37°C±1°C throughout with gentle mixing of
the cultures. A humidified atmosphere of 5% CO2 in air may be introduced at the
culture
• Lymphocytes are grown as suspension cultures in sterile, disposable centrifuge
tubes
• A healthy donor who do not smoke and willing to donate blood on a regular
basis is selected , absence of viral diseases such as hepatitis and HIV are
important both for protection of operators and for the success of the assay
• The Lymphocytes are tested for a good response to PHA giving an acceptable
mitotic index(MI) and AGT.
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7. WHITE BLOOD CULTURE
• The Lymphocytes should have a normal karyotype and exhibit levels of
chromosome aberrations that falls within a normal range with references to the
historical negative control database
• Prior to vein puncture, it should be ascertained that the potential donor has not
knowingly been exposed to ionizing radiation, hazardous chemicals or has
suffered from viral infections in the 2 weeks period before giving blood
• Heparinised blood, 0.4 ml is added to the warmed supplemented RPMI1640
medium so that the final volume following addition of S-9 mix/KCl and the dosing
formulation is 10ml
• These suspension cultures are incubated for approximately 48 hrs
• Even small but prolonged reductions in incubator temperature can affect the AGT
and this may not be obvious as it is likely to be measured peroidically
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8. PREPARATION, ADDITION & REMOVAL OF DOSING
FORMULATION
• Add approximately 10 mg CPA and add sufficient amount of DMSO to prepare the
stock solution that is 100x strong
• Prepare a alternative stock solution that is 10x strong and dissolve in water for
injection then sterilize to avoid microbial contamination
• The stock solution is diluted to provide six concentration of CPA , in the presence
of S-9 mix it
• The S-9 fraction is thawed just before required and the S-9 mixture is prepared
• The CPA dosing formulation(0.1 ml) is added to each culture to achieve the
required final concentration of CPA in a total of 10ml, culture exposed to CPA in
absence of S-9 receives 0.5 ml of KCl
• Incubation is continued for 3 hrs
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9. PREPARATION, ADDITION & REMOVAL OF DOSING
FORMULATION
• Culture tubes are centrifuged at approximately 300x g for 10 mins, the
supernatant is carefully removed and cells resuspended in 10 ml RPMI medium
without supplements, prewarmed to 37°C. This process is repeated so that there
have been two changes of medium, after the third centrifugation, the cells are
resuspended in 10ml complete RPMI medium, prewarmed at 37°C and
incubation is continued
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10. SAMPLING, SLIDE PREPARATION AND STAINING
• Colcemid are added 2-3 hrs before sampling to arrest dividing cells at metaphase
• At the sampling time, cultures are centrifuged at 300x g for 10 mins, the
supernatant carefully removed and cells resuspended in 4ml warmed KCl at 37°C
for 15 mins to allow cells to grow
• Cells are fixed by gently mixing suspension of cells with 6 ml fixative . It is very
important to avoid clumping of the lymphocytes, the tubes containing cells
suspension in fixative and hypotonic solution are centrifuged at approximately
300 x g for 10mins
• The supernatant is removed and discarded into a labelled container for disposal
• The cells are resuspended by slow addition of fresh, cold fixative, mixing using a
vortex mixer to avoid cell clumping
• The tubes containing the suspension are centrifuged, this process is repeated
until the cells pallets no longer contain traces of red blood cells
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11. SAMPLING, SLIDE PREPARATION AND STAINING
• Lymphocytes are usually kept at 1-10°C at least overnight to ensure adequate
fixation
• Cells suspension are centrifuged and resuspended in a minimal amount of freshly
prepared fixative , several drops of cell suspension are placed on cold microscope
• After the slides have dried the cells are stained for 5 mins in filtered 4% Giemsa in
pH 6.8 Buffer
• The slides are rinsed, dried and mounted with coverslips
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12. STAINING
• Stain the slides in a solution of Hoechst 33258 for 25 mins protected from
sunlight
• Rinse slides thoroughly in MacIlvaine's buffer pH 8.0 twice, the second time
being in the buffer prewarmed to 40°C , place the slides flat in a tray and add
sufficient prewarmed buffer to cover the slides causing the chromatid arm to
swell
• Expose the slides to the UV light at approximately 366 nm for 25-40 mins
• Remove the slides and rinse thoroughly in PBS at pH 6.8
• Stain for 10 mins in filtered 4% Giemsa stain in PBS at pH 6.8, rinse the slides
thoroughly, first in PBS at pH6.8 and then in water, shake off excess moisture, air
dry and mount with coverslip
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13. ANALYSIS OF CELLS IN FIRST, SECOND AND THIRD
DIVISION
• The AGT is calculated as follows:
proliferative index(PI)= %cells M1 2(%cellsM2) 3(%cells M3)
100
Average Generation time= Hours in BrdU
PI
The AGT should be in the region of 12-14hrs for human lymphocytes, longer than
16hrs indicates that culture conditions are suboptimal and the source of the
problem should be identified and rectified
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14. ASSESSMENT OF MITOTIC INHIBITION
• Mitotic index = number of cells in mitosis X 100
Total no of cells observed
Mitotic inhibition%=[1-{mean Mlt/Mean Mlc}] X 100
Where Mlt= MI in cell exposed to test substance
Mlc= MI in solvent control cells
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15. ANALYSIS AND INTERPRETATION OF RESULTS
• After completion of analysis and decoding, the proportions of aberrant cells per
culture (often expressed as a percentage, since 100 cells are routinely analyzed)
are tabulated as:
• 1.Cells with structural aberrations including gaps
• 2.Cells with structural aberrations excluding gaps (this information is used to
draw a conclusion as to the clastogenic potential of a test substance as gaps may
occur by non-genotoxic modes of action)
• 3.Polyploid, endoreduplicated, or hyper diploid cells (An increase in polyploidy
may indicate that a chemical has the potential to induce numerical aberrations,
when use of the in vitro micronucleus test should be considered.)
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16. The acceptance and evaluation criteria to determine the outcome of the test
should be specified in advance. An acceptable test, assuming good cell growth,
would be likely to have:
1.Homogeneity between replicate cultures
2.The proportion of cells with structural aberrations (excluding gaps) in vehicle
control cultures should fall within the historical negative control range. In the
absence of an historical negative control range, an approximate guide would be
that the frequency of cells with aberrations should be <5%. This low frequency
means that there may not be any aberrations seen in 50 or even 100 cells.
3.At least 160 cells out of an intended 200 should be suitable for analysis at each
concentration of CPA, unless 10 or more cells per slide showing structural
aberrations other than gaps only have been observed during analysis.
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17. The following evaluation criteria may be used for ascribing the potential of a test
article to induce chromosome aberrations in a valid assay.
• A proportion of cells with structural aberrations at one or more concentrations
exceeds the concurrent vehicle control values and the historical negative control
(normal) range (if available) in both replicate cultures.
• A statistically significant increase in the proportion of cells with structural
aberrations (excluding gaps) is observed (p≤0.05).
• Evidence of a concentration-related trend in the proportion of cells with
structural aberrations (excluding gaps) will be judged to support the conclusion
• Results that only partially satisfy the above criteria need to be dealt with on a
case-by-case basis..
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18. • A test article that satisfies none of the above criteria may be considered not to
have the potential to induce chromosome aberrations. The criteria to establish a
negative response are more stringent, and two independent experiments are
required currently.
• The distinction between an indirect-acting and a direct-acting genotoxin is
important in predicting in vivo risk arising from a positive in vitro assay
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