Chromosome Aberration Test
Chromosome Aberration Test in vitro
The purpose of the in vitro chromosome aberration (CA) test is to identify agents that cause structural chromosome aberrations (clastogenesis)2)3)4) which are produced by a variety of mechanisms. Many of these changes will be lethal to the cell during the first few cell cycles after their induction but are used as indicators of the presence of non-lethal changes such as reciprocal translocations, inversions and small deletions. These more subtle changes may have important consequences in both germ and somatic cells. Thus, chromosomal mutations and related events are the cause of many human genetic diseases and there is substantial evidence that these changes are involved in cancer in humans and experimental systems.
The test is performed in accordance with the OECD Guideline for Testing of Chemicals, Section 4, No. 473 at Eurofins BioPharma Product Testing Munich GmbH 1) and part of the test battery for chemicals, cosmetics, pharmaceuticals or medical devices.
Another assay to test for clastogenicity is the Micronucleus Assay.
Experimental Performance
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Protocol |
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Cell lines |
Chinese Hamster V79 cells or stimulated cultured human peripheral blood lymphocytes |
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Endpoint |
Detection of clastogenicity by microscopic evaluation of metaphase cells Cell viability determination by relative increase in cell count (V79 cells) or mitotic index (human lymphocytes) |
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Concentrations |
At least 3 test item concentrations evaluated in duplicat Maximum concentrations: · Chemicals: 2 mg/mL, 2 µL/mL or 10 mM, whichever is the lowest (5 mg/mL for not defined components/ UVCB) · Medical device: 100% extract · Pharmaceuticals: 0.5 mg/mL or 1 mM, whichever is the lowest |
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Exposure time |
Experiment I: short-term incubation for 4 h Experiment II: long-term incubation for 21 h (V79 cells) or 24 h (human lymphocytes) |
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Metabolic activation |
5% Rat liver homogenate S9 |
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Quality controls |
Negative control: cell culture medium Positive controls: ethylmethanesulfonate, cyclophosphamide |
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Vehicles of test chemicals |
Cell culture medium, 1% dimethylsulfoxid, 10% physiological saline, 0.5% tetrahydrofuran, 0.5% ethanol |
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Data capture |
Microscopic evaluation of chromosomal aberrations in 300 metaphase cells per concentration Cell viability determined by relative increase in cell count (V79 cells) or mitotic index, statistical evaluation |
Test procedure (human lymphocytes)
The assay is considered as acceptable, when all three experimental conditions are conducted: short-term treatment without and with metabolic activation and long-term treatment without metabolic activation in case of negative results.
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First Experiment (short-term incubation) and Second Experiment (long-term incubation) |
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1. Day: Preparation day |
Blood collection from a single donor and seeding of the cells. Cultures of peripheral blood lymphocytes are stimulated to divide by the addition of a mitogen (e.g. phytohemagglutinin: PHA) to the culture medium. |
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3. Day: Test Item Incubation |
Treatments should commence at around 48 h after culture initiation when the cells are actively proliferating. The culture medium will be replaced by 25% serum-containing medium containing different concentrations of the test item and S9 mix (only with metabolic activation). Beginning of the treatment Cytotoxicity and precipitation will be determined after the treatment period of the cultures. |
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4. Day: Preparation of the Cultures |
Cells should be sampled first at about 24 h later (1 - 1.5 fold of the normal cell cycle time). |
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5. Day: Staining of the Cells with Giemsa |
After preparation, the cells are spread on the slides. The air dried slides will be stained with Giemsa solution and afterwards analysed microscopically. |
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6. Day: Start of Analysis of Metaphase Cells |
Chromosomal aberrations: All structural chromosome aberrations such as breaks, fragments, deletions, exchanges and chromosomal disintegration will be recorded. Gaps will be recorded as well but are not included in the calculation of the aberration rates. Relative Mitotic Index: Cytotoxicity will be assessed by the mitotic index (% cells in mitosis, by counting the number of mitotic cells in 1000 cells) and values are compared with negative/solvent controls. Proliferation Index: The cell cycle of the actual lymphocyte cultures is monitored using a BrdU-labeling technique. |
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1. Day: Preparation day |
Blood collection from a single donor and seeding of the cells. Cultures of peripheral blood lymphocytes are stimulated to divide by the addition of a mitogen (e.g. phytohemagglutinin: PHA) to the culture medium. |
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Historical Laboratory Control Data of the Negative Control (human lymphocytes) |
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Negative Control Number of aberrant cells |
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short-term |
long-term |
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-S9 |
+S9 |
-S9 |
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mean [%] |
2.0 |
1.9 |
1.6 |
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SD [%] |
0.89 |
1.04 |
0.98 |
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RSD [%] |
43.6 |
55.7 |
60.1 |
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min [%] |
0.0 |
0.0 |
0.0 |
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max [%] |
3.3 |
4.0 |
4.2 |
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n |
33 |
40 |
26 |
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LCL |
0.26 |
-0.21 |
-0.33 |
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UCL |
3.84 |
3.94 |
3.60 |
| NC: | Negative Control (cell culture medium) | max.: | maximum number of aberrant cells |
| mean: | mean number of aberrant cells | n: | Number of assays |
| SD: | Standard Deviation | LCL: | Lower control limit (95%, mean-2SD) |
| RSD: | relative Standard Deviation | UCL: | Upper control limit (95%, mean+2SD) |
| min.: | minimum number of aberrant cells |
Evaluation of Results
A test chemical is considered clearly positive if, in all experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is dose-related when evaluated with an appropriate trend test,
- any of the results are outside the 95% control limits of the historical negative control data.
The test chemical is then considered able to induce chromosomal aberrations in cultured mammalian or human peripheral blood lymphocyte cells in this test system.
A test chemical is considered clearly positive if, in all experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is dose-related when evaluated with an appropriate trend test,
- any of the results are outside the 95% control limits of the historical negative control data.
The test chemical is then considered able to induce chromosomal aberrations in cultured mammalian or human peripheral blood lymphocyte cells in this test system.
Significance
The Chromosome Aberration Test is part of a test battery for testing genotoxicity, together with the Reverse Mutation Assay, the Mouse Lymphoma Assay/HPRT-Test, the micronucleus test or the in vivo comet assay.
References
- Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 473, "In vitro Mammalian Chromosome Aberration Test", adopted 29 July, 2016
- Kirkland, D. (1998). Chromosome aberration testing in genetic toxicology- past, present and future. Mutation Research,: 404 (1998), 173-185
- Galloway, S.M., Aardema, M.J., Ishidate, M. Jr., Ivett, J.L., Kirkland, D.J., Morita, T., Mosesso, P. and Sofuni, T. (1994). Report from Working Group on in Vitro Tests for Chromosomal Aberrations. Mutat. Res. 312, 241-261
- Galloway, S., Lorge, E., Aaardema, M.J., Eastmond, D., Fellows, M., Heflich, R., Kirkland, D., Levy, D.D., Lynch, A.M., Marzin, D., morita, T., Schuler, M., Speit, G. (2011). Workshop summary: Top concentration for in vitro mammalian cell genotoxicity assays; and report from working group on toxicity measures and top concentration for in vitro cytogenetics assays (chromosome aberrations and micronucleus). Mutat. res.: 723 (2), 77-83