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Chromosome Aberration Test in vitro

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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

Protocol

Cell lines

Chinese Hamster V79 cells or

stimulated cultured human peripheral blood lymphocytes

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)

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

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)

Metabolic activation

5% Rat liver homogenate S9

Quality controls

Negative control: cell culture medium

Positive controls: ethylmethanesulfonate, cyclophosphamide

Vehicles of test chemicals

Cell culture medium, 1% dimethylsulfoxid, 10% physiological saline, 0.5% tetrahydrofuran, 0.5% ethanol

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.

 First Experiment (short-term incubation) and

Second Experiment (long-term incubation)

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.

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
Incubation for 4 h for short-term treatment and 24 h for long-term treatment. Additional negative and/or solvent and positive controls are treated in the same way.

Cytotoxicity and precipitation will be determined after the treatment period of the cultures.

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).

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.

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.

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.

 

 

Historical Laboratory Control Data of the Negative Control (human lymphocytes)

 

Negative Control Number of aberrant cells

short-term

long-term

-S9

+S9

-S9

mean [%]

2.0

1.9

1.6

SD [%]

0.89

1.04

0.98

RSD [%]

43.6

55.7

60.1

min [%]

0.0

0.0

0.0

max [%]

3.3

4.0

4.2

n

33

40

26

LCL

0.26

-0.21

-0.33

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

  1. Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 473, "In vitro Mammalian Chromosome Aberration Test", adopted 29 July, 2016
  2. Kirkland, D. (1998). Chromosome aberration testing in genetic toxicology- past, present and future. Mutation Research,: 404 (1998), 173-185
  3. 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
  4. 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
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