J. M. Parry

The following text is based upon:-

Further comments upon the current draft are welcome by the 31st October, 1998.

In vitro mammalian micronucleus assay

Introduction

1. The in vitro micronucleus assay is a mutagenicity test system for the detection of chemicals which induce the formation of small membrane bound DNA fragments i.e. micronuclei in the cytoplasm of interphase cells. These micronuclei may originate from acentric fragments (chromosome fragments lacking a centromere) or whole chromosomes which are unable to migrate with the rest of the chromosomes during the anaphase of cell division (1 and referees therein)

2. Chromosome mutations of both structure and number are implicated in many human diseases. There is substantial evidence that chromosome mutations and related events in oncogenes and tumour suppressor genes of somatic cells are involved in the induction and/or progression of some types of cancer in humans and experimental animals. Thus, the purpose of the in vitro micronucleus assay is to detect those agents which modify chromosome structure and segregation in such a way as to lead to induction of micronuclei in interphase cells.

3. Because micronuclei in interphase cells can be assessed much more objectively than chromosomal aberrations in metaphase cells, there is not as rigorous a requirement for detailed training before a person is competent to score the slides. Also, as there is no requirement to count the chromosomes in a metaphase preparation, nor to evaluate subtle chromatid and chromosome damage, but only to determine whether or not a cell contains a micronucleus, the preparations can be scored much more quickly. This makes it practical to score thousands instead of hundreds of cells per treatment, and thus imparts greater accuracy to the assay. Finally, as micronuclei may contain whole (lagging) chromosomes there is the potential to detect aneuploidy-inducing agents which are currently very difficult to study in conventional chromosomal aberration tests.

4. The in vitro micronucleus assay may employ cultures of established cell lines, cell strains or primary cell cultures. The cells used are selected on the basis of their growth ability in culture and their spontaneous micronuclei frequency.

5. Definitions used are set out in the Annex

INITIAL CONSIDERATIONS

6. Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. This metabolic activation system cannot entirely mimic in vivo conditions. Care should be taken to avoid conditions which would lead to positive results which do not reflect intrinsic mutagenicity and may arise from changes in pH, osmolality or high levels of cytotoxicity (5, 6).

7. This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian mutagens and carcinogens. However, the correlation is dependant on chemical class and there is increasing evidence that there are some carcinogens that are not detected by this test because they appear to act through other mechanisms.

8. Analysis of the induction of micronuclei in human lymphocytes cultures has indicated in this cell system that the most convenient stage to score micronuclei is the binucleate interphase stage (2,3). Such cells have completed one cell division after chemical treatment and are therefore capable of expressing micronuclei. Treatment of cultures with the inhibitor of actin polymerisation cytochalasin B results in the "trapping" of cells at the binucleate stage where they can be easily identified (2, 3). The measurement of the relative frequencies of binucleate to mononucleate cells within a culture also provides a simple method of measuring the toxicity of a treatment (4).

PRINCIPLE OF THE TEST METHOD

9. Cell cultures are exposed to the tests substances both with and without metabolic activation. After exposure to a test substance, cell culture are grown for a period sufficient to allow chromosome damage to lead to the formation of micronuclei in interphase cells. Harvested and stained interphase cells are then analysed microscopically for the presence of micronuclei. Micronuclei should only be scored in those cells that complete nuclear division following exposure to the test chemical.

10. A variety of cell lines, strains or primary cell cultures may be used. These include human and Chinese hamster fibroblasts, mouse lymphoma cells, human or other mammalian peripheral blood lymphocytes.

When human peripheral lymphocytes are used the donors should be chosen keeping in mind that age and sex influence baseline micronucleus frequencies. Older women should not be used as they tend to have high micronucleus counts largely due to increased micronucleation of the X chromosome.

When using human lymphocytes, either whole blood or separated lymphocytes may be used.

Media and culture conditions

11. Appropriate culture medium and incubation conditions (culture vessels, CO₂ concentration, temperature and humidity) should be used in maintaining cultures. Established cell lines and strains should be checked routinely for the stability of the modal chromosome number and the absence of mycoplasma contamination and cultures should not be used if contaminated. The normal cell cycle time for the cell and culture conditions used should be known. If the cytokinesis block method is used then concentrations of the cytokinesis inhibitor used must give an adequate yield of binucleate cells.

12. Established cell lines and strains: cells are propagated from stock cultures, seeded in culture medium at a density such that the cultures will not reach confluency before the time of harvest, and incubated at 37'C.

13. Lymphocytes: whole blood treated with an anti-coagulant (e.g. heparin) or separated lymphocytes obtained from healthy subjects are added to culture medium containing a mitogen (e.g. phytohaemagglutinin) and incubate at 37'C.

14. Cells should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a co-factor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (7, 8) or a combination of phenobarbitone and b-naphthoflavone (9, 10). The post-mitochondrial fraction is usually used at concentrations in the range from 1-10% v/v in the final test medium. The selection of a metabolic activation system may depend upon the class of chemical being tested. In some cases it may be appropriate to utilize more than one concentration of post-mitochondrial fraction.

A number of developments, including the construction of genetically engineered cell lines expressing specific activating enzymes, may provide the potential for endogenous activation. The choice of the cell lines used should be scientifically justified (e.g., by relevance of the cytochrome P450 isoenzyme for the metabolism of the test substance). (ref...)

Human lymphocytes have been shown to have a limited capacity to activate some indirect genotoxins (reference necessary). Developments in the understanding of the influence of genetic polymorphisms of xenobiotic-metabolizing enzymes on the genotoxic response of human cells may require that donors are chosen considering their genotype when used in the testing of specific chemical types.

Test substance/Preparation

15. Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if appropriate prior to treatment of the cells. Liquid test substances may be added directly to the test systems and/or diluted prior to treatment. Fresh preparations of the test substance should be employed unless stability data demonstrate the acceptability of storage.

Solvents/vehicle

16. The solvent/vehicle should not be suspected of chemical reaction with the test substance and should be compatible with the survival of the cells and with the maintenance of S9 activity. If other than well-known solvent/vehicles are used, their inclusion should be supported by data indicating their compatibility with the test. It is recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing water-unstable substances, the organic solvents should be free of water. Water can be removed by adding a molecular sieve.

17. Among the criteria to be considered when determining the highest test concentration are cytotoxicity and solubility in the test system. It is not known at this time whether changes in pH and osmolality can indirectly produce positive responses as they can in chromosomal aberration tests, but it is considered prudent to maintain similar physiological controls and limit the highest concentration to 10 mM, 5mg/ml or 5ml/ml when pH varies significantly from physiological conditions,

18. Cytotoxicity should be determined with and without metabolic activation concurrently in the main experiment using an appropriate indication of cell integrity. This could be degree of confluency, viable cell counts, mitotic index or the ratio of binucleate: mononucleate cells (for experiments with cytochalasin B). It may be useful to determine cytotoxicity and solubility in a preliminary experiment.

Use of cytochalasin B

19. Cytochalasin B inhibits microfilament assembly and cytokinesis and thus prevents separation of daughter cells after mitosis and leads to binucleated cells (11). The evaluation can thus be limited to proliferating cells and a reduction of cell proliferation can be measured simultaneously. This is generally considered necessary for human lymphocytes, but not for established cell lines. The appropriate concentration of cytochalasin B is usually between 3 and 6 Fg/ml and has to be tested for each cell line to achieve at least 50% binucleated cells in the control culture. If the test protocol involves removal of the test substance, cytochalasin B should be added after the washing to remove the test chemical. For continuous treatment, cytochalasin B can be added simultaneously with the test substance approximately 20hrs after treatment initiation.

Exposure concentrations

20. At least three analysable test concentrations should be used. Where cytotoxicity occurs, the concentrations should cover the range from the maximum to little or no toxicity; this will usually mean that the concentrations should be separated by no more than a factor of %10. At the time of harvesting, the highest concentration should show a significant reduction in degree of confluency, cell count or proliferation index such as binucleate: mononucleate cell ratio (all less than 50%).

21. For relatively insoluble substances that are not toxic at concentrations lower than the insoluble concentration, the highest dose used should be a concentration above the limit of solubility in the final culture medium at the end of the treatment period. In some cases (e.g. when toxicity occurs only at higher than the lowest insoluble concentration) it is advisable to test at more than one concentration with visible precipitation. It may be useful to assess solubility both at the beginning and the end of the treatment, as solubility can change during the course of exposure in the test system due to presence of cells, S9, serum, etc. Insolubility can be detected by using the unaided eye. The precipitate should not interfere with the scoring.

Controls

22. Concurrent positive and negative (solvent or vehicle) controls both with and without metabolic activation should be included in each experiment. When metabolic activation is used, the positive control chemical should be one that requires activation to give a mutagenic response.

23. Positive controls should employ a known inducer of micronuclei at exposure levels expected to give a reproducible and detectable increase over background which demonstrates the sensitivity of the test system. Positive control concentrations should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. Examples of positive control substances include:

Positive Control Chemicals

24. Other appropriate positive control reference substances may be used. The use of chemical class-related positive control chemicals may be considered, when available.

25. Negative controls, consisting of solvent or vehicle alone in the treatment medium, and treated in the same way as the treatment cultures, should be included for every harvest time. In additiion, untreated control (lacking solvent) should also be used unless there are historical control data demonstrating that there is little variation between individual experiments and the acceptability of the solvent/vehicle.

Evaluators please note. Section 26 is new in this draft. Please consider carefully the ollowing points.

Note. References will be placed in final numerical order at final stage of draft.

Culture harvest time

26. The aim of an experiment will be to expose actively growing cell cultures to a test substance and/or its metabolites for a period equivalent to about 1.5 normal cell cycles. In the case of cell types requiring growth stimulation (such as human lymphocytes by phytohaemoglutamin) treatment would start approximately 24 hrs after culture initiation. In a first experiment actively growing cells should be exposed to the test substance both with and without metabolic activation for 3-6 hours and then sampled at a time equivalent to at least 1.5 cell cycles to allow time for micronuclei to be expelled into the cytoplasm. When Cytochalasin B is used, cells are cultured for a further 44 hours in the presence of this actin filament inhibitor. If this protocol gives negative results both with and without metabolic activation, an additional experiment should be performed with continuous treatment without activation for a period of about 1.5 cycle lengths. All negative results should be confirmed using modified conditions as appropriate.

Preparation of cells

27. Each culture is harvested and processed separately. Cell preparation may involve hypotonic treatment, but this step is not necessary if adequate cell spreading is otherwise achieved. Different techniques can be used in slide preparation, providing that high-quality preparations are obtained. Slides can be prepared by a cyto-centrifuge, or the cells can be dropped, spread, or smeared on slides. Cell cytoplasm should be retained but well-spread to allow the detection of micronuclei and (in the cytokinesis-block method) reliable identification of binucleate cells. The slides can be stained using various methods. Fluorescent DNA-specific dyes are preferred to less specific stains (such as Giemsa), as there will facilitate the detection of even very small micronuclei. Antikinetochore antibodies, fluorescence in situ hybridization with pancentromeric DNA probes, or primed in situ labelling with pancentromere-specific primers together with appropriate DNA counterstaining, can be used to identify the contents (whole chromosome/chromosomal fragment) of micronuclei.

Analysis

28. All slides, including those of positive and negative controls, should be independently coded before the microscopic analysis. At least 1000 cells per duplicate cell culture should be scored to assess the frequency of cells with one, two, or more than two micronuclei.

If the cytokinesis-block technique is applied, the analysis of micronuclei should generally be restricted to binucleate cells, and at least 1000 lymphocytes per duplicate culture should additionally be classified as mononucleates, binucleates or multinucleates (more than two nuclei), to estimate cytokinesis-block proliferation index. The cytokinesis-block proliferation index (CBPI) is a measure of cell cycle delay and should be expressed as:

CBPI = [number binucleate cells + 2 (number multinucleate cells)]/(total number of cells)

30. In both type of assays (with or without cytochalasin B) the frequencies of mitoses may provide additional measure of cell toxicity. Micronuclei should be identified according to the criteria of Heddle (1973) (15) with some modifications. Only micronuclei not exceeding 1/3 of the main nucleus diameter, not overlapping with the main nucleus and with distinct borders, will be included in the scoring. If pancentrometric fluorescence in situ hybridization or antikinetochore antibodies are used, the frequency of micronuclei should be scored as explained above, but the proportion of the fluorescence signal (representing centromeres or kinetochores, respectively) in micronuclei should be evaluated only in cells where the main nuclei show clear signals.

31. The advantage of the cytokinesis-block assay is that it allows the differentiation between cells that have not divided (mononucleates) from those that have undergone one (binucleates) or more nuclear divisions (multinucleates) in the presence of Cytochalasin B. It should, however, be kept in mind that some cells may escape the cytokinesis block. Thus some mononucleate cells may actually have divided in the culture, while some binucleate cells may have divided more than once. Care should be taken not to include binucleate cells with irregular shapes or sizes of the main nuclei, as these cells may represent the latter category. Binucleate cells should not either be confused with poorly spread multinucleate cells. A further division of a binucleate lymphocyte is highly unuasal and results in a high baseline rate of micronuclei in multinucleate cells.

DATA AND REPORTING

32. The experimental unit is the culture to be analysed, and therefore the percentage of micronucleated cells within a culture is the basic parameter. Additionally, cells with one, two and more than two micronuclei can be considered separately. If the cytokinesis-block technique is applied, the frequencies of binucleate cells with micronuclei (and with one, two and more than two micronuclei) should exclusively be used in the evaluation of micronucleus induction.

33. The proliferation index, based on the frequencies of mononucleate, binucleate and multinucleate cells (see above), should be provided for all treated and control cultures as a measure of cell cycle delay in the cytokinesis-block method. The frequency of mitoses can be used as a measure of toxicity when cytochalasin B is not used and as an additional parameter in the cytokinesis block method.

34. Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment(s) should also be recorded.

35. Individual culture data should be provided. Additionally, all data should be summarised in tabular form.

36. There is no requirement for verification of a clear cut positive response. Equivocal results should be clarified by further testing using modification of experimental conditions. Negative results should be confirmed. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments for either equivocal or negative results. Study parameters that might be modified include the test concentration spacing, the method of treatment and the metabolic activation conditions.

37. Chemicals which induce micronuclei in the in vitro assay may do so by a variety of mechanisms such as chromosome breakage and chromosome loss. Analyses of micronuclei using kinetochore antibodies or centromere specific in situ probes allow the determination as to whether micronuclei contain whole or broken chromosomes and may be useful for chemicals inducing a positive response in the assay.

Evaluation and interpretation of results

38. There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells containing micronuclei. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determination of a positive response.

39. Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. These equivocal or questionable responses may occur regardless of the number of times the experiment is repeated.

40. Positive results from the in vitro micronucleus test indicate that the test substance induces chromosome damage or damage to the cell division apparatus in cultured mammalian somatic cells. Negative results indicate that, under the test conditions, the test substance does not induce chromosome structural and/or numerical aberrations in cultured mammalian somatic cells.

41. When a test substance has been shown to induce micronuclei containing whole chromosomes due to loss of chromosomes from the mitotic spindle further studies can be performed to determine whether the substance induces non-disjunction. Antikinetochore antibodies, pancentromeric and chromosome specific centromere probes are particularly convenient for this purpose.

Test Report

42. The test report must include the following information:

Test substance:

- identification data and CAS no., if known

- physical nature and purity

- physicochemical properties relevant to the conduct of the study

- stability of the test substance, if known

Solvent/Vehicle:

- justification for choice of solvent/vehicle

- solubility and stability of the test substance in solvent/vehicle, if known

Cells:

- type and source of cells

- suitability of the cell type used

- absence of mycoplasma, if applicable

- information on cell cycle length, doubling time or proliferation index

- sex and age of blood donors, whole blood or separated lymphocytes

- number of passages, if applicable

- methods for maintenance of cell cultures if applicable

Test conditions:

- identity of cytokinesis blocking substance (e.g. cytochalasin B), if used, its concentration and duration of cell exposure

- rationale for selection of concentrations and number of cultures including, e.g. cytotoxicity data and solubility limitations, if available

- composition of media, CO₂ concentration if applicable

- concentration of test substance

- volume of vehicle and test substance added

- incubation temperature

- incubation time

- duration of treatment

- cell density at seeding, if appropriate

- type and composition of metabolic activation system, including acceptability criteria

- positive and negative controls

- methods of slide preparation

- criteria for micronuclei identification

- number of cells analysed

- methods for the measurements of toxicity

- criteria for considering studies as positive, negative or equivocal

- methods, such as use of kinetochore antibody to characterise whether micronuclei contain whole or fragmental chromosomes

Results:

- signs of toxicity, e.g. degree of confluency, cell cycle data, cell counts, proliferation index

- signs of precipitation

- data on pH and osmolality of the treatment medium, if determined

- definition of what constitutes a micronucleus

- definition of acceptable cells for analysis

- number of cells with micronuclei and number of micronuclei per cell, given separately for each treated and control culture

- dose-response relationship, where possible

- statistical analyses, if any

- concurrent negative (solvent/vehicle) and positive control data

- historical negative (solvent/vehicle) and positive control data, with ranges, means and standard deviations

Discussion of the results:

Conclusion:

Literature: