Toxicological Principles for the Safety Assessment of Food Ingredients
Chapter IV.C.1.b. In vitro Mammalian Chromosomal Aberration Test
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- Good Laboratory Practice
- Initial Considerations
- Principle of the Test Method
- Description of the Method
- Data and Reporting
The purpose of the in vitro chromosomal aberration test is to identify agents that cause structural chromosomal aberrations in cultured mammalian cells 1, 2, 3. Structural aberrations may be of two types, chromosome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but chromosome type aberrations also occur. The in vitro chromosomal aberration test may employ cultures of established cell lines, cell strains or primary cell cultures. Chromosomal aberrations are the cause of many human genetic diseases and there is substantial evidence that chromosomal damage and related events causing alterations in oncogenes and tumor suppressor genes of somatic cells are involved in cancer induction in humans and experimental animals.
An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations 25. However, the protocol prescribed in this guidance document is not intended to provide an adequate method for the detection of agents that cause numerical chromosomal aberrations. Thus, a lack of polyploidy should not be considered adequate evidence that the test material does not have the potential to induce numerical aberrations, including aneuploidy.
This guidance document is based on the guidelines published by the Organization for Economic Cooperation and Development (OECD) and/or those published by the United States Environmental Protection Agency (US EPA). At the date of publication of this chapter, these documents are available at:
II. Good Laboratory Practice
Nonclinical laboratory studies must be conducted according to U.S. FDA good laboratory practice (GLP) regulations, issued under Part 58. Title 21. Code of Federal Regulations. This document may be obtained from the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C., 20402, (toll free 866-512-1800).
Aneuploid: Having an abnormal number of chromosomes not an exact multiple of the haploid number.
Centromere: A region(s) of a chromosome with which spindle fibers are associated during cell division, allowing orderly movement of daughter chromosomes to the poles of the daughter cells.
Chromatid-type aberration: Structural chromosomal damage expressed as breakage of single chromatids or breakage and reunion between chromatids.
Chromosome-type aberration: Structural chromosomal damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.
Clastogen: An agent that induces chromosome breaks, an essential step in the formation of structural chromosomal aberrations.
Endoreduplication: A process in which after the S phase of DNA replication, the nucleus does not go into mitosis but starts another S phase. The result is chromosomes with 4, 8, 16,...chromatids.
Gap: An achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the chromatid(s).
Mitotic index: The ratio of cells in metaphase divided by the total number of cells observed in a population of cells; an indication of the degree of proliferation of that population.
Numerical aberration: A change in the number of chromosomes from the normal number characteristic of the cells utilized.
Polyploidy: A multiple of the haploid chromosome number (n) greater than the diploid number (i.e., 3n, 4n and so on).
Structural aberration: A change in chromosomal structure detectable by microscopic examination of the metaphase stage of cell division, observed as intrachanges, or interchanges, or deletions and fragments.
IV. Initial Considerations
Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. This metabolic activation system cannot mimic entirely the mammalian in vivo metabolic and pharmacokinetic conditions. Care should be taken to avoid extreme conditions of pH or osmolality which would lead to positive results which do not reflect intrinsic mutagenicity 4, 5.
This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, correlation is dependent on chemical class and there is increasing evidence that there are carcinogens that are not detected by this test or other genetic toxicity tests because they appear to act through mechanisms other than direct DNA damage.
V. Principle of the Test Method
Cell cultures are exposed to the test substance both with and without metabolic activation. At predetermined intervals after exposure of cell cultures to the test substance, they are treated with a metaphase-arresting substance (e.g., Colcemid® or colchicine), harvested, stained and metaphase cells are analyzed microscopically for the presence of chromosomal aberrations.
VI. Description of the Method
A variety of mammalian cell lines, strains or primary cell cultures, including human cells, may be used (e.g., Chinese hamster fibroblasts, human or other mammalian peripheral blood lymphocytes). Cells are selected for use in the assay on the basis of experience with the cell type, growth ability in culture, stability of the karyotype, chromosome number, chromosomal morphological diversity, and spontaneous frequency of chromosomal aberrations. Established cell lines and strains should be checked routinely for stability in the modal chromosome number.
2. Media and Culture Conditions
Appropriate culture media, and incubation conditions (culture vessels, CO2 concentration, temperature and humidity) should be used in maintaining cultures. Cultures should be monitored routinely for the absence of mycoplasma contamination and should not be used if contaminated. The normal cell cycle time for the cells and culture conditions used should be known.
3. Preparation of Cultures
Established cell lines and strains: cells are propagated from stock cultures, seeded in an appropriate culture medium at a density such that the cultures will not reach confluency before the time of harvest, and incubated at 37°C.
Lymphocytes: whole blood treated with an anti-coagulant (e.g., heparin) or separated lymphocytes are added to an appropriate culture medium containing a mitogen (e.g., phytohemagglutinin) and incubated at 37°C. Lymphocytes from different individuals may respond differently to culture conditions or the test materials. Therefore, lymphocytes from at least two healthy donors should be used.
4. Preparation of Doses
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. The volume of the test material plus solvent or vehicle should be the same in all cultures, including negative and vehicle controls. Fresh preparations of the test substance should be employed unless stability data demonstrate the acceptability of storage.
B. Test Conditions
The solvent/vehicle should not interfere in any way with the performance of the test, e.g., by reacting chemically with the test substance, by affecting the metabolism of the test substance by S9, by altering the response of the cell to the test substance, or by inducing changes in the cell. The suitability of non-standard vehicles/solvents must be demonstrated according to these criteria. It is recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing water-unstable substances, the organic solvents used should be free of water. Water can be removed by adding a molecular sieve.
2. Metabolic Activation
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 rats treated with enzyme-inducing agents such as Aroclor 1254 6, 7, 8, 9, or a mixture of phenobarbitone and ß-naphthoflavone 10, 11, 12. The post-mitochondrial fraction is usually used at concentrations in the range from 1-10% v/v in the final test medium. The condition 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 prudent use of engineered cell lines in place of exogenous metabolic activation should be scientifically justified (e.g., by the relevance of the cytochrome P450 isoenzyme for the metabolism of the test substance).
Concurrent positive and negative (solvent or vehicle) controls both with and without metabolic activation should be included in each experiment.
Positive controls should employ a known clastogen 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 positive control slide to the reader. When metabolic activation is used, the positive control chemical should be the one that requires activation to give a clastogenic response. Examples of positive control substances include:
|Metabolic activation condition||Chemical and CAS No.|
|Absence of exogenous metabolic activation||Methyl methanesulfonate [CAS no. 66-27-3]|
|Ethyl methanesulfonate [CAS no. 62-50-0]|
|Ethylnitrosourea [CAS no. 759-73-9]|
|Mitomycin C [CAS no. 56-57-7]|
|4-Nitroquinoline-N-Oxide [CAS no. 56-57-5]|
|Presence of exogenous metabolic activation||Benzo(a)pyrene [CAS no. 50-32-8]|
|Cyclophosphamide (monohydrate)* [CAS no. 50-18-0 (CAS no. 6055-19-2)]|
*There are literature reports (compiled in reference 26) showing that, in some studies but not in others, cyclophosphamide can be clastogenic in vitro in the absence of metabolic activation. If cyclophosphamide is used as a positive control, it should also be tested in the absence of S9 to demonstrate its S9-dependence in the cell line being used in that laboratory.
4. Dose Levels
Among the criteria to be considered when determining the highest concentration are cytotoxicity, solubility in the test system, and changes in pH or osmolality.
It may be useful to determine cytotoxicity and solubility in a preliminary dose-range finding assay using the same treatment regimen and metabolic activation to be used in the definitive chromosomal aberration study. Cytotoxicity should be determined using an appropriate indication of cell integrity and growth, such as degree of confluency, viable cell counts, plating efficiency, or mitotic index. Cytotoxicity should also be determined with and without metabolic activation in the definitive assay.
Analyzable cells must be obtained from a minimum of three test concentrations. Where cytotoxicity occurs, these concentrations should cover a 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 between 2 and the square root of 10. At the time of harvesting, the highest concentration should show a reduction in degree of confluency, cell count, or plating efficiency, of at least 50%. The use of mitotic index as a measure of cytotoxicity is acceptable for mixed cell cultures, such as whole blood cultures where peripheral lymphocytes are used as the genotoxic target cell. In these types of cultures other toxicity estimators are not possible or are technically impractical. A reduction in mitotic index can indicate a cytostatic rather than cytotoxic response and may be affected by the length of time between treatment and harvest. It is therefore not recommended as a measure of cytotoxicity in experiments with cell lines in which other measures of cytotoxicity are possible. Information on cell cycle kinetics, such as average generation time (AGT), could be used as supplementary information. AGT, however, is an overall average that does not always reveal the existence of delayed subpopulations, and even slight increases in average generation time can be associated with very substantial delay in the time of optimal yield of aberrations.
For relatively non-cytotoxic compounds the maximum concentration should be 5 µl/ml, 5 mg/ml, or 0.01M, whichever is the lowest.
For relatively insoluble substances that are not toxic at soluble concentrations, the highest dose tested should be a concentration above the limit of solubility in the culture medium at the end of the treatment period. It has been suggested that in some cases (e.g., when toxicity occurs only at insoluble concentrations) it may be advisable to test at more than one concentration with visible precipitation. In considering this recommendation, caution should be exercised to minimize conditions that complicate quantification of results. It may be useful to assess solubility 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.
In some cases, it may be possible to use lower chemical concentrations and to increase the treatment time (without metabolic activation) so that the test can be performed under conditions where the chemical is soluble. Fresh chemical preparations should be employed unless stability data demonstrate the acceptability of storage.
Proliferating cells are treated with the test substance in the presence and absence of a metabolic activation system. Treatment of lymphocytes should commence at about 48 hours after mitogenic stimulation.
Duplicate cultures should normally be used at each concentration, and are strongly recommended for negative/solvent control cultures. Where minimal variation between duplicate cultures can be demonstrated 13, 14 from historical data, it may be acceptable for single cultures to be used at each concentration.
D. Harvest of Cultures
In the first experiment, cells should be exposed to the test substance both with and without metabolic activation for 3-6 hours, and sampled at a time equivalent to about 1.5 normal cell cycle length after the beginning of treatment 12. If this protocol gives negative results both with and without activation, an additional experiment without activation should be done, with continuous treatment until sampling at a time equivalent to about 1.5 normal cell cycle lengths. Certain chemicals may be more readily detected by treatment/sampling times longer than 1.5 cycle lengths12. Negative results with metabolic activation need to be confirmed by additional testing. Modification of the test protocol, such as variation of S9 source or concentration, should be considered for this confirmatory test.
E. Chromosome Preparation
Cell cultures are treated with Colcemid® or colchicine usually for one to three hours prior to harvesting. Each cell culture is harvested and processed separately for the preparation of chromosomes. Chromosome preparation involves hypotonic treatment of the cells, fixation and staining 27. Hypotonic treatment should be adjusted to provide optimal separation of chromosomes but without loss of chromosomes. Staining should allow accurate discrimination of chromosome structures 27.
All slides, including those of positive and negative controls, should be independently coded before microscopic analysis. Since fixation procedures often result in the disruption of a proportion of metaphase cells with loss of chromosomes, the cells scored should contain a number of centromeres equal to the modal number ± 2 for all cell types. At least 200 well-spread metaphases should be scored per concentration and control equally divided among the duplicates, if applicable. This number can be reduced when high numbers of aberrations are observed. Though the purpose of the test is to detect structural chromosomal aberrations, it is important to record polyploidy and endoreduplication.
VII. Data and Reporting
A. Treatment Results
The experimental unit is the cell, and therefore the percentage of cells with structural chromosomal aberration(s) should be evaluated. Different types of structural chromosomal aberrations should be listed with their numbers and frequencies for experimental and control cultures. Gaps are recorded separately and reported but generally not included in the total aberration frequency. Various schemes involving varying degrees of detail have been used to classify chromosomal aberrations. For most tests, it is adequate to classify aberrations into four main categories; chromosome breaks, chromosome exchanges, chromatid breaks and chromatid exchanges. In addition, other events such as polyploidy, endoreduplication, heavily damaged cells (for example, more than ten aberrations in one cell), and cells with shattered or pulverized chromosomes should also be recorded.
Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment(s) should also be recorded.
Individual culture data should be provided. Additionally, all data should be summarised in tabular form.
There is no requirement for verification of a clear positive response. Marginally or weakly positive results should be verified by additional testing. Equivocal results should be clarified by further testing, preferably using modification of experimental conditions. The need to confirm negative results has been discussed in section V.D. "Harvest of Cultures". Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration spacing and the metabolic activation conditions.
B. Evaluation and Interpretation of Results
There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosomal aberrations at a single test concentration. Statistical methods should be used as an aid in evaluating the test results 3, 13 but should not be the only determining factor for a positive response. Biological relevance should be considered.
An increase in the number of polyploid cells may indicate that the test substance has the potential to interfere with the mitotic processes and to induce numerical chromosomal aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test substance has the potential to inhibit cell cycle progression 17, 18. Induction of numerical aberrations should be confirmed.
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. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.
Positive results from the in vitro chromosomal aberration test indicate that the test substance induces structural chromosomal aberrations in cultured mammalian somatic cells. Negative results indicate that, under the test conditions, the test substance does not induce chromosomal aberrations in cultured mammalian somatic cells.
There are several conditions under which positive data may be a result of the test conditions rather than any intrinsic clastogenicity of the test material. Changes in pH or osmolality have been shown to induce aberrations 5, 19. There is also evidence that some chemicals induce an increase in structural aberrations only at high levels of cytotoxicity 20, 21. In this case, the aberrations may be the result of disruption of cellular processes that would not be expected at lower doses, and may therefore have no relevance to clastogenic risk at physiologically relevant concentrations. However, before it can be concluded that the test agent presents no clastogenic hazard, it must be demonstrated that the material is not genotoxic in any other in vitro assays, it does not interact directly with the DNA, it is not a topoisomerase inhibitor, it is not related structurally to known clastogens, and that the concentrations inducing aberrations in vitro cannot be achieved in vivo.
Caution should also be exercised when interpreting data from studies using Chinese hamster cells. Certain chemicals appear to induce a high frequency of damage at a specific location, or fragile site, on the long arm of the X chromosome 22, 23, 24. In tests where it can be documented that a large percentage of aberrations are at this site and there is no significant increase in other aberrations, the relevance of this phenomenon to effects in human cells is unclear.
C. Test Report
The test report should include the following information:
1. 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.
- justification for choice of solvent/vehicle.
- solubility and stability of the test substance in solvent/vehicle, if known.
3. Dosing Solutions
- Time interval between stock solution and dosing solution preparation and use, and storage conditions.
- Data that verify concentration of the dosing solution, if available.
4. Test cultures
- type and source of cells;
- karyotype features and suitability of the cell type used;
- absence of mycoplasma, if applicable;
- information on cell cycle length;
- sex of blood donors, whole blood or separated lymphocytes, mitogen used;
- number of passages, if applicable;
- methods for maintenance of cell cultures if applicable;
- modal number of chromosomes.
5. Test Conditions
- identity of metaphase arresting substance, 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 scoring aberrations;
- number of metaphases analyzed;
- methods for the measurements of toxicity;
- criteria for considering studies as positive, negative or equivocal.
- signs of toxicity, e.g., degree of confluency, cell cycle data, cell counts, mitotic index;
- signs of precipitation;
- data on pH and osmolality of the treatment medium, if determined;
- definition for aberrations, including gaps;
- number of cells with chromosomal aberrations and type of chromosomal aberrations given separately for each treated and control culture;
- changes in ploidy if seen;
- 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.
7. Discussion of the Results
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