Redbook 2000: IV.C.1.d Mammalian Erythrocyte Micronucleus Test
Toxicological Principles for the Safety Assessment of Food Ingredients
Chapter IV.C.1.d. Mammalian Erythrocyte Micronucleus Test
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Micronuclei are cytoplasmic chromatin-containing bodies formed when acentric chromosome fragments or chromosomes lag during anaphase and fail to become incorporated into daughter cell nuclei during cell division. Because genetic damage that results in chromosome breaks, structurally abnormal chromosomes, or spindle abnormalities leads to micronucleus formation, the incidence of micronuclei serves as an index of these types of damage. It has been established that essentially all agents that cause double strand chromosome breaks (clastogens) induce micronuclei. Because enumeration of micronuclei is much faster and less technically demanding than is scoring of chromosomal aberrations, and because micronuclei arise from two important types of genetic damage (clastogenesis and spindle disruption), the micronucleus assay has been widely used to screen for chemicals that cause these types of damage.
This guidance addresses the most widely used in vivo micronucleus assay: the mammalian erythrocyte micronucleus assay. This in vivo micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents.
The purpose of the micronucleus test is to identify substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes.
When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; micronuclei that have been formed may remain behind in the otherwise enucleated cytoplasm. Visualization of micronuclei is facilitated in these cells using specific staining techniques and because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage.
Centromere (Kinetochore) is 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.
Micronuclei are small nuclei, separate from and additional to the main nuclei of cells, produced during telophase of mitosis (meiosis) by lagging chromosome fragments or whole chromosomes.
Normochromatic erythrocyte is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by stains selective for ribosomes.
Polychromatic erythrocyte is an immature erythrocyte, in an intermediate stage of development, that still contains ribosomes and therefore can be distinguished from mature, normochromatic erythrocytes by stains selective for ribosomes.
III. Initial Considerations
The bone marrow of rodents is routinely used in this test since polychromatic erythrocytes are produced in that tissue. The measurement of micronucleated immature (polychromatic) erythrocytes in peripheral blood is equally acceptable in any species in which the inability of the spleen to remove micronucleated erythrocytes has been demonstrated, or which has shown an adequate sensitivity to detect agents that cause structural and/or numerical chromosome aberrations. Micronuclei can be distinguished by a number of criteria. These include identification of the presence or absence of a kinetochore or centromeric DNA in the micronuclei. The frequency of micronucleated immature (polychromatic) erythrocytes is the principal endpoint. The number of mature (normochromatic) erythrocytes in the peripheral blood that contain micronuclei among a given number of mature erythrocytes can also be used as the endpoint of the assay when animals are treated continuously for a period that exceeds the lifespan of the erythrocyte in the species under consideration (e.g., 4 weeks in the mouse), provided that significant splenic selection against micronucleated erythrocytes does not occur in that species/strain. The consequences of splenic selection, if it occurs, should be fully addressed.
This mammalian in vivo micronucleus test is especially relevant to assessing mutagenic hazard in that it allows consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes although these may vary among species, among tissues and among genetic endpoints. An in vivo assay is also useful for further investigation of a mutagenic effect detected by an in vitro system.
If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test.
IV. Principle of the Test Method
Animals are exposed to the test substance by an appropriate route. If bone marrow is used, the animals are sacrificed at appropriate times after treatment, the bone marrow extracted, and preparations made and stained.(16),(17),(18),(26),(32),(41) When peripheral blood is used, the blood is collected at appropriate times after treatment and smear preparations are made and stained.(4),(5),(14),(16),(27),(28),(29),(32) Preparations are analyzed for the presence of micronuclei.
V. Description of the Method
1. Selection of Animal Species
Historically, mice or rats have been used routinely for this assay. If bone marrow is the tissue sampled, any appropriate mammalian species may be used (see section III., above). As with any toxicology study, selection of the appropriate species should be justified. When peripheral blood is used, mice are recommended. However, any appropriate mammalian species may be used provided it is a species in which the spleen does not remove micronucleated erythrocytes or is a species which has shown an adequate sensitivity to detect agents that cause structural and/or numerical chromosome aberrations. Commonly used laboratory strains of young healthy animals should be employed. At the commencement of the study, the weight variation of animals should be minimal and not exceed ±20% of the mean weight of each sex.
2. Housing and Feeding Conditions
The temperature in the experimental animal room should be appropriate for the species used; for mice and rats this should be 22°C (±3°C). Although the relative humidity should be at least 30% and preferably not exceed 70% other than during room cleaning, the aim should be 50-60%. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to ensure a suitable admixture of a test substance when administered by this route. Animals may be housed individually, or caged in small groups of the same sex.
3. Preparation of the Animals
Healthy young adult animals should be randomly assigned to the control and treatment groups. The animals should be identified uniquely. The animals should be acclimated to the laboratory conditions for at least five days. Cages should be arranged in such a way that possible effects due to cage placement are minimized.
4. Preparation of Doses
Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted, if appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted prior to dosing. 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 produce toxic effects at the dose levels used, and should not be suspected of chemical reaction with the test substance. If other than commonly employed solvents/vehicles are used, their use should be supported with reference data indicating their compatibility with the test substance and the animals. It is recommended that, wherever appropriate, the use of an aqueous solvent/vehicle should be considered first.
Concurrent positive and negative (solvent/vehicle) controls should generally be included for each sex in each test conducted with rodents. However, when the micronucleus assay is conducted as part of a general toxicity study according to GLP guidelines, then verification of appropriate dosing will be performed by chemical analysis. In such cases, concurrent treatment of animals with a positive control agent may not be necessary and control of staining and scoring procedures may be accomplished by including appropriate reference samples obtained previously from animals that are not part of the current experiment. In studies with higher species, such as primates or dogs, positive controls may be omitted provided that an acceptable response to positive control substances of the species used has been demonstrated previously by the testing laboratory. In all cases, concurrent negative controls are an obligatory study component. Except for treatment with the test substance, animals in the control groups should be handled in an identical manner to animals of the treatment groups.
Positive controls should produce micronuclei in vivo at exposure levels expected to give a detectable, statistically significant increase over background. Positive control doses should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. It is acceptable that the positive control be administered by a route different from the test substance and sampled at only a single time. In addition, the use of chemical class-related positive control chemicals may be considered, when available. Examples of positive control substances include:
|Cyclophosphamide (monohydrate)||50-18-0 (6055-19-2)|
Negative control animals, treated with solvent or vehicle alone and otherwise treated in the same way as the treatment groups, should be included for every sampling time, except that under appropriate circumstances it may be possible to use an animal as its own control by comparing pre-treatment and post-treatment samples. If single sampling is applied for negative controls, the sampling time chosen should be justified. In addition, untreated controls should also be used unless there are (a) data available from the test laboratory, or (b) historical or published control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent/vehicle.
If peripheral blood is used, a pre-treatment sample may also be acceptable as a concurrent negative control, but only in the short peripheral blood studies (e.g., 1-3 treatment(s)) when the resulting data are in the expected range for the historical control and when the absence of a solvent effect has been demonstrated.
VI. Procedure for Rats and Mice
The following sections provide guidance for procedures in mice and rats, the species used most commonly in this assay.
A. Number and Sex of Animals
Each treated and control group should include at least 5 analyzable animals per sex.(12) If at the time of the study there are data available from studies in the same species and using the same route of exposure that demonstrate that there are no substantial differences between sexes in toxicity, then testing in a single sex will be sufficient.
B. Treatment Schedule
Several different treatment schedules (i.e., 1, 2, or more treatments at 24 hr intervals) can be recommended. Samples from extended dose regimens are acceptable as long as a positive effect has been demonstrated for this study or, for a negative study, as long as toxicity has been demonstrated or the limit dose (see section "D", below) has been used, and dosing continued until the time of sampling. This is based on studies showing that repeated exposures of mice and rats of up to subchronic duration produced effects of a magnitude similar to those obtained with the traditional acute assay.(1),(2),(8),(11),(19),(21),(22),(23),(25),(29),(37),(38),(44),(48),(50) However, because there is some concern that sensitivity might be reduced in longer term studies due to a failure to achieve a true MTD, or because adaptation may occur, it is currently considered that the duration treatment should be limited to four weeks until the sensitivity of the assay is confirmed when longer treatments are used.(12)
Test substances may also be administered as a split dose, i.e., two or more treatments on the same day separated by no more than a few hours, to facilitate administering a large volume of material or to minimize fluctuations in blood levels of the test article.
Two ways in which the test may be performed are:
- Animals are treated with the test substance once, or twice at an interval of not more than 24 hours. Samples of bone marrow are taken at least twice between 24 and 48 hr after the last dose, with appropriate interval(s) between samples. The use of sampling times earlier than 24 hours after treatment should be justified. Samples of peripheral blood are taken at least twice between 36 and 72 hours after the last treatment, with appropriate interval(s) between samples. When a positive response is recognized at one sampling time, additional sampling is not required.
- If three or more daily treatments are used (e.g., three or more treatments at 24 hour intervals), samples may be collected once no later than 24 hours following the final treatment for the bone marrow and once no later than 40 hours following the final treatment for the peripheral blood.(12), (20)
Additional sampling times may be used, when relevant and scientifically justified.
C. Dose Levels
If a dose range finding study is performed because there are no suitable data available, it should be performed in the same laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study.(7) If there is toxicity, three dose levels should be used for the first sampling time. These dose levels should cover a range from clear toxicity to little or no toxicity. At the later sampling time only the highest dose needs to be used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. Substances with specific biological activities at low non-toxic doses (such as hormones and mitogens) may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as a dose that produces some indication of toxicity of the bone marrow (e.g., a reduction in the proportion of immature erythrocytes among total erythrocytes in the bone marrow or peripheral blood).
D. Limit Test
If no observable toxic effects result from a single treatment with one dose level of at least 2000 mg/kg body weight, or from two treatments on the same day, and if genotoxicity would not be expected based upon data from structurally related substances, then a full study using 3 dose levels may not be necessary. For studies of a longer duration, the limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.
E. Administration of Doses
The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The maximum volume of liquid that can be administered by gavage or injection at one time depends on the size of the test animal. The volume should not exceed 2 ml/100g body weight. The use of volumes higher than these must be justified. Except for irritating or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimized by adjusting the concentration to ensure a constant volume at all dose levels.
F. Bone Marrow/Blood Preparation
Bone marrow cells are usually obtained from the femurs or tibias immediately following sacrifice. Commonly, cells are removed from femurs or tibias, prepared and stained using established methods. Peripheral blood is obtained from the tail vein or other appropriate blood vessel. Blood cells are immediately stained supravitally(4),(5),(14) or smear preparations are made and then stained. The use of a DNA specific stain (e.g., acridine orange(15) or Hoechst 33258 plus pyronin-Y(30)) can eliminate some of the artifacts associated with using a non-DNA specific stain. This advantage does not preclude the use of conventional stains (e.g., Giemsa). Additional systems (e.g., cellulose columns to remove nucleated cells(36)) can also be used provided that these systems have been shown to work adequately for micronucleus preparation in the laboratory.
The proportion of immature among total (immature + mature) erythrocytes is determined for each animal by counting a total of at least 200 erythrocytes for bone marrow and 1000 erythrocytes for peripheral blood.(9) All slides, including those of positive and negative controls, should be independently coded before microscopic analysis. At least 2000 immature erythrocytes per animal are scored for the incidence of micronucleated immature erythrocytes. Additional information may be obtained by scoring mature erythrocytes for micronuclei. When analyzing slides, the proportion of immature erythrocytes among total erythrocytes should not be less than 20% of the control value. When animals are treated continuously for 4 weeks or more, at least 2000 mature erythrocytes per animal can also be scored for the incidence of micronuclei. Systems for automated analysis (image analysis or flow cytometric analysis of cell suspensions) are acceptable alternatives to manual evaluation if appropriately justified and validated relative to classical microscopic scoring.(12)
VII. Procedure for Species Other than Rats and Mice
Published information based on studies in mice, rats, hamsters, swine, dogs, nonhuman primates, and humans(3),(6),(18),(28),(31),(32),(39),(40),(45) indicate that spontaneous and induced frequencies of micronucleated erythrocytes are similar in most mammalian species, and suggests that measurement of the incidence of micronucleated immature erythrocytes in the bone marrow is appropriate for assessing chromosomal or spindle damage in those species studied to date. Appearance and disappearance of micronucleated erythrocytes in the bone marrow is a function of the kinetics of erythrogenesis and the lifespan of erythrocytes in each species, and therefore dosing and sampling regimens must be modified in accordance with the appropriate parameters of erythrocyte kinetics for each species. Species other than mice or rats may be used when appropriate, but the following information should be included:
- Justification of the selected species, and the dosing and sampling schedules used in relation to the kinetics of erythropoiesis and the lifespan of erythrocytes in the species used;
- Evidence that the spontaneous micronucleus frequency is consistent with published information, and/or consistent within the laboratory conducting the study;
- Evidence that known genotoxicants produce an increase in micronucleus frequency in the species used, and reference values for the magnitude of the response induced;
- Impact of splenic selective removal of micronucleated cells from peripheral blood (when the latter serves as the tissue being monitored).
VIII. Data and Reporting
A. Treatment Results
Individual animal data should be presented in tabular form. The experimental unit is the animal. The number of immature erythrocytes scored, the number of micronucleated immature erythrocytes, and the number of immature among total erythrocytes should be listed separately for each animal analyzed. When animals are treated continuously for 4 weeks or more, the data on mature erythrocytes should also be given if it is collected. The proportion of immature among total erythrocytes and, if considered applicable, the percentage of micronucleated mature erythrocytes should be given for each animal. If there is no evidence for a difference in response between the sexes, the data from both sexes may be combined for statistical analysis.
B. Evaluation and Interpretation of Results
There are several criteria for determining a positive result, such as a dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. Statistical methods should be used to evaluate the test results.(24),(35) The statistical criteria for a positive, negative, or equivocal result should be stated clearly in the protocol. Since biological factors may modify the interpretation, statistical significance should not be the only determining factor for reaching a conclusion. Equivocal results should be clarified by further testing, using a modification of experimental conditions if appropriate.
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 in the micronucleus test indicate that a substance induces micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species. Negative results indicate that, under the test conditions, the test substance does not produce chromosomal or spindle damage leading to the formation of micronuclei in the immature erythrocytes of the test species.
The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g., systemic toxicity) should be discussed. The demonstration of adequate target tissue exposure in a negative micronucleus assay is a particularly important consideration when there is positive evidence of genotoxicity in one or more other test systems.
C. Test Report
The test report should also 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 vehicle
- solubility and stability of the test substance in the solvent/vehicle, if known
3. Dosing solutions
- times dosing solutions were prepared and used (or interval between preparation and usage), and storage conditions
- data that verifies the concentration of the dosing solution, if available
4. Test animals
- species/strain used, including justification
- number, age and sex of animals
- source, housing conditions, diet, etc.
- individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group
- information regarding the potential influence of splenic selection on the incidence of micronucleated cells in the peripheral blood, if applicable
5. Test Conditions
- positive and negative (vehicle/solvent) control data
- data from range-finding study, if conducted
- rationale for dose level selection
- details of test substance preparation
- details of the administration of the test substance
- rationale for route of administration and dosing regimen
- methods for verifying that the test substance reached the general circulation and/or target tissue, if applicable
- conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable
- details of food and water quality
- detailed description of treatment and sampling schedules
- methods of slide preparation
- methods for measurement of toxicity
- criteria for scoring micronucleated immature erythrocytes and, if appropriate and applicable, mature erythrocytes
- number of cells analyzed per animal
- criteria for considering studies as positive, negative or equivocal
- signs of toxicity
- proportion of immature erythrocytes among total erythrocytes
- number of micronucleated immature erythrocytes among total immature erythrocytes, given separately for each animal
- if appropriate and applicable, number of micronucleated mature erythrocytes among total mature erythrocytes, given separately for each animal
- mean ± standard deviation of micronucleated immature and, if applicable, mature erythrocytes per group
- dose-response relationship, where possible
- statistical analyses and justification for method applied, with appropriate literature citation
- concurrent and historical negative control data
- concurrent and historical positive control data
7. Discussion of the Results
XI. Addendum: Identification of Micronuclei Derived from Acentric Fragments vs. Centromeric Chromosomes
Micronuclei can be formed by acentric fragments or entire chromosomes lagging in mitosis. These latter micronuclei were first recognized by their large size,(49) by C-banding(47) or by measurement of DNA content.(46) However, these methods were not very reliable. Therefore, two molecular cytogenetic methods were developed to identify the presence of centromeres in micronuclei and thereby differentiate between micronuclei of clastogenic and aneugenic origin:(12) 1) immunofluorescent CREST-staining and 2) fluorescence in situ hybridization (FISH) with pancentromeric DNA-probes. When it is mechanistically important to determine the presence of the kinetochore or centromere in the micronuclei, these methods can be applied.
The CREST method applied to the bone marrow micronucleus test is described in detail by Miller and Adler.(33) Cells on slides (normal bone marrow smears) are fixed, dehydrated, incubated in two steps with SDS and Triton-X, and then stained with antibody. DNA is counterstained by Hoechst 33258.
With FISH, the minor satellite DNA-probe which hybridizes close to the centromere(43) is used to identify the centromeric region, if present. A method for FISH with the centromeric DNA-probes has been described by Pinkel et al.(34) This method can be applied to flow sorted micronuclei-containing erythrocytes(10) or to isolated micronuclei obtained from peripheral blood samples.(13)
In control slides, the rate of labeled micronuclei containing the centromeric region is about 50%.(43) Approximately 70% of micronuclei induced by known aneugens (colchicine and vinblastine) are labeled,(33) whereas those induced by clastogens (hydroquinone and mitomycin C) only show 5-15% labeled micronuclei.(33) To characterize the relative clastogenic vs aneugenic activity of a chemical, it is useful to use as an index the number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes that contain the centromeric region.(42)
The main deficiency in the FISH methods described to date is that they do not differentiate between normochromatic and polychromatic erythrocytes.(12) Thus, only preparations in which a large fraction of the micronuclei present are induced by the test article are suitable for analysis. For example, peripheral blood samples from experiments with acute exposures in adult animals are essentially never suitable for analysis because the target cell population (immature erythrocytes) is only 3-5% of the erythrocytes in the sample.
In conclusion, CREST- or FISH-labeling are considered reliable methods to detect aneugenic properties of chemicals in the in vivo micronucleus assay, provided attention is paid to ensuring that appropriate samples are used for analysis.(12) However, the complexity of the current methods limits their use to those cases in which a chemical is suspected of causing spindle impairment (e.g., due to the presence of large micronuclei, the induction of polyploidy, etc.) or when there are other specific reasons to obtain this mechanistic information.
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