Table Of ContentDRAFT OECD GUIDELINE FOR THE TESTING OF CHEMICALS
Rodent alkaline single cell gel electrophoresis (Comet) assay
INTRODUCTION
1. OECD Test Guidelines (TGs) are available for a wide range of in vitro genotoxicity assays
that are able to detect DNA damage, gene mutation and/or chromosomal aberrations. There
are TGs for in vivo endpoints (i.e. chromosomal aberrations, gene mutations and DNA
repair as unscheduled DNA synthesis); however, these do not directly measure DNA
damage. The alkaline single cell gel electrophoresis (Comet) assay fulfills the need for
practical and widely available in vivo tests for measurement of DNA damage induction in
multiple tissues.
2. The Comet assay has been reviewed and recommendations have been published by various
international groups (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11). The initial event of a
mutation is modification of DNA. Failure to repair or error-prone repair of modified DNA
can lead to DNA strand breakage, and the Comet assay is able to visualize the strand breaks
and alkali-labile lesions by alkaline treatment to single strand DNA followed by
electrophoresis. Based on the molecular size, undamaged DNA remains in the nucleus
during electrophoresis but smaller fragments move from the nucleus and assume the shape
of a comet (the nucleus) with a tail.
3. Despite the international recommendations referred to above, a formal validation of the in
vivo rodent Comet assay was not undertaken until recently. This Test Guideline is
therefore based on the Comet assay method protocol (12), which was developed
during the MHLW (the Ministry of Health, Labour and Welfare) -sponsored validation
study conducted between 2006 and 2012. This validation study was coordinated by the
Japanese Center for the Validation of Alternative Methods (JaCVAM), in conjunction with
the European Centre for the Validation of Alternative Methods (ECVAM), the Interagency
Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the
Korean Center for the Validation of Alternative Methods (KOCVAM).
4. The Comet assay method was evaluated by the OECD coordinated peer review panel
supported by JaCVAM. This Test Guideline includes the recommended use and limitations
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of the Comet assay method based on these evaluations.
5. Definitions of key terms are set out in the Annex.
INITIAL CONSIDERATIONS
6. The Comet assay is a simple method for measuring DNA strand breaks in eukaryotic cells.
Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to
form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix.
Electrophoresis at high pH results in structures resembling comets, observed by
fluorescence microscopy; the intensity of the comet tail relative to the head reflects the
number of DNA breaks and the size of the resulting fragments (12) (13) (14).
7. These rodent in vivo Comet assays are especially relevant to assessing genotoxic hazard in
that the assays’ responses are dependent upon in vivo metabolism, pharmacokinetics, DNA
repair processes, and translesion DNA synthesis, although these may vary among species,
among tissues and among the types of DNA damage. The DNA lesion is the initial event of
gene mutation and/or chromosomal aberrations, and an in vivo assay for DNA damage is
therefore useful for further investigation of genotoxic effects detected by an in vitro
system, and for following up results of tests using other in vivo endpoints. Whilst TG 488
(46) and TG 474 (47) are useful follow-up in vivo tests for chemicals inducing gene
mutations or chromosomal aberrations in vitro, respectively, the Comet assay is a useful in
vivo follow-up for chemicals inducing both gene mutations and chromosomal aberrations in
vitro.
8. If there is evidence that the test substance, or a relevant metabolite, will not reach any of
the tissues of interest, it is not appropriate to perform an in vivo Comet assay.
9. The Comet assay has applications in testing new chemicals for genotoxicity, monitoring
environmental contamination with genotoxins, human biomonitoring for occupational
exposure and molecular epidemiology, and fundamental research in DNA damage and
repair (14) (15) (16). Several modifications, e.g, neutral or enzyme method have been
developed (15) (17) (18) (19) (20) (21). However, this guideline focuses on the alkaline
assay for assessing DNA-damaging potential of chemicals for regulatory purposes.
10. Recent strategies for genotoxicity testing have suggested that the Comet assay would, as a
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second in vivo assay, be preferable to the rodent liver UDS assay in genotoxicity
evaluation of chemicals for regulatory purposes (22) (23) (24). A single study can combine
the analysis of micronuclei in erythrocytes with the Comet assay in appropriately selected
tissues (22) (23) (25) (26) (27) (28). The selected tissue(s) will be determined by
knowledge of metabolism and distribution, potential for site-of-contact effects, or
knowledge of the mechanism of genotoxicity/carcinogenicity.
11. Only the liver and stomach were used in the international validation study, because the liver
is the most active organ to metabolize chemicals and also most frequently a target for
carcinogenicity, and the stomach is usually first site of contact for chemicals after oral
exposure, and therefore, this guideline is constructed based on data generated with these
two tissues. However, the technique is equally applicable to any tissues from which single
cell or nuclei suspensions can be made. Therefore any tissues from chemically treated
animals can be investigated based on existing knowledge of the chemical or purpose of the
study. Thus the genotoxic potential of the chemical can be assayed in the target organs of
carcinogenic and/or other toxic effects. Such information is important for the risk
characterization of the chemical. However the use of tissues other than liver and stomach
should be justified.
12. There are many publications of the Comet assay using organs other than liver and stomach,
e.g., multiple organs (29) (30), kidney (31) (32), trachea (33), skin (34), urinary bladder
(35) (36), or testis/sperm (20) (37) (38). It has been shown that there was no essential
difference in the assay result whether isolated cells or nuclei were used (39). Also the
method to isolate cells/nuclei (e.g., homogenizing, mincing and mesh filtration) did not
give different results (40). The effects on DNA migration of altering other aspects of the
methodology have been also investigated (41) (42) (43).
13. There are several endpoints for the Comet assay but the DNA content in the tail (% tail
DNA) is recommended to assess DNA damage (16). After analysis of sufficient nuclei, the
data are analyzed with appropriate methods to judge the assay results, e.g., in the JaCVAM
validation study the mean of percent tail DNA was statistically compared with that of the
vehicle control group to assess the genotoxicity of the test chemical (45). It should be noted
that fragmentation of the DNA can be induced not only by chemical genotoxicity but also
during the process of cell death, i.e., apoptosis and necrosis. Sometimes, however, it is
difficult to distinguish between genotoxicity and apoptosis/necrosis by the shape of nucleus
and comet tail after electrophoresis, and investigators have to make a subjective decision.
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To help with such subjective decisions, an atlas of the Comet assay with many examples
has been published (48).
14. Sufficient data are available to support the use of the Comet assay in any organs of treated
animals. The method has been shown to be reproducible within and between laboratories,
to apply to the intended domain (i.e. detection of DNA damage), and to be appropriately
predictive of the carcinogenicity of test chemicals.
15. This assay is recommended as an in vivo genotoxicity method in the ICH S2(R1) (24) for
pharmaceutical drugs for human use, and is also recommended by EFSA (57) and UK
COM (58). The method, although without any standard methodological document, has
been widely used, including for regulatory purposes, for many years to assess genotoxicity
in vivo.
PRINCIPLE OF THE METHOD
16. Animals are treated with a test chemical through an appropriate administration route once,
twice or three times separated by 24 h intervals, or they can be treated for even longer
period, e.g., for 28 days (49)(23). Animals are killed and tissues are sampled at 2 to 6 hours
after the last treatment of two or more daily administrations, or at both 2-6 and 16-26 hours
after a single administration (16). Organs of interest are dissected and free cell/nucleus
suspensions are prepared. The use of an early sampling time is essential because the lesions
in the DNA are repaired quickly and efficiently. To fulfill animal welfare requirements, this
assay can be incorporated into other toxicological studies, e.g., repeated dose toxicity study,
or the endpoint can be combined with other genotoxicity endpoints, e.g., with the rodent
micronucleus assay (25)(26)(49).
17. Single cell and/or nucleus suspensions from the tissues of interest are embedded in soft
agar to so as to immobilize them on glass slides. To remover cellular and/or nuclear
membrane, cells/nuclei are treated with detergent to produce naked nuclei. DNA strand
breaks and alkali-labile lesions form smaller DNA fragments when treated with strong
alkali e.g., pH13 or above. The nuclei in the agar on glass slide are then subjected to
electrophoresis. By adjusting the voltage, current, and electrophoresis period, normal DNA
molecules remain in the position where the nucleus had been on the slide, while smaller
fragments migrate towards the anode, and the extent of migration is determined by the size
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of the fragments.
18. After electrophoresis, the DNA is visualized using a fluorescent nucleic acid-specific stain
e.g., SYBR Gold, Green I, or ethidium bromide, followed by image analysis under a
fluorescent microscope. Images are analyzed by an image-analyzer to measure several
parameters, e.g., length of tail, DNA content in the tail, and from these other measures such
as tail moment can be calculated (50). The most frequently used parameter, and
recommended by the validation study, is DNA content in the tail (% DNA in tail) although
other parameters can also be used. After analysis of nuclei from control and treated groups,
histograms of the measured parameters from each cell are made so as to provide an
overview of the effect of treatment. The mean (or possibly median) value of % tail DNA
represents treatment to be compared with that of negative control.
DESCRIPTION OF THE METHOD
Preparations
Selection of animal species
19. Commonly used laboratory strains of healthy young adult animals should be used. Rats are
most often the experimental animals of choice as they are most commonly used in toxicity
assessment of chemicals, although mice can be also selected. Rats may be considered as the
first choice in order to correlate data with that from a rat toxicity study, or to investigate the
mechanism of carcinogenesis for a tumour seen in rats, and are relevant if rat metabolism is
known to be representative of human metabolism. Other animal species including human
beings can theoretically and practically be used if justified, but this TG is focused on
rodents.
Housing and feeding conditions
20. The temperature in the experimental animal room ideally should be 22oC (±3oC). Although
the relative humidity should be at least 30% and preferably not exceed 70% other than
during room cleaning, the goal should be to maintain a relative humidity of 50-60%.
Lighting should be artificial, with a daily sequence of 12 hours light, followed by 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
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admixture of a test substance when administered by this route. Animals should be housed in
small groups (no more than five) of the same sex if no aggressive behavior is expected.
Animals may be housed individually if scientifically justified.
Preparation of the animals
21. Healthy young adult animals (usually 6-10 weeks old at start of treatment) are randomly
assigned to the control (negative and positive) and treatment groups. The animals are
identified uniquely. The animals are 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. 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.
Preparation of doses
22. Solid test substances should be dissolved or suspended in appropriate solvents or vehicles
or admixed in diet or drinking water prior to dosing of the animals. Liquid test substances
may be dosed directly or diluted prior to dosing. For inhalation exposures, test materials
can be administered as gas, vapour, or a solid/liquid aerosol, depending on their
physicochemical properties. Fresh preparations of the test substance should be employed
unless stability data demonstrate the acceptability of storage.
Test Conditions
Solvent/vehicle
23. The solvent/vehicle should not produce toxic effects at the dose volumes used, and should
not be suspected of chemical reaction with the test substance. If other than well-known
solvents/vehicles are used, their inclusion should be supported with reference data
indicating their compatibility. It is recommended that wherever possible, the use of an
aqueous solvent/vehicle should be considered first.
Positive controls
24. Concurrent positive control animals should normally be used. When concurrent positive
controls are used, it is not necessary to administer them by the same route as the test
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substance; however, the positive controls should be known to induce DNA lesions in one or
more tissues of interest for the test substance. The doses of the positive control chemicals
should be selected so as to produce weak or moderate effects that critically assess the
performance and sensitivity of the assay. Examples of positive control substances and some
of their target tissues are included in Table 1.
Table 1: Examples of positive control substances and some of their target tissues
Chemicals and CAS No.
Ethyl methanesulfonate (CAS RN 62-50-0) for liver and stomach
Ethyl nitrosourea (CAS RN 759-73-9) for liver and stomach
Methyl methanesulfonate (CAS RN 66-27-3) for liver and stomach
N-Nitrosodimethylamine (CAS RN 62-75-9) for liver
Negative controls
25. Negative controls, treated with solvent or vehicle alone, and otherwise treated in the same
way as the treatment groups, should be included for every sampling time. In the absence of
historical or published control data showing that no deleterious or mutagenic effects are
induced by the chosen solvent/vehicle, untreated or established vehicle controls should also
be included for every sampling time in order to establish acceptability of the vehicle
control.
Verification of laboratory proficiency
26. Competency in these assays should be established by demonstrating the ability to
reproduce expected results from published validation study data (45) for mean % tail DNA
of positive control substances (including weak responses) such as those listed in Table 1,
and vehicle controls. Dose-effect relationships should also be demonstrated, where
appropriate. During the course of these investigations, the laboratory can establish a
historical positive control range and distribution, and a historical negative control range and
distribution. Re-evaluation of laboratory proficiency is recommended if major changes to
the experimental conditions are proposed for the assay.
PROCEDURE
Number and Sex of Animals
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27. The number of animals per group should be predetermined to be sufficient to provide the
statistical power necessary to detect at least a doubling in genotoxic effect. Group sizes will
consist of a minimum of 5 animals; however, if the statistical power is insufficient, the
number of animals should be increased as required. Male animals should normally be used.
There may be cases where testing females alone would be justified; for example, when
testing human female-specific drugs, or when investigating female-specific metabolism. If
there are significant differences between the sexes in terms of toxicity or metabolism, then
both males and females will be required.
Administration Period
28. Based on observations made in the validation study, three daily treatments is generally
considered preferable for producing sufficient sensitivity to detect genotoxic effects,
whilst at the same time allowing the assay to be combined with the micronucleus test.
Alternative treatment regimens may be appropriate for some evaluations, and these
alternative-dosing schedules should be scientifically justified in the protocol. Samples from
extended dose regimens (e.g., 28-day daily dosing) are acceptable as long as a positive
effect has been demonstrated for this study or, for a negative study, as long as toxicity to
the target tissue(s) has been demonstrated or the limit dose has been used, and dosing
continued until the time of sampling. Test substances also may be administered as a split
dose, i.e., two treatments on the same day separated by no more than a few hours, to
facilitate administering a large volume of material, although adjustments may need to be
made to accommodate the early sampling time following the last dose.
Dose Levels
29. If a preliminary range-finding study is performed because there are no suitable data
available to aid in dose selection, it should be performed in the same laboratory, using the
same species, strain, sex, and treatment regimen to be used in the main study. The
range-finding study should be started with the most likely dose to cause toxicity, using a
small number of animals (e.g. 2 per sex). If the MTD is not defined, a further group of
animals should be exposed to a higher or lower dose depending on the clinical effects of
the first dose. This strategy should be repeated until the appropriate MTD is found. The
highest dose level should be chosen with the aim of inducing toxic effects but not death or
severe suffering (i.e., the maximum tolerated dose [MTD]). The highest dose may also be
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defined as a dose that produces toxicity in the target tissue(s).
30. Substances with specific biological activities at low non-toxic doses (such as hormones
and mitogens), and substances that exhibit saturation of toxicokinetic properties may be
exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis.
31. If the test substance produces toxicity, the MTD and a descending sequence of at least
two additional appropriately (less than SQR(10)) spaced dose levels should be selected for
each sampling time, with a view to demonstrating any dose-related response. Studies
intending to more fully characterize the quantitative dose-response information may require
additional dose groups. The dose levels used should preferably cover a range from the
maximum to little or no toxicity.
Limit test
32. If dose range-finding experiments, or existing data from related animal strains, indicate that
a treatment regime of at least the limit dose (described below) produces no observable toxic
effects (including no toxicity in the tissue[s] of interest), and if genotoxicity would not be
expected based upon in vitro genotoxicity studies or data from structurally related
substances, then a full study using three dose levels may not be considered necessary. In
such cases, a single dose of the test substance, at the limit dose, may be sufficient. For an
administration period of 1-3 day(s) daily treatment up to 14 days, the limit dose is 2000
mg/kg body weight/day and of more than 14 days that is 1000 mg/kg/day.
Administration of Doses
33. The test substance is usually administered by gavage using a stomach tube or a suitable
intubation cannula. However, the anticipated route of human exposure should be
considered when designing an assay. Therefore, other routes of exposure (such as, drinking
water, subcutaneous, intravenous, topical, inhalation, intratracheal, dietary, or implantation)
may be acceptable where they can be justified. Intraperitoneal injection is not
recommended since it is not a physiologically relevant route of human exposure. 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 greater than this should be justified. Except for irritating or
corrosive substances, which will normally reveal exacerbated effects at higher
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concentrations, variability in test volume should be minimized by adjusting the
concentration to ensure a constant volume at all dose levels.
Sampling Time
34. The sampling time is a critical variable because it is determined by the period needed for
DNA damage to be induced but before that damage is removed, repaired or leads to cell
death This period may be chemical-specific. A suitable compromise for the measurement of
genotoxicity at 2-6 h after the last treatment for two or more treatments, or at both 2-6 and
16-26 h after a single administration.
Observations
35. General clinical observations should be made at least once a day preferably at the same
time(s) each day and considering the peak period of anticipated effects after dosing. and the
health condition of the animals should be recorded. At least twice daily, all animals should
be observed for morbidity and mortality. For longer duration studies, all animals should be
weighed at least once a week, and at sacrifice. Measurements of food consumption should
be made at least weekly. If the test substance is administered via the drinking water, water
consumption should be measured at each change of water and at least weekly. Animals
exhibiting non-lethal indicators of excess toxicity should be euthanized prior to completion
of the test period.
Tissue Collection
36. The rationale for tissue collection should be defined clearly although the most frequently
used tissues for the Comet assay are liver and glandular stomach or duodenum. Since it is
possible to study genotoxicity induction in virtually any tissue, the selection of tissues to be
collected should be based upon the reason for conducting the study and any existing
genotoxicity, carcinogenicity or toxicity data for the test chemical under investigation.
Important factors for consideration should include the route of administration (based on
likely human exposure route(s)), the predicted tissue distribution, and the possible
mechanism of action. In the absence of any background information, several somatic
tissues as may be of interest should be collected.
Preparation of specimen
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Description:2 7/11/2012 7:38 PM of the Comet assay method based on these evaluations. 5. Definitions of key terms are set out in the Annex. INITIAL CONSIDERATIONS
