DRAFT OECD GUIDELINE FOR THE TESTING OF CHEMICALS

Rodent alkaline single gel ()

INTRODUCTION

1. OECD Test Guidelines (TGs) are available for a wide range of 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 d u r i n g the MHLW (the Ministry of Health, Labour and Welfare) -sponsored validation study conducted be t we en 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

1 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 6. The Comet assay is a simple method for measuring DNA strand breaks in eukaryotic cells. Cells embedded in on a slide are lysed with and high to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling , 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

2 7/11/2012 7:38 PM 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.

3 7/11/2012 7:38 PM 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

4 7/11/2012 7:38 PM 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

5 7/11/2012 7:38 PM 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

6 7/11/2012 7:38 PM 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

8 7/11/2012 7:38 PM 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

9 7/11/2012 7:38 PM 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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37. Animals will be humanely killed at the appropriate time (or times) after the last treatment with a test substance, consistent with animal welfare and 3Rs. Tissues removed will be placed into ice-cold mincing buffer, rinsed sufficiently with the cold mincing buffer to remove residual blood, and stored on ice until processed. For histopathology in case of positive findings, samples will be obtained and fixed (45). . 38. Single cell/nuclei preparations should be made within one hour after animal sacrifice. The liver and the stomach, as examples, should be processed as follows:

Liver: A portion of the left lateral lobe of the liver should be removed and washed in the cold mincing buffer until as much blood as possible has been removed. The portion should be minced with a pair of fine scissors to release the cells. The cell suspension should be stored on ice to allow large clumps to settle (or, the cell suspension should be strained through a Cell Strainer to remove lumps and the remaining suspension should be placed on ice), and the supernatant should be used to prepare comet slides.

Stomach: The stomach should be cut open and washed free from food using cold mincing buffer. The forestomach should be removed and discarded. The glandular stomach should then be placed into cold mincing buffer and incubated on ice for from 15 to 30 minutes. After incubation, the surface epithelia should be gently scraped two times using a scalpel blade or a Teflon scraper. This layer should be discarded and the gastric mucosa rinsed with the cold mincing buffer. The stomach epithelia should be carefully scraped 4-5 times (or more, if necessary) with a scalpel blade or Teflon scraper to release the cells. The cell suspension should be stored on ice to allow large clumps to settle (or, the cell suspension should be strained with a Cell Strainer to remove clumps and the remaining suspension should be placed on ice), and samples of the supernatant used to prepare comet slides.

39. Slide preparation should be done within one hour after single cell/nuclei preparation. Comet slides should be prepared using laboratory specific procedures. The volume of the cell suspension added to 0.50% low melting point agarose to make the slides should not decrease the percentage of low melting point agarose by more than 10% (i.e., not below 0.45%) .

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40. Once prepared, the slides should be immersed in chilled lysing solution overnight in a refrigerator under a light proof condition. After this incubation period, the slides should be rinsed in purified water or neutralization solution to remove residual detergent and prior to the alkali unwinding step.

Unwinding and electrophoresis

41. Slides should be randomly placed onto the platform of a submarine-type electrophoresis unit and the electrophoresis solution added. A balanced design should be used. The electrophoresis solution should be poured until the surfaces of the slides are completely covered with the solution. The slides should be left to unwind for 20 minutes. The slides should be subjected to electrophoresis under controlled conditions, e.g., at 0.7 V/cm during at least 20 minutes, with a constant voltage at approximately 300 mA. The current at the start and end of the electrophoresis period should be recorded. The temperature of the electrophoresis solution through unwinding and electrophoresis should be maintained at a constant temperature, usually <10oC. The temperature of the electrophoresis solution at the start of unwinding, the start of electrophoresis, and the end of electrophoresis should be recorded.

42. After completion of electrophoresis, the slides should be immersed in the neutralization buffer for at least 5 minutes. All slides should be dehydrated by immersion into absolute ethanol for at least 5 minutes if slides will not be scored soon, allowed to air dry, and then stored until scored at room temperature. Once scored, slides should be retained and stored under low humidity conditions (e.g., in a desiccator) for potential rescoring.

Methods of Measurement

43. Coded slides will be scored. The slides will be stained with nucleic acid specific fluorochrome. The comets should be measured via a digital (e.g. CCD) camera linked to an image analyzer system using a fluorescence microscope at suitable magnification, e.g., 200X. For each sample (animal/tissue), fifty nuclei per slide should be analyzed, with 2 slides scored per sample when five animals per group are used. Approximately 10-areas/slide should be observed at 5 cells or less/field, taking care to avoid any selection bias, overlap counting of cells, and edge areas of slides.

12 7/11/2012 7:38 PM 44. Heavily damaged cells (commonly referred to as “hedgehogs”) that exhibit a microscopic image consisting of a small or non-existent head, and large diffuse tails, should be excluded from data collection if the image analysis system cannot properly score them. However, the frequency of such comets should be determined per sample, based on the visual scoring of 100 cells per sample.

45. The comet endpoints collected should be % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode, and, if possible for a particular image analysis system, tail moment.

DATA AND REPORTING

Treatment of Results

46. Individual animal data should be presented in tabular form. The experimental unit is the animal. The report should include the individual % tail DNA for each tissue from each animal.

Evaluation and Interpretation of Results

47. Negative control values should be well controlled in each experiment, and the data acceptance criteria to judge acceptability of each assay should be set in advance based on historical negative control values. Recommended means of % tail DNA in negative control groups are 1-8% in the liver and 1-20% in the stomach based on data collected in the JaCVAM validation trial. Mean of negative control values below 1% are not recommended because such low values often mislead interpretation of Comet assay results (45) and do not allow for the detection of cross-linking agents.

48. There are several criteria for determining a positive result, such as a dose-related increase in the % tail DNA or a clear increase in the % tail DNA in a single dose group compared to the solvent/vehicle control group. Except where a limit test is performed, at least three treated dose groups should be analysed in order to provide sufficient data for dose-response analysis. Consideration of whether the observed values are within or outside of the historical control range can provide guidance when evaluating the biological significance of the response (55). While biological relevance of the results should be the primary consideration, appropriate statistical methods may be used as an aid in evaluating the test

13 7/11/2012 7:38 PM results (51)(52)(53)(54). Statistical tests used should consider the animal as the experimental unit.

49. A test substance for which the results do not meet the above criteria in any tissue is considered non-genotoxic in this assay.

50. For biological relevance of a positive result, cytotoxicity at the target tissue should also be discussed. Histopathological information can help in the interpretation of positive result in the Comet assay. Whilst low or moderate cytotoxicity is often seen with known genotoxins, severe cytotoxicity (seen through histopathology) might be expected to increase % tail DNA in the liver but might decrease it in the stomach. It is clear that any changes in % tail DNA would require very careful interpretation if they were seen alongside severe cytotoxic changes observed through histopathology (45, 56). Careful interpretation of increased or decreased % tail DNA in the presence of severe cytotoxicity is therefore essential.

51. Positive results in the Comet assay indicate that the test substance induces DNA damage in terms of DNA single strand breaks in the specific organs of the test species. Negative results indicate that, under the test conditions, the test substance does not induce DNA damage in terms of DNA single strand breaks in the specific organs of the test species.

Test Report

52. The test report should include the following information:

Test substance: - identification data and CAS RN, if known; - source, lot number if available; - 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 vehicle; - solubility and stability of the test substance in the solvent/vehicle, if known; - preparation of dietary, drinking water or inhalation formulations;

14 7/11/2012 7:38 PM - analytical determinations on formulations (e.g., stability, homogeneity, nominal concentrations);

Test animals: - species/strain used and justification for the choice; - 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;

Test conditions: - positive and negative (vehicle/solvent) control data; - data from the range-finding study; - rationale for dose level selection; - details of test substance preparation; - details of the administration of the test substance; - rationale for route of administration; - methods for sample preparation, where available, histopathological analyses, especially for the positive chemical - methods for verifying that the test substance reached the target tissue, or general circulation, if negative results are obtained; - actual dose (mg/kg body weight/day) calculated from diet/drinking water test substance concentration (ppm) and consumption, if applicable; - details of food and water quality; - detailed description of treatment and sampling schedules and justifications for the choices; - method of euthanasia; - procedures for isolating and preserving tissues; - methods for single cell/nucleus suspension; - source and lot numbers of all reagents (where applicable); - methods for enumeration of parameters of the assay; and - electrophoresis conditions:

Results: - animal condition prior to and throughout the test period, including signs of toxicity; - body and organ weights at sacrifice;

15 7/11/2012 7:38 PM - dose-response relationship, where possible; - for each tissue/animal, the % tail DNA and mean values; - concurrent and historical negative control data with ranges, means and standard deviations; - concurrent positive control (or non-concurrent DNA positive control) data; - statistical analyses and methods applied;

Discussion of the results

Conclusion

LITERATURE

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(16) Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P, Collins A, Smith A, Speit G, Thybaud V, Tice RR, Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop., Mutagenesis. 2003;18:45-51.

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(18) Klaude M, Eriksson S, Nygren J, Ahnström G., The comet assay: mechanisms and technical considerations., Mutat Res. 1996;363:89-96.

(19) Olive PL., The role of DNA single- and double-strand breaks in cell killing by ., Radiat Res. 1998;150(5 Suppl):S42-51.

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(23) Rothfuss A, Honma M, Czich A, Aardema MJ, Burlinson B, Galloway S, Hamada S, Kirkland D, Heflich RH, Howe J, Nakajima M, O'Donovan M, Plappert-Helbig U, Priestley C, Recio L, Schuler M, Uno Y, Martus HJ. Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing. Mutat Res. 2011; 723(2):108-20.

(24) ICH S2(R1)

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(25) S. Kushwaha, D.N. Tripathi, A. Vikram, P. Ramarao, G.B. Jena, Evaluation of multi-organ DNA damage by comet assay from 28 days repeated dose oral toxicity test in mice: A practical approach for test integration in regulatory toxicity testing, Regulatory Toxicology and Pharmacology 58 (2010) 145–154

(26) Recio L, Hobbs C, Caspary W, Witt KL., Dose-response assessment of four genotoxic chemicals in a combined mouse and rat micronucleus (MN) and Comet assay protocol., J Toxicol Sci. 2010;35:149-62.

(27) Rothfuss A, et al., 2012

(28) Vasquez MZ., Combining the in vivo comet and micronucleus assays: a practical approach to genotoxicity testing and data interpretation., Mutagenesis. 2010;25:187-99.

(29) Sasaki YF, Sekihashi K, Izumiyama F, Nishidate E, Saga A, Ishida K, Tsuda S. The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP Carcinogenicity Database. Crit. Rev. Toxicol. 2000; 30:629-799.

(30) Sekihashi K, Yamamoto A, Matsumura Y, Ueno S, Watanabe-Akanuma M, Kassie F, Knasmüller S, Tsuda S, Sasaki YF. Comparative investigations of multiple organs of mice and rats in the comet assay. Mutat. Res. 2002; 517:53-74.

(31) Nesslany F, Zennouche N, Simar-Meintières S, Talahari I, NKili-Mboui EN, Marzin D. In vivo Comet assay on isolated kidney cells to distinguish genotoxic carcinogens from epigenetic carcinogens or cytotoxic compounds. Mutat. Res. 2007; 630:28-41.

(32) Brendler-Schwaab SY, Herbold BA., A new method for the enrichment of single renal proximal tubular cells and their first use in the comet assay., Mutat Res. 1997;393:175-8.

(33) Naya M, Kobayashi N, Endoh S, Maru J, Honda K, Ema M, Tanaka J, Fukumuro M, Hasegawa K, Nakajima M, Hayashi M, Nakanishi J., In vivo genotoxicity study of single-wall carbon nanotubes using comet assay following intratracheal instillation in rats., Regul Toxicol Pharmacol. 2012;64:124-129.

19 7/11/2012 7:38 PM (34) Toyoizumi T, Ohta R, Nakagawa Y, Tazura Y, Kuwagata M, Noguchi S, Yamakage K., Use of the in vivo skin comet assay to evaluate the DNA-damaging potential of chemicals applied to the skin., Mutat Res. 2011;726:175-80.

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(40) Nakajima M, Ueda M, Yamakage K, Nakagawa Y, Nakagawa M, Ohyama W, Omori T, Asano N, Hayashi M, Uno Y. Tissue sample preparation for in vivo rodent alkaline comet assay. Genes Environ. 2012; 34:50-4.

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20 7/11/2012 7:38 PM (43) Lykke Forchhammer, Clara Johansson, Steffen Loft, Lennart Möller, Roger W. L. Godschalk, Sabine A. S. Langie, George D. D. Jones, Rachel W. L. Kwok, Andrew R. Collins, Amaya Azqueta, David H. Phillips, Osman Sozeri, Maciej Stępnik, Jadwiga Palus, Ulla Vogel, Håkan Wallin, Michael N. Routledge, Catherine Handforth, Alessandra Allione, Giuseppe Matullo, João Paulo Teixeira, Solange Costa, Patrizia Riso, Marisa Porrini, and Peter Møller, Variation in the measurement of DNA damage by comet assay measured by the ECVAG† inter-laboratory validation trial, Mutagenesis (2010) 25: 113-123.

(44) Wiklund SJ, E Agurell., Aspects of design and statistical analysis in the Comet assay. Mutagenesis 18(2):167-175, 2003.

(45) JaCVAM

(46) OECD TG488

(47) OECD TG474

(48) Comet Atlas

(49) Rothfuss A, O'Donovan M, De Boeck M, Brault D, Czich A, Custer L, Hamada S, Plappert-Helbig U, Hayashi M, Howe J, Kraynak AR, van der Leede BJ, Nakajima M, Priestley C, Thybaud V, Saigo K, Sawant S, Shi J, Storer R, Struwe M, Vock E, Galloway S., Collaborative study on fifteen compounds in the rat-liver Comet assay integrated into 2- and 4-week repeat-dose studies., Mutat Res. 2010;702:40-69.

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(51) Lovell DP, G Thomas G, R Dubow., Issues related to the experimental design and subsequent statistical analysis of in vivo and in vitro comet studies. Teratog Carcinog Mutagen. 19(2), 109-119, 1999.

(52) Wiklund SJ, E Agurell., Aspects of design and statistical analysis in the Comet assay. Mutagenesis 18:167-175, 2003.

21 7/11/2012 7:38 PM (53) Bright J, Aylott M, Bate S, Geys H, Jarvis P, Saul J, Vonk R., Recommendations on the statistical analysis of the Comet assay., Pharm Stat. 2011;10:485-93.

(54) David P. Lovell and Takashi Omori, Statistical issues in the use of the comet assay, Mutagenesis (2008) 23: 171-182

(55) Hayashi M, Dearfield K, Kasper P, Lovell D, Martus HJ, Thybaud V. Compilation and use of genetic toxicity historical control data. Mutat. Res. 2011; 723(2):87-90.

(56) Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, Escobar P, Honma M, Kumaravel TS, Nakajima M, Sasaki YF, Thybaud V, Uno Y, Vasquez M, Hartmann A. Fourth International Workgroup on Genotoxicity Testing: result of the in vivo comet assay workgroup. Mutat. Res. 2007; 627:31-5. (57) EFSA Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment, EFSA Journal 9 (2011) 2379; www.efsa.europa.eu/efsajournal (58) UK COM guidelines – www.iacom.org.uk/guidstate/index.htm

Additional list:

Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, Escobar P, Honma M, Kumaravel TS, Nakajima M, Sasaki YF, Thybaud V, Uno Y, Vasquez M, Hartmann A, Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup., Mutat Res. 2007;627:31-5.

Andreas Hartmann, Martin Schumacher, Ulla Plappert‐Helbig, Phil Lowe, Willi Suter, and Lutz Mueller, Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations, Mutagenesis (2004) 19: 51-59

Kirkland D, Speit G. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens III. Appropriate follow-up testing in vivo. Mutat. Res. 2008; 654:114-132.

Lovell DP, G Thomas G, R Dubow., Issues related to the experimental design and subsequent

22 7/11/2012 7:38 PM statistical analysis of in vivo and in vitro comet studies. Teratog Carcinog Mutagen. 19, 109-119, 1999.

Merk O, Speit G., Detection of crosslinks with the comet assay in relationship to genotoxicity and cytotoxicity., Environ Mol Mutagen. 1999;33:167-72.

Nakajima M, Ueda M, Yamakage K, Nakagawa Y, Nakagawa M, Ohyama W, Omori T, Asano N, Hayashi M, Uno Y. Tissue sample preparation for in vivo rodent alkaline comet assay. Genes Environ. 2012; 34:50-4.

Nesslany F, Zennouche N, Simar-Meintières S, Talahari I, NKili-Mboui EN, Marzin D. In vivo Comet assay on isolated kidney cells to distinguish genotoxic carcinogens from epigenetic carcinogens or cytotoxic compounds. Mutat. Res. 2007; 630:28-41.

Sasaki YF, Sekihashi K, Izumiyama F, Nishidate E, Saga A, Ishida K, Tsuda S., The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP Carcinogenicity Database., Crit Rev Toxicol. 2000;30:629-799.

Sekihashi, K., A. Yamamoto, Y. Matsumura, S. Ueno, M. Watanabe-Akanuma, F. Kassie, S. Knasmuller, S. Tsuda, Y.F. Sasaki, Comparative investigation of multiple organs of mice and rats in the comet assay, Mutat Res., 517, 53-74, 2002.

23 7/11/2012 7:38 PM Annex 1 DEFINITIONS

Alkaline single cell gel electrophoresis: Comet: Predictivity: UDS: % tail DNA

Annex 2 Solutions

The following are examples which were used in JaCVAM trials. Other preparation methods are acceptable if justified. 1) Agarose 1.0-1.5% (w/v) standard agarose gel for the bottom layer (if used) Regular melting agarose will be dissolved at 1.0-1.5% (w/v) in Dulbecco’s phosphate buffer (Ca++, Mg++ free and phenol free) by heating in a microwave. 0.5 % (w/v) low-melting agarose (Lonza, NuSieve GTG Agarose) gel for the cell-containing layer and, if used, a top layer Low-melting agarose will be dissolved at 0.5% (w/v) in Dulbecco’s phosphate buffer (Ca++, Mg++ free and phenol free) by heating in a microwave. During the study this solution will be kept at 37-45°C and discarded afterward.

2) Lysing solution The lysing solution will consist of 100 mM EDTA (disodium), 2.5 M sodium chloride, and 10 mM tris hydroxymethyl aminomethane in purified water, with the pH adjusted to 10.0 with 1 M sodium hydroxide and/or hydrochloric acid. This solution may be refrigerated at <10C until use. On the same day of use, 1 % (v/v) of triton-X100 and 10 % (v/v) DMSO will be added to this solution and the complete lysing solution will be refrigerated at <10C for at least 30 minutes prior to use.

3) Alkaline solution for unwinding and electrophoresis The alkaline solution consists of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water, pH >13. This solution will be refrigerated at <10C until use. The pH of the solution will be measured just prior to use.

4) Neutralization solution

24 7/11/2012 7:38 PM The neutralization solution consists of 0.4 M tris hydroxymethyl aminomethane in purified water, pH 7.5. This solution will be either refrigerated at <10C or stored consistent with manufacturer’s specifications until use.

5) Mincing buffer The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO will be added immediately before use). This solution will be refrigerated at <10C until use.

6) solution The fluorescent DNA stain is SYBR Gold (Invitrogen-Molecular Probes), prepared and used according to the manufacturer’s specifications.

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