Mutagenesis Advance Access originally published online on May 17, 2005
Mutagenesis 2005 20(4):245-254; doi:10.1093/mutage/gei033
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REVIEW |
The in vivo comet assay: use and status in genotoxicity testing
Federal Institute for Drugs and Medical Devices, Bonn, Germany, 1Novartis Pharma AG, Basel, Switzerland, 2Wella AG, Darmstadt, Germany and 3Universität Ulm, Abteilung Humangenetik, D–89069, Ulm, Germany
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Abstract |
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The in vivo comet assay (single cell gel electrophoresis assay) in its alkaline version (pH >13) is being increasingly used in genotoxicity testing of substances such as industrial chemicals, biocides, agrochemicals, food additives and pharmaceuticals. Recommendations for an appropriate performance of the test using OECD guidelines for other in vivo genotoxicity tests have been published. In this review, we critically discuss the biological significance of comet assay effects in general and the status of the test in current strategies for genotoxicity testing. Examples for practical applications of the in vivo comet assay and potential consequences of positive and negative test results are given. The significance of comet assay results for hazard identification and risk assessment is discussed. In accordance with international guidelines for genotoxicity testing the in vivo comet assay is recommended for follow-up testing of positive in vitro findings. It is particularly useful as a tool for the evaluation of local genotoxicity, especially for organs/cell types which cannot easily be evaluated with other standard tests. A positive result in an appropriately performed in vivo comet assay indicates genotoxicity of the test compound in the tissue tested and gains particular significance when a mutagenic potential of the test compound has already been demonstrated in vitro. Such findings will have practical consequences in the risk assessment processes and further development of substances.
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Introduction |
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TopAbstractIntroductionDNA damage detected by...Status of the in...Scenarios for the application...Use of in vivo...References |
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The in vivo comet assay (single cell gel electrophoresis) is increasingly being used in genotoxicity testing. The advantages of the in vivo comet assay include its applicability to various tissues and/or special cell types, its sensitivity for detecting low levels of DNA damage, its requirement for small numbers of cells per sample, general ease of test performance, the short time needed to complete a study and its relatively low cost (1
). Following current OECD guidelines for other in vivo genotoxicity tests, international expert groups have published recommendations describing standards aiming at high quality protocols in order to obtain valid and reliable data (1,2). Therefore, aspects of the comet assay protocol will not be discussed here. However, it has to be emphasized that the quality of the study protocol and the experimental performance is of crucial importance for the scientific and regulatory acceptance of comet assay results. In this paper, the biological significance of comet assay results is discussed, the status of the in vivo comet assay in current strategies for genotoxicity testing is described and examples of current applications of the in vivo comet assay in industrial genotoxicity testing are given. Using these examples, possible consequences of in vivo comet assay results for hazard identification and risk assessment are discussed.
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DNA damage detected by the comet assay |
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The alkaline version (pH >13), which is exclusively discussed here, can be used to detect DNA damage such as strand breaks, alkali-labile sites (ALS), DNA–DNA and DNA–protein crosslinks. In contrast to other DNA alterations, cross-links may stabilize chromosomal DNA and inhibit DNA migration (3
,4). Thus, reduced DNA migration in comparison to the negative control (which should show some degree of DNA migration) may indicate the induction of crosslinks, which are relevant lesions with regard to mutagenesis and should be further investigated. Increased DNA migration indicates the induction of DNA strand breaks and/or ALS. Furthermore, enhanced activity of excision repair may result in increased DNA migration. DNA excision repair can influence comet assay effects in a complex way (5). While DNA repair generally reduces DNA migration by eliminating DNA lesions, ongoing excision repair may increase DNA migration due to incision-related DNA strand breaks. Thus, the contribution of excision repair to the DNA effects seen in the comet assay depends on the types of induced primary DNA damage and the time point of analysis (6). DNA lesions leading to effects in the comet assay can not only be strand breaks, which may be relevant for the formation of chromosome aberrations, but also DNA modifications such as abasic sites (AP sites) with relevance for the induction of gene mutations. However, it has to be recognized that the primary lesions detected by the comet assay may also be correctly repaired without resulting in permanent genetic alterations. Neither the extent of DNA migration in the comet assay nor the shape of the comet can reveal the mode of action or the mutagenic potential of a test substance.
There are only few limitations of the comet assay with regard to its application and interpretation. Short-lived primary DNA lesions such as single strand breaks, which may undergo rapid DNA repair, could be missed when using inadequate sampling times. However, an appropriate study design including one early preparation time point (i.e. at 2–6 h) should ensure that these lesions are captured. A general issue with ‘DNA strand break assays’ such as the comet assay is that indirect mechanisms related to cytotoxicity can lead to positive effects. However, an advantage of the comet assay compared with other techniques analysing DNA damage in tissues (such as the alkaline elution method) is that DNA damage is assessed on the level of individual cells. Thus, dead or dying cells may be identified on microscopic slides by their specific image. Necrotic or apoptotic cells can result in comets with small or non-existent head and large, diffuse tails (7) commonly described as ‘hedgehogs’, ‘ghost cells’, ‘clouds’ or ‘non-detectable cell nuclei’ (NDCN). Experience with the in vitro comet assay shows that such cells can appear upon treatment with cytotoxic, non-genotoxic agents (8–11). However, such microscopic images may also be detected after treatment with high doses of radiation or high concentrations of strong mutagens, and are, therefore, not uniquely diagnostic for apoptosis/necrosis (12). For the in vivo comet assay only limited data are available to establish whether cytotoxicity results in increased DNA migration in tissues of experimental animals. Several in vivo studies with rats did not show elevated DNA migration despite decreased viability, necrosis or apoptosis in target organs such as kidneys (13), testes (14), liver or duodenum (15). In contrast, enhanced DNA migration was observed in homogenized liver tissue of mice dosed with carbon tetrachloride (16). In the latter study, histopathological examination showed evidence of necrosis in the liver and the authors concluded that the increased DNA migration in the comet assay was secondary to cytotoxicity and represented a false positive effect (16). To avoid potential false positive effects resulting from cytotoxicity, specific recommendations regarding a concurrent assessment of cytotoxicity have been made. These include dye viability assays, histopathology and a neutral diffusion assay (1,2).
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Status of the in vivo comet assay within a test battery |
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According to the recommendations of international guidelines, mutagenicity testing is based on a battery of in vitro and in vivo tests aimed at different genetic endpoints. This test battery mainly consists of mutagenicity tests that detect gene, chromosome or genome mutations. Mutations can lead to inherited diseases and are considered an essential step in the multi-step process of carcinogenesis. Therefore, the determination of a mutagenic risk mainly relies on results from mutagenicity tests. Results from indicator tests (i.e. tests measuring effects related to the process of mutagenesis, such as DNA damage, recombination and repair) can provide additional useful information in the context of extended genotoxicity testing.
The comet assay is an indicator test for the detection of DNA damage and is primarily used as a supplemental in vivo test for substances with positive results from in vitro mutagenicity tests and/or for mechanistic studies. The in vivo comet assay has some advantages over other in vivo indicator tests with regulatory acceptance, such as the unscheduled DNA synthesis (UDS) test or the alkaline elution method. The in vivo UDS test is generally performed in liver tissue only, while the comet assay can be applied to virtually any organ of interest provided that an appropriate cell preparation has been established for each organ and cell type. In addition, the comet assay detects a broader spectrum of primary DNA lesions, including single strand breaks and oxidative base damage, which may not sensitively be detected by the UDS test because they are not repaired by nucleotide excision repair (17). In cases where the UDS test has been used for the analysis of first site-of-contact tissue such as stomach epithelia, it seemed to be rather insensitive for detecting direct-acting or short-lived mutagens compared with the comet assay (18). The advantages of the comet assay over the alkaline elution test are the detection of DNA damage on a single cell level and the requirement of only small numbers of cells per sample. In contrast, when using the alkaline elution assay, high amounts of cells are necessary for the determination of genotoxic effects and, therefore, only a limited number of organs/tissues can be evaluated using this technique. As a consequence of its methodological advantages and its increasing acceptance within the scientific and regulatory community, the comet assay is gaining in importance and already has a significant role in genotoxicity testing strategies. Some examples are listed below:
In the second edition of the ‘Technical Guidance Document on Risk Assessment by the European Commission’ (19) different scenarios are outlined for further in vivo genotoxicity testing in cases of positive findings in vitro. Emphasis is laid on test systems that enable the investigation of genotoxicity in specific target tissues. It is argued that in vivo tests should be performed only in cases where an adequate exposure of the target tissue by the test substance and/or its metabolites can be reasonably expected. Consequently, it is proposed to apply genotoxicity tests to a variety of organs for test substances with low systemic availability. Additionally, alternative tests to the commonly used in vivo MNT and the UDS tests should be chosen when it is appropriate to evaluate genotoxicity in systemically exposed tissues other than the bone marrow or liver. Each case should be judiciously considered so that the most appropriate test is used. The in vivo comet assay is mentioned here as one of the main options.
The latest survey document of the German ‘Federal Institute for Risk Assessment’ (BfR) on strategies for genotoxicity testing of substances (20) outlines that in the case of a negative in vitro battery, in vivo tests are not necessary for base set testing with the exception of feed additives and pharmaceuticals. With respect to the in vivo assessment of compounds with positive results in mammalian cells in vitro, the TGD is cited and the above described role of the comet assay is mentioned. In addition, the use of the comet assay is recommended in cases of poor systemic availability, i.e. when in the in vivo MNT the bone marrow may not be sufficiently exposed. It is pointed out that in these cases ‘testing for local genotoxicity may be useful, especially in directly exposed tissues’.
The ‘United Kingdom Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment’ (COM) (21) points out the comet assay's ‘particular value in evaluating directly acting genotoxins at their initial site of contact’. According to the COM strategy, the comet assay can be considered as a Stage 2 test ‘required for compounds that are positive or equivocal in any of the Stage 1 (in vitro) tests to ascertain whether mutagenic activity can be expressed in vivo’. Although not directly mentioned, it is implied that the comet assay can be useful if negative results are obtained with the initial in vivo test and ‘additional testing will be required using other tissue(s) before definite conclusions can be drawn regarding the absence of activity in vivo’. In this context, the comet assay is defined as a test for ‘measurement of induction of DNA lesions, i.e. measure of exposure, uptake and reactivity to DNA’.
The ‘International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use’ (ICH) does not mention the comet assay directly but suggests to use, among others, ‘DNA strand-breakage assays’ for further in vivo testing as an alternative to the in vivo liver UDS test (22).
Taken together, the in vivo comet assay is currently regarded as a useful test for follow-up testing of positive in vitro findings. It is particularly useful as a second in vivo test (e.g. as an alternative to the in vivo UDS test) and a tool for the evaluation of local genotoxicity, especially for organs/cell types which cannot easily be evaluated with other standard tests.
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Scenarios for the application of the in vivo comet assay |
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Besides the applications according to the recommendations mentioned above, there are other situations in which the in vivo comet assay is used in the industrial environment. In mechanistic studies the comet assay is applied in order to distinguish between genotoxic and non-genotoxic modes of action as a cause for neoplastic changes observed in long-term rodent studies. Furthermore, new in vivo comet assay data on commercially available compounds may be published in the scientific literature. In particular, positive results may trigger further comet assay studies and/or other additional genotoxicity testing. Therefore, in an industrial environment, for a useful application of the in vivo comet assay four scenarios seem to be relevant:
- Follow-up testing of positive in vitro findings (second in vivo test subsection);
- Evaluation of local genotoxicity (evaluation of local genotoxicity subsection);
- Studies on the underlying mechanism of neoplastic changes observed in long-term rodent studies (evaluation of genotoxicity contribution subsection); and
- Further evaluation of positive in vivo genotoxicity data from the scientific literature (further evaluation of published positive comet assay data subsection).
These different scenarios are discussed below in the context of applicable examples summarized in Tables I–IV. The second column of each table contains a selection of available genotoxicity data with an emphasis on the most routinely performed assays. It is not intended to provide a review of all genotoxicity data of the respective compound.
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(i) Follow-up testing of positive in vitro genotoxicity findings
The minimal base set for an in vitro testing battery for industrial chemicals (new chemicals and existing chemicals) as well as for agrochemicals, biocides and pharmaceuticals comprises a bacterial gene mutation test and a test capable of detecting chromosomal aberrations in mammalian cells. In addition, for agrochemicals and biocides an in vitro gene mutation test in mammalian cells must be available as well. Positive results in at least one of the in vitro tests trigger in vivo tests. Frequently, an in vivo MNT with bone marrow cells of rodents is performed as the first in vivo test.
Further in vivo testing is usually performed to support a first negative in vivo test in case of (a) positive in vitro test(s). When applying the comet assay as a second in vivo test, it has been proposed to investigate the liver as the major organ for the metabolism of absorbed compounds, as well as one site-of-first-contact tissue (1,2). In the case of industrial chemicals an in vivo comet assay might be recommended to further evaluate target organs.
Table I summarizes examples of datasets for drug candidates for which in vivo comet assay studies were performed as a second in vivo test (15). The first two compounds showed an isolated positive in vitro-result obtained in a clastogenicity test in V79 cells (Chinese hamster cell line). In the case of the first drug, a second chromosome aberration test with human lymphocytes as well as an in vitro MNT in V79 cells were negative, indicating that the positive effect was of limited relevance. In both cases, an in vivo MNT yielded a negative result and so did the in vivo comet assay. Both datasets were accepted by European health authorities, allowing the progression into human trials. The acceptance of negative results in the in vivo comet assay by health authorities demonstrates that the comet assay was regarded as at least equivalent to existing tests with regulatory acceptance (e.g. the in vivo UDS test). The situation in the third example is more complex. Several in vitro tests were performed of which some yielded positive or equivocal results. An in vivo MNT and an in vivo UDS test were clearly negative. The in vivo comet assay performed with liver cells and leukocytes of rodents was also negative. A ‘weight-of-evidence’-analysis with this dataset allowed us to conclude that the various in vitro positive results should not preclude the progression of the compound into human trials as a topical application.
(ii) Evaluation of local genotoxicity
One of the major applications of the in vivo comet assay is to evaluate local genotoxicity in e.g. (first) site of contact-tissue for compounds with poor systemic bioavailability and for very short-lived compounds or their metabolites (1,2). Furthermore, the evaluation of genotoxic effects in directly exposed organs may address certain human exposure scenarios. Target organs in this respect include nasal or oral cavity, lung, oesophagus, stomach mucosa, duodenum or skin.
In Table II, examples are given for datasets of drug candidates for which in vivo comet assay studies at (first) site of contact-tissues were performed. The first example is an aromatic nitro compound showing positive results in a bacterial reverse mutation assay in nitro-reductase proficient Salmonella strains only. The complete genotoxicity data are discussed in detail in Suter et al. (23). Since the gastrointestinal (GI)-tract of humans contains bacteria potentially capable of producing the genotoxic metabolite, a comet assay using tissue of the GI-tract of rodents was performed for hazard identification purposes. From the negative comet assay result along with negative data from an in vivo mutation test in the GI-tract tissue of transgenic animals, the authors concluded that a genotoxic hazard for humans is not likely (23). This line of argumentation was accepted by the health authorities. The second example is a compound exhibiting positive effects in various in vitro tests but a negative in vivo MNT in bone marrow cells of mice. In the in vivo comet assay, stomach mucosa was investigated as a site of contact-tissue. Decreased DNA migration was observed in stomach mucosa of treated animals indicating a crosslinking potential of the compound in the target cells (15). A crosslinking potential of this compound was also found in in vitro tests and the induction of DNA–protein crosslinks was demonstrated. Based on the positive in vivo results the compound was not developed further (9).
(iii) Evaluation of the contribution of genotoxicity to the induction of tumor development in chronic rodent studies
The comet assay has been used to elucidate a possible contribution of genotoxicity to the induction of pre-neoplastic/neoplastic changes, which were detected in long-term toxicity or carcinogenicity studies. A typical reason to apply the comet assay is a carcinogenicity test in rodents which yielded evidence for a tumorigenic response of a compound that was negative in a standard genotoxicity test battery in vitro and in vivo. Therefore, the in vivo comet assay with cells of the respective tumor target organ was used as a supplementary mechanistic test to distinguish between genotoxic and non-genotoxic mechanisms of tumor induction. Again, in such cases, the possibility of evaluating virtually any tissue of experimental animals is a major advantage of the in vivo comet assay although there may be limitations with regard to the identifiction of different cell types within one organ. In some cases, where there is suspicion of the involvement of only a certain cell type in tumorigenesis, cell enrichment procedures can be employed. In these cases the small number of cells needed for the comet assay is a special advantage.
A negative result from such a study would provide evidence that tumor induction may rather result from an epigenetic mechanism than from organ-specific genotoxicity. However, the suspected epigenetic mechanism should be further characterized to support such a hypothesis.
In Table III, examples are given for compounds for which in vivo comet assay studies were performed as tools to evaluate organ-specific genotoxicity in relation to tumor development. The first two examples are compounds with a negative genotoxicity dataset for which a 2-year rodent bioassay indicated tumorigenicity in certain organs. In these cases, the in vivo comet assays performed with tissue from the respective target organ did not reveal genotoxicity.
In the first example, the main tumor, cholangiocarcinoma, usually representing a very rare tumor type, developed after a short time period and was visible in 
80% of the mice. However, the main target cells, cholangiocytes, could not be evaluated in the comet assay due to difficulties in enriching this cell type and to obtaining sufficient cells for evaluation. Mice additionally developed tumors in the lung. An in vivo comet assay carried out with lung cells was negative. However, despite the negative comet assay result with lung tissue, further development of the compound was stopped because no mechanistic studies with cholangiocytes were possible and an epigenetic mechanism for the tumor development could not be identified.
In the case of the second compound (Table III) negative data from a UDS test in rat liver was already available. However, the evaluation of the target organ (jejunum) in the comet assay provided more convincing evidence that the compound did not induce organ-specific genotoxicity. Thus, the comet assay data provided significant evidence that genotoxicity was an unlikely cause of tumor development.
The third compound, Alachlor (Table III), showed a weak genotoxic effect in a bacterial mutagenicity test and equivocal results in an in vivo UDS test with rat liver. The negative comet assay result from the target organ, in context with other genotoxicity data, as well as an identified epigenetic mechanism led to the conclusion that this chemical does not pose a genotoxic hazard to humans (24).
Trichloroethylene (TRI) was reported to induce a low incidence of renal tumors in rats (25). It was evaluated in the in vivo comet assay, after inhalation for 6 months, and was found to be negative in kidney cells (13). In addition, TRI was described as negative in an in vivo comet assay with proximal tubular cells of rats treated via 6-h inhalations for 5 days. A negative result was also obtained for the TRI metabolite, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), after oral application (26). Based on the results of these two studies, the authors concluded that the tumorigenic activity of TRI is most probably related to a non-genotoxic mechanism.
Chlorination, a standard method for disinfection, causes the formation of chlorinated by-products. One of the major by-products is dichloroacetic acid (DCA), which in mammals is also found to be a metabolite of TRI. DCA was reported to induce liver tumors in mice when applied in the drinking water (27,28). Therefore, an in vivo comet assay with mice was performed after a 28-day exposure to DCA at a concentration of 3.5 g/l in drinking water. Detection of retarded DNA migration indicated the presence of DNA–DNA crosslinks in blood leukocytes. In combination with the results of a weakly-positive in vivo MNT, it was concluded that DCA might be an extremely weak inducer of chromosomal damage (29).
The sixth compound shown in Table III, the hair dye HC Red 3, showed equivocal evidence of carcinogenicity for male B6C3F1 mice as indicated by an increased incidence of hepatocellular adenomas or carcinomas (combined) (30). In vitro tests performed with this hair dye (i.e. in vitro chromosome aberration test with CHO cells and a mouse lymphoma assay) revealed clastogenic effects and induction of gene mutations. However, in an in vivo MNT in mice and a UDS test performed with rat liver, no genotoxic activity of HC Red 3 was found. To reconfirm the absence of a genotoxic effect in vivo, a comet assay in mice was performed and liver, bladder and blood cells were evaluated. The negative results in the possible target organ for carcinogenicity (liver) and in other organs, urinary bladder and blood added additional weight to the assumption that HC RED 3 is genotoxic in vitro but has no genotoxic potential in vivo (31).
The seventh example, the herbicide, Linuron, was shown to induce adenomas in Leydig cells of rats and hepatocellular carcinoma in mice (32). An in vivo comet assay carried out with rat testicular cells was negative while liver samples were found to exhibit increased DNA migration at doses resulting in a slight reduction of hepatocyte viability (14). However, an alkaline elution assay was also carried out with the same liver samples and was borderline positive. The authors concluded that the carcinogenic activity might, in part, be related to DNA reactivity with concomitant cytotoxicity, which could increase tumorigenicity by inducing cell death and consequent regenerative proliferation (14).
The last example is dicyclanil which was shown to cause hepatocarcinoma in mice. In an in vivo comet assay assessing the contribution of genotoxicity in relation to tumorigenicity, no genotoxic effects were seen in a range of organs from mice. The authors concluded that the carcinogenic potential is attributable to a non-genotoxic mechanism (33).
Taken together, these examples demonstrate that the comet assay has successfully been used to provide useful evidence for the mode of action of rodent carcinogens. Such information about organ-specific genotoxicity is assuming greater importance in follow-up genotoxicity testing for the assessments of carcinogenic risks.
(iv) Further evaluation of published positive comet assay data
Positive comet assay data may be produced for compounds that have been previously assessed as neither genotoxic nor carcinogenic. Table IV lists examples of such datasets reported recently from a study investigating food additives in the in vivo comet assay (34). In these studies, positive comet assay data were obtained with some compounds at doses lower than those usually used in carcinogenicity studies. Compounds such as thiabendazole (fungicide) or saccharin (sweetener) were shown to induce DNA effects in the comet assay in various organs of mice. The authors proposed several hypotheses to explain why the apparently genotoxic effects in several organs did not lead to carcinogenic effects in mice. However, this discrepancy has, as yet, not been satisfactorily explained. But it should be borne in mind that the relationship between genotoxic effects in vivo and carcinogenic effects is complex. Even at dose levels associated with tumor induction, DNA damage is induced in multiple organs, not all of which are target sites for carcinogenicity, and even if all exposed animals exhibit an increased level of DNA damage in the target organ(s), not all animals will develop tumors. In the case of thiabendazole the positive comet assay data (34,35) are surprising since thiabendazole has been reported to induce aneugenicity in vitro (36) and in vivo (37) but was not found to be clastogenic.
For the food dye tartrazine, induction of DNA effects in the comet assay was seen in cells isolated from the colon of mice (34) at a dose being only slightly higher than the acceptable daily intake for humans recommended by the Joint FAO/WHO Expert Committee on Food Additives (38). The dose where this effect was seen lies far below the doses used in carcinogenicity studies in rats and mice, which were clearly negative (39–41). Furthermore, the genotoxicity profile supports the absence of a genotoxic hazard for tartrazine. Except for the contradictory results for tartrazine seen in the chromosomal aberration assays with CHO cells (42,43), all other tests revealed negative results (BMT and MLA: Ref. 43; CAT: Ref. 42; UDS in vitro: Ref. 44; MNT and CAT BM: Ref. 45). Taken together, the findings of Sasaki et al. (35) seems to be an isolated positive result with limited relevance. Therefore, additional comet assay studies performed according to the latest protocol recommendations (2) might be useful to clarify these discrepancies.
An example of a compound that revealed conflicting data in the in vivo comet assay is ortho-phenylphenol (OPP). While Sasaki et al. (35) observed positive effects in several organs of mice treated with a single dose of 2000 mg/kg OPP, a study reported by Bomhard et al. (46) following the protocol recommendations of Tice et al. (1) and using the same dose and application route of the previous study showed a negative result (Table IV). In such cases, differences in the comet assay protocol used as well as technical differences in the whole study performance may account for a conflicting comet assay result. In the study carried out by Sasaki et al. (35), animals did not show clinical signs of toxicity while the same dose caused strong clinical signs and mortality in the study of Bomhard et al. (46). The differences with regard to the systemic effects may be due to the fact that OPP in the latter study was bioavailable at high (toxic) concentrations, while the bioavailability seems to be lower in the experiments performed by Sasaki et al. (35). Among other differences, there was a difference in cell preparation. While Sasaki et al. (35) subjected a suspension of isolated nuclei to the comet assay procedure, Bomhard et al. (46) used intact cells and determined cytotoxicity in the isolated cells. However, it has not yet been established whether such technical differences may influence the test result.
Isolated positive comet assay results should first of all be critically evaluated in the light of current recommendations (1,2) to exclude methodological shortcomings and potential artifacts. Because a positive result in the in vivo comet assay may have a significant impact for the further development or marketing of the compound, decisions should only be made on the basis of appropriately performed tests and verifiable data. In the cases where negative carcinogenicity data are already available and the in vivo comet assay result represents an isolated positive finding in the context of exisiting genotoxicity data, the biological significance of the effect seen in the comet assay should be assessed with caution.
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Use of in vivo comet assay results for hazard identification and risk assessment |
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Genotoxicity testing in vivo is performed for hazard identification and is part of the risk assessment process. The aim is to determine risks for heritable mutations (germ cells) and cancer development (somatic cells). Results from the in vivo comet assay contribute to hazard identification (i.e. how likely an agent is to be genotoxic/mutagenic to humans) and to dose–response assessment (i.e. the relationship between the dose of a substance and the probability of induction of an adverse effect) and mechanistic understanding of a substance's mode of action. The comet assay belongs to the group of indicator tests (as opposed to mutagenicity tests) because it detects DNA damage that may result in mutations.
Due to the assay's methodological advantages and its increasing acceptance within the scientific community, in vivo comet assay data are increasingly considered by regulatory agencies in the process of risk assessment and may be requested under specific circumstances. In cases where it is relevant to assess for DNA damage in specific organs like stomach, kidney, bladder, etc, the comet assay at present is the most feasible method. If appropriately performed, it has been shown to be a reliable test system with high sensitivity to detect DNA damage in organs that cannot be investigated in the classical assays such as the MNT or UDS test (15). However, the quality of the study performance should be critically evaluated. Incompletely described studies or studies with obvious methodological shortcomings should not be considered for risk assessment.
A negative result indicates the absence of genotoxic activity of the test compound detectable by the comet assay in the organ(s) tested. If this compound was shown to induce genotoxic effects in vitro, a negative in vivo comet assay—generally in combination with other negative in vivo genotoxicity tests—provides further evidence that genotoxic effects detected in vitro have no relevance for the in vivo situation. According to the authors' experience with agrochemicals, pharmaceuticals and hair dyes, the supporting information of a negative in vivo comet assay would allow the further development of a compound. However, to fulfil regulatory requirements, further testing may be necessary.
A positive result in an appropriately performed comet assay indicates a genotoxic effect of the test compound in the respective organ(s) and species. It has to be taken as an indication for a mutagenic potential of the test compound in cells of the organ(s) investigated and may have practical consequences. The positive in vivo comet assay result gains particular significance when the mutagenic potential of the test substance has already been demonstrated in vitro. Besides the biological significance of the observed effect (e.g. the exclusion of secondary effects due to cytotoxicity), the quality of the test performance (e.g. GLP compliance) and the plausibility of the result should be critically evaluated in the context of existing genotoxicity data for this compound as well as available toxicokinetic data.
An isolated positive in vivo comet assay result should lead to a critical re-evaluation of the existing genotoxicity data and the need for further testing has to be defined. For substances in development stages, a positive in vivo comet assay represents a major hurdle and will frequently result in discontinuing further development. If further testing is indicated, the strategy has to be decided ‘case by case’ and depends on the nature of the compound and the existing data. Further testing should be performed to enable a careful risk assessment of the compound by means of the ‘weight of evidence’ approach.
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Acknowledgments |
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We wish to thank the ‘Verband der Chemischen Industrie (VCI) e.V.’ for the encouragement to write this publication and for hosting meetings of the authors.
Conflict of interest statement. The ‘Verband der Chemischen Industrie (VCI) e.V.’ initiated this publication and hosted meetings of the authors. However, the views expressed in this report are completely independent of any industrial influence.
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Notes |
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* To whom correspondence should be addressed. Tel.: +49 731 5002 3429; Fax: +49 731 5002 3438; Email: guenter.speit{at}medizin.uni-ulm.de
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References |
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