SECTION IV:
REGULATORY, IMAGING ANDSTATISTICAL CONSIDERATIONS
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CHAPTER 15
Comet Assay – Protocols andTesting Strategies
ANDREAS HARTMANNa,* AND GUNTER SPEITb
aNovartis Pharma AG, Preclinical Safety, WKL105.4. 09, CH-4002 Basel,Switzerland; bUniversitat Ulm, Institut fur Humangenetik, D-89069 Ulm,Germany
15.1 Introduction
The assessment of a genotoxic hazard of chemicals and pharmaceuticals is animportant component of the preclinical safety assessment. Experience withgenetic toxicology testing over the past several decades has demonstrated thatno single assay is capable of detecting all genotoxic effects. Therefore, thepotential for a compound to cause genotoxicity is typically determined througha battery of in vitro and in vivo genotoxicity tests. In the case of pharmaceu-ticals, these assays are typically conducted at an early time point in thedevelopment of a new drug as they are relatively short in duration, inexpensive,and provide an early means to identify potential genotoxic carcinogens, whichotherwise would not be detected until the completion of carcinogenicity assays.Internationally harmonised genotoxicity testing guidance ICHS2A and S2B
that have been in operation since 1995 and 19971,2 are under revision as genetictoxicology testing has evolved since. Recently, a number of changes has beenproposed by an ICH expert working group that has issued a new guidance draft,termed ‘‘S2R: Guidance on Genotoxicity Testing and Data Interpretation for
Issues in Toxicology No 5
The Comet Assay in Toxicology
Edited by Alok Dhawan and Diana Andersonr Royal Society of Chemistry 2009
Published by the Royal Society of Chemistry, www.rsc.org
*Corresponding author
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Pharmaceuticals Intended for Human Use’’. One major concern driving therevision of the guidance takes into account accumulating evidence demonstratingthat in vitro cytogenetic assays are oversensitive towards positive results asso-ciated with cytotoxicity.3–5 As a consequence, the limitation of test concentra-tions and cytotoxicity levels were proposed for the mammalian cell tests toaddress concerns over growing numbers of nonrelevant positive findings. Thebasis for this proposal is built by an extensive review of results obtained within vitro hazard identification testing and in vivo risk assessment testing in thepharmaceutical industry as well as regulatory review.In the context of the revision of the ICH guidance, supplementary in vivo
assays such as the Comet assay are becoming more important in the assessmentof genotoxicity. The options for a standard battery of genotoxicity tests will beexpanded by the possibility to choose to conduct an in vivo test with investi-gation of genotoxic damage in two tissues instead of conducting an in vitro testwith mammalian cells followed by an in vivo test. As pharmaceuticals aregenerally tested for toxicity in rodent repeat-dose toxicity tests and as there isno requirement for an acute high dose rodent toxicity test any longer, theassessment of genotoxicity (e.g., bone marrow micronucleus test or other tissue/endpoint) is proposed to be integrated into the rodent repeat-dose toxicitystudy to optimise animal usage. Finally, in the context of the Food and DrugAdministration (FDA) Critical Path Initiative and the European MedicinesAgency Road Map to 2010, opportunities for more efficient drug developmentare sought that include abbreviated genotoxicity testing. One of the initiativesthat has emerged in this context is the elaboration through guidance ofexploratory investigational new drugs (INDs)/clinical trial applications(CTAs).6,7 With regards to genetic toxicology testing it is acceptable to conducta standard bacterial mutation assay as well as a test for chromosomal aber-rations either in vitro or in vivo. Furthermore, according to the new guidances itis acceptable to perform the in vivo cytogenetics assessment in conjunction withthe repeat-dose toxicity study in the rodent.6,7 This approach reflects a proposalin which repeated daily treatments with subchronic duration of exposures wereshown to produce similar results (magnitude of response) in comparison toacute treatment.8
15.2 Applications of the In Vivo Comet Assay
for Regulatory Purposes
The use of the in vivo Comet assay for regulatory purposes mainly focused onapplications as a supplementary test to follow up on in vitro positives or toinvestigate potential target organ genotoxicity.9,10 As such, the Comet assayhas potential advantages over other in vivo genotoxicity test methods, which arereliably applicable to rapidly proliferating cells only or have been validatedpreferentially in a single tissue only. The Comet assay may detect a broaderspectrum of primary DNA lesions, including single-strand breaks and oxidativebase damage, which may not be detected in the UDS test because they are not
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repaired by nucleotide excision repair.11 The advantages of the Comet assayover the alkaline elution test include the detection of DNA damage at thesingle-cell level and the requirement for only small numbers of cells per sample.In contrast, when using the alkaline elution assay, large quantities of cells arenecessary for the determination of genotoxic effects and, therefore, only alimited number of organs/tissues can be evaluated using this technique. Inparticular, this seems important for the investigation of suspected tissue specificgenotoxic activity, which includes ‘‘site-of-contact’’ genotoxicity (cases of highlocal versus low systemic exposure).The main focus of this chapter is the regulatory use of the in vivo Comet assay
for genotoxicity testing of pharmaceuticals with special emphasis on recom-mendations on test performance that have been issued by international expertpanels. As part of the International Workshop on Genotoxicity Testing(IWGT), expert working groups on the Comet assay were convened to reviewand discuss the procedures and methods and to issue recommendations for theComet assay in vitro and in vivo in 199912 and more focused on refinement ofrecommendations for the in vivo assay at the IWGT in 2005.13 In the lattermeeting, protocol areas that were unclear or that required more detail in orderto produce a standardised protocol with maximum acceptability by interna-tional regulatory agencies were discussed with regards to guidance for con-ducting the in vivo Comet assay that had been issued following exert paneldiscussions at the 4th International Comet Assay Workshop in 2001.11
15.3 Recommendations for Test Performance
For the application of the in vivo Comet assay in genetic toxicity it is importantto understand under which circumstances data from this test system can con-tribute to hazard identification and risk assessment. A review on the use andstatus of the Comet assay in current strategies for genotoxicity testing sum-marised the state-of-the-art and is recommended for further reading. The reviewlists specific examples for practical applications of the in vivo Comet assay andpotential consequences of positive and negative test results are provided.9
15.3.1 Genetic Endpoint of the Comet Assay
For regulatory use of the Comet assay it is important to understand that thisassay is an indicator test that detects primary DNA lesions and, therefore, cannot be used as a primary in vivo genotoxicity test such as the micronucleus test.Indicator tests (or supplemental tests) do not directly measure mutations butdetect effects related to the process of mutagenesis, such as DNA damage,recombination and repair. These assays differ with respect to the endpointsassessed. Induction of primary DNA lesions, that is, measurement of exposure,uptake and reactivity to DNA can be measured by the Comet assay, the 32P-postlabeling assay, the alkaline elution or unwinding assays.14,15 For thedetermination of the repair of DNA lesions the unscheduled DNA synthesis
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(UDS) test is being used. Finally, measurement of induction of genetic changesusing transgenic animal assays for point mutations is utilised. Results of sup-plemental tests can provide additional useful information in the context ofextended genotoxicity testing. It has to be emphasised that primary DNAlesions may be repaired error-free and do not necessarily result in formation ofmutations. Neither the magnitude of DNA migration in the Comet assay northe shape of the comet can reveal the types of DNA damage causing the effector their biological significance, that is, their mutagenic potential. Therefore,conclusions regarding the mutagenicity of a test compound cannot be madesolely on the basis of Comet assay effects. However, a negative Comet result canbe considered as a strong indicator for the absence of a mutagenic potential.Among the various versions of the Comet assay, the alkaline (pH of the
unwinding and electrophoresis buffer Z 13) method enables detection ofthe broadest spectrum of DNA damage16 and is, therefore, recommended (inthe first instance) for regulatory purposes.9,11,12 The alkaline version detectsDNA damage such as strand breaks, alkali-labile sites (ALS), and single-strandbreaks associated with incomplete excision repair. Under certain conditions,the assay can also detect DNA–DNA and DNA–protein crosslinking, which (inthe absence of other kinds of DNA lesions) appears as a relative decrease inDNA migration compared to concurrent controls. In contrast to other DNAalterations, crosslinks may stabilise chromosomal DNA and inhibit DNAmigration.17 Thus, reduced DNA migration in comparison to the negativecontrol (which should show some degree of DNA migration) may indicate theinduction of crosslinks, which are relevant lesions with regard to mutagenesisand should be further investigated. Increased DNA migration indicates theinduction of DNA-strand breaks and/or ALS. Furthermore, enhanced activityof excision repair may result in increased DNA migration, which can influenceComet assay effects in a complex way. While DNA repair generally reducesDNA migration by eliminating DNA lesions, ongoing excision repair mayincrease DNA migration due to incision-related DNA-strand breaks. Thus, thecontribution of excision repair to the DNA effects seen in the Comet assaydepends on the types of induced primary DNA damage and the time point ofanalysis.18
15.3.2 Basic Considerations for Test Protocol
The recommendations issued by Tice et al.12 and Hartmann et al.11 were refinedby Burlinson et al.13 and describe aspects of the test procedure regarding testanimals, test substance, use of concurrent negative and positive control animalsas well as dose selection for the design of a cytogenetic assay in much detail. Inbrief, either a single treatment or repeated treatments (generally at 24-hintervals) are equally acceptable. In both experimental designs, the study isacceptable as long as a positive effect has been demonstrated or, for a negativeresult, as long as an appropriate level of animal or tissue toxicity has beendemonstrated or the limit dose with appropriate tissue exposure has been used.
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For repeated treatment schedules, dosing must be continued until the day ofsampling. On a daily basis, test substances may be administered as a split dose(i.e. two treatments separated by no more than a few hours), to facilitateadministering a large volume of material. The test may be performed in twoways. If animals are treated with the test substance once, then tissue/organsamples are obtained at 2–6 h and 16–26 h after dosing. The shorter samplingtime is considered sufficient to detect rapidly absorbed as well as unstable ordirect acting compounds. In contrast, the late sampling time is intended todetect compounds that are more slowly absorbed, distributed and metabolised.When a positive response is identified at one sampling time, data from the othersample time need not be collected although it might be useful for the inter-pretation of the test result. Alternatively, if multiple treatments at 24-h intervalsare used, tissue/organ samples need be collected only once. The sampling timeshould be 2–6 h after the last administration of the test substance. Alternativesampling times may be used when justified on the basis of toxicokinetic data.One important aspect for the validity of a study is the inclusion of positive
and negative controls. A positive control substance needs to demonstrate thatthe conditions of the study, in particular, the electrophoresis, were appropriateto demonstrate the induction of DNA damage. In addition, the stability of thenegative/positive control data over time and criteria for determining theacceptability of new studies, based on historical control data, should beestablished for each tissue.Finally, minimal reporting standards should follow current OECD standards
for the in vivo genotoxicity test and should ensure that all studies can beindependently evaluated. Previous recommendations have covered someaspects of reporting standards.11,12
15.3.3 Selection of Tissues and Cell Preparation
In principle, any tissue of the experimental animal, provided that a high-qualitysingle-cell/nucleus suspension can be obtained can be used for a Comet assay.Selection of the tissue(s) to be evaluated should be based, wherever possible, ondata from absorption, distribution, metabolism, excretion studies, and/or othertoxicological information. A tissue should not be evaluated unless there isevidence of, or support for, exposure of the tissue to the test substance and/orits metabolite(s). In the absence of such information and, unless scientificallyjustified, two tissues should be examined. Recommended tissues are liver, whichis the major organ for the metabolism of absorbed compounds, and a site offirst contact tissue, e.g. gastrointestinal for orally administered substances,respiratory tract for substances administered via inhalation, or skin for der-mally applied substances. Which tissue is evaluated first is at the discretion ofthe investigator and both tissues need not be evaluated if a positive response isobtained in the first tissue evaluated.Single-cell suspensions can be obtained from solid tissue by mincing briefly
with a pair of fine scissors,19 incubation with digestive enzymes such as
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collagenase or trypsin,20 or by pushing the tissue sample through a meshmembrane. In addition, cell nuclei can be obtained by homogenisation.21
During mincing or homogenisation, EDTA can be added to the processingsolution to chelate calcium/magnesium and prevent endonuclease activation. Inaddition, radical scavengers (e.g., DMSO) can be added to prevent oxidant-induced DNA damage. Any cell-dissociation method is acceptable as long as itcan be demonstrated that the process is not associated with inappropriatebackground levels of DNA damage.
15.3.4 Image Analysis
DNA migration in individual cells can be assessed by using image analysis or bymanual scoring. While the use of image analysis enables various parameters to beanalysed, manual scoring usually determines the length of DNA migration, thepercentage of cells with and without migration, or the proportion of comets thatcan be ‘‘binned’’ into various migration categories.22 However, a limitation ofmanual scoring may be a potential inability to take into account the density orshape of tails that can include short but dense tails and long but sparse tailsdepending on the effects of compounds tested. With image-analysis systems, themost common parameters analysed are the tail intensity, i.e. the percentage DNAin the tail (% tail DNA), tail moment, and tail length and/or image length(referring to nucleus plus migrated DNA). Some parameters (e.g., tail moment)may be calculated differently among image-analysis systems that can lead toquantitative differences which can be problematic when comparing inter-laboratory data. The percentage DNA in the tail is considered the parameter thatcan best be compared between laboratories. The consensus of the IWGT was thatimage analysis is preferred but not required and that the parameter % tail DNAappeared to be the most linearly related to dose and the easiest to intuitivelyunderstand.14 There was no consensus that % tail DNA must be the onlyparameter used. However, if some measure of tail moment is used, than % tailDNA and tail length data must be provided as well. Heavily damaged cellsexhibiting a specific microscopic image (commonly referred to as hedgehogs)consisting of small or nonexistent head and large, diffuse tails23 potentiallyrepresent dead or dying cells and may be excluded from data collection. However,determining their frequency may be useful for data interpretation. Data on thedistribution of migration among cells should also be presented. This is accom-plished by sorting cells within ‘‘bins’’ based on the metric used to evaluate DNAmigration and presenting the data as the percentage of cells within each bin.
15.3.5 Assessment of Cytotoxicity – A Potential
Confounding Factor
A general issue with DNA-strand break assays such as the Comet assay is thatindirect mechanisms related to cytotoxicity may lead to enhanced strand-breakformation. However, since DNA damage in the Comet assay is assessed on the
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level of individual cells, dead or dying cells may be identified on microscopicslides by their specific image. Necrotic or apoptotic cells can result in cometswith small or nonexistent head and large, diffuse tails24 as observed in vitroupon treatment with cytotoxic, nongenotoxic articles.25–27 However, suchmicroscopic images are not uniquely diagnostic for apoptosis or necrosis sincethey may also be detected after treatment with high doses of radiation or highconcentrations of strong mutagens.28 For the in vivo Comet assay, only limiteddata are available to establish whether cytotoxicity results in increased DNAmigration in tissues of experimental animals. In a comprehensive investigationwith genotoxic and nongenotoxic kidney carcinogens, the ability of the Cometassay to distinguish genotoxicity versus cytotoxicity was assessed by investi-gating five known genotoxic renal carcinogens acting through diversemechanisms of action and two rodent renal epigenetic carcinogens. The authorsconcluded that the Comet assay using kidney cells of rats is not prone to false-positive results due to cytotoxicity.29 Other investigations showed that despitenecrosis or apoptosis in target organs of rodents such as kidneys,30 stomach,31
liver or duodenum,10 no elevated DNA migration was observed. However,enhanced DNA migration was seen in homogenised liver tissue of mice dosedwith carbon tetrachloride32 when histopathological examination showed evi-dence of necrosis in the liver. Therefore, to avoid potential false-positive effectsresulting from cytotoxicity, recommendations regarding a concurrent assess-ment of target organ toxicity have been made, including dye viability assays,histopathology and a neutral diffusion assay.11,12
15.3.6 Ongoing Validation Exercises
The Japanese Center for the Validation of Alternative Methods (JaCVAM) isorganising an international validation study of the in vivo Comet assay, incooperation with the US National Toxicology Program Interagency Center forthe Evaluation of Alternative Toxicological Methods (NICEATM) and theInteragency Coordinating Committee on the Validation of Alternative Meth-ods (ICCVAM), the European Centre for the Validation of AlternativeMethods (ECVAM), and the Mammalian Mutagenicity Study Group(MMSG)/Japanese Environmental Mutagen Society (JEMS). The purpose ofthis validation study is to evaluate the ability of the in vivo Comet assay toidentify genotoxic chemicals as a potential predictor of rodent carcinogenicity.A more validation-type study investigated several aspects of an experimental
design such as positives controls, tissue toxicity and sources of experimentalvariability.31 To examine cytotoxicity the neutral diffusion assay and histo-pathological/haematological analysis were used. Based on analyses of pooleddata from several studies tissue preparations were identified as a source of highvariability. The authors’ conclusion was that a higher number of samples/slidesmay be required to achieve sufficient power to detect a positive effect in certaintissues.31 Clearly, more such validation exercises are required to better defineoptimised protocols.
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15.4 Applications of the In Vivo Comet Assay
for Regulatory Purposes
A comprehensive review on the use and status of the in vivo Comet assay hasbeen issued recently.9 For regulatory purposes, the in vivo Comet assay iscurrently being used (1), to follow up on positive findings in one or more testsof the standard genotoxicity battery; (2) to elucidate a potential contribution ofgenotoxicity to the induction of preneoplastic and/or neoplastic changesdetected in long-term tests in rodents; (3) to investigate local genotoxicity.Additional areas that have been proposed are testing of industrial chemicals33
or assessment of photochemical genotoxicity.34,35 In addition, the Comet assaycan be applied as a mechanistic tool to distinguish clastogenic from aneugeniceffects. Since aneugenicity is well accepted to result from mechanisms of actionfor which thresholds exist, demonstration that micronucleus formation is aresult of chromosome loss should allow an acceptable level of human exposureto be defined.36 No matter the trigger for conducting supplemental in vivogenotoxicity testing, it is critical that the approach utilised, for example theendpoint and target tissue assessed, is scientifically valuable, such that theresults will aid in interpreting the relevance of the initial finding of concern.Ultimately, the goal of supplemental genotoxicity testing is to determine if amutagenic risk is posed to humans under the intended use of a compound.
15.4.1 Follow-Up Testing of Positive In VitroCytogenetics Assays
An analysis of positive and negative in vitro chromosomal aberration resultsin various cell types amongst data that had been submitted to the GermanFederal Institute for Drugs and Medical Devices (BfArM) between 1995 and2005 showed that approximately 30% of the compounds had positive in vitrogenotoxicity data.37 The dataset consisted of 804 chromosomal aberrationstudies on nearly 600 pharmaceuticals and showed that the frequency ofpositive results in four different cell types studied for chromosomal aberra-tions and in the mouse lymphoma assay (detecting gene mutations as well aschromosomal damage) was very similar. It is noteworthy that such a highpercentage of positive mammalian cell results is seen in submission dossiersassuming companies have already screened out compounds that are notconsidered suitable for development. In contrast to the high percentage ofpositive in vitro studies, results from bone marrow cytogenetic assays arefrequently negative. This discrepancy may result from a number of majordifferences that exist when testing in cultured cells versus the intact animals.One important difference is the external metabolic system that is used inin vitro systems while in the intact organism, intact metabolic pathways exist.Metabolic inactivation can occur in the intact animal, the parent compoundor active metabolite may not reach the target cell in vivo, rapid detoxificationand elimination may occur, or plasma levels in vivo may not be comparable to
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concentrations that generate positive responses in the in vitro assays thatare often accompanied by high levels of cytotoxicity. It is also worth notingthat positive results generated in vitro may be secondary to effects, such ascytotoxicity, which may never be achieved under in vivo exposure conditions.At present, data from in vivo experiments are therefore essential before defi-nitive conclusions are drawn regarding the potential mutagenic hazardto humans from chemicals that produce positive results in one or morein vitro tests.There has been much discussion in recent years regarding the most appro-
priate follow-up testing in vivo when positive results are obtained in vitro butthe in vivo micronucleus test is negative. A recent analysis compared the use-fulness of the in vivo Comet assay as a second in vivo test in comparison with theUDS test and the transgenic mutation (TG) assay.38 While the UDS test gaveonly positive results witho20% of carcinogens, the TG assay gave positiveresults with 450% of the carcinogens, but the Comet assay detected almost90% of the micronucleus negative or equivocal carcinogens. Although moredata are needed before a general recommendation can be made, this studyclearly indicated that the Comet assay should play a more prominent role inregulatory testing strategies in the future.
15.4.2 Follow-Up Testing of Tumourigenic Compounds
Carcinogenicity testing of compounds such as pharmaceuticals negative in thestandard in vitro and in vivo genotoxicity assays may yield evidence of atumourigenic response in rodents. The ICH guidance S2B2 stipulates that suchcompounds shall be investigated further in supplemental genotoxicity tests, ifrodent tumourigenicity is not clearly based on a nongenotoxic mechanism.Typically, supplemental in vivo genotoxicity tests are performed with cellsof the respective tumour target organ to distinguish between genotoxic andnongenotoxic mechanisms of tumour induction. For such purposes it isimportant that the method applied has a high specificity to distinguish geno-toxic from nongenotoxic modes of action. In a study investigating genotoxicand nongenotoxic kidney carcinogens in rats, the in vivo Comet assaydemonstrated the induction of DNA damage in the target organ by genotoxiccarcinogens but not by kidney carcinogens that act through nongenotoxicmodes of action.29 In a comprehensive summary of investigations of morethan 200 compounds, a high specificity for Comet assay data was demonstratedby a high positive response ratio for rodent genotoxic carcinogens and a highnegative response ratio for rodent genotoxic noncarcinogens.39 Finally, arecent review summarised published data and concluded that one of themajor advantages of the in vivo Comet assay was the ability to evaluate vir-tually any tissue of experimental animals. It was concluded that a negativeresult from such a study would provide strong evidence that tumour inductionmay rather result from an epigenetic mechanism than from organ-specificgenotoxicity.9
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15.4.3 Assessment of Local Genotoxicity
The Comet assay is considered a very useful tool to investigate genotoxicity atthe first site of contact, such as oral or stomach mucosa cells or the nasal cavityof rodents. This approach is of interest for investigations of compounds with alow systemic bioavailability or for very short-lived compounds or their meta-bolites.11,12 Furthermore, the evaluation of genotoxic effects in directly exposedorgans may address certain human exposure scenarios. Target organs in thisrespect include nasal or oral cavity, lung, oesophagus, stomach mucosa, duo-denum or skin. A comprehensive review of applications and available data hasbeen published.9
15.4.4 Assessment of Germ Cell Genotoxicity
A recent review by Speit et al. assessed the use of the Comet assay for investi-gating germ cell genotoxicity.40 While current test strategies focus on somaticcells from different organs to detect the genotoxic activity, the Comet assay alsohas the potential to be a useful tool for investigating germ cell genotoxicity. The‘‘Globally Harmonised System of Classification and Labeling of Chemicals(GHS)’’ has published classification criteria for germ cell mutagens, i.e. chemi-cals that may cause mutations in the germ cells of humans that can be trans-mitted to the progeny.41 Category 1 B defines ‘‘chemicals which should beregarded as if they induce heritable mutations in the germ cells of humans’’.Among the criteria that are given for this category, one requires ‘‘positiveresults(s) from in vivo somatic cell mutagenicity tests in mammals, in combina-tion with some evidence that the substance has the potential to cause mutationsin germ cells. This supporting evidence may, for example, be derived frommutagenicity/genotoxicity tests in germ cells in vivo, or by demonstrating theability of the substance or its metabolite(s) to interact with the genetic material ofgerm cells’’. Although GHS does not explicitly mention the Comet assay butonly lists the Sister Chromatid Exchange (SCE) analysis in spermatogonia andthe Unscheduled DNA Synthesis test (UDS) in testicular cells as examples forgenotoxicity tests in germ cells, the Comet assay might play an important role inthis context in the future. GHS requires that classification for heritable effects inhuman germ cells has to be made on the basis of well-conducted, sufficientlyvalidated tests, preferably as described in OECD test guidelines. The standardalkaline in vivo Comet assay can easily be adapted to investigations with cellsfrom the gonads (testis and ovary) for demonstrating that a test compoundreaches the gonads and is able to interact with the genetic material of germ cells.However, standardisation and validation studies are necessary before the Cometassay can be usefully applied in risk assessment of germ cell mutagens.
15.4.5 Assessment of Photogenotoxicity
The assessment of photochemical genotoxicity (‘‘photogenotoxicity’’) is animportant part of photosafety testing that has become a regulatory requirement
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for medicinal products, dermatological and sunscreen products. Photochemicalgenotoxicity of drugs was described to be involved in the generation of skintumours42 and can lead to injuries to the eye.43 In the European Union (EU),pharmaceutical products are regulated under the European Agency for theEvaluation of Medicinal products (EMEA), whilst dermatological and sun-screen products are regulated under the EU Cosmetics Directive. The condi-tions for the photosafety evaluation of pharmaceuticals and cosmetics are laidout by The EMEA Committee for Proprietary Medicinal Products ‘‘Notes forGuidance on Photosafety Testing’’44 and the FDA/CDER Guidance onindustry photosafety testing.45 The main objective of photogenotoxicity testingis to make an assessment of the potential of a compound to turn into a pho-tochemical carcinogen upon activation with UV or visible light.Several standard genotoxicity assays such as the photo-Ames, photo-
chromosome aberration (CA) and photo-Comet assays have been described inthe literature and are based on standard ‘‘dark’’ versions of regulatory assaysused for genotoxicity assessment. The tests have been adapted towards use inphotogenotoxicity testing, such as the photoclastogenicity test in CHO cellsand tentative guidelines have been issued.46 Considerable concern regarding thebiological plausibility of the response of certain chemicals in the in vitrophotoclastogenicity assay has been raised, suggesting that this assay is over-sensitive and lacks specificity.47 Specifically, given that more than 55% of allsubstances tested yielded positive results in these tests, the definition of in vitrophotogenotoxicity for substances that are clastogenic in the dark requiresreconsideration, especially taking into account the absence of validated in vivotests that could distinguish genuine from pseudophotoclastogens.47 In addition,several compounds that did not absorb UV light were shown to elicit a pho-toclastogenic response in the photochromosome aberration assay using a CHOcell line.47,48 Therefore, the biological significance of in vitro photo-clastogenicity data for hazard identification and risk assessment remainsquestionable and alternative methods need to be considered. In vivo methodsmay, therefore, be considered an alternative. Compared to in vitro testson isolated cells, additional parameters may influence the photogenotoxicityin vivo such as the metabolism of a compound, systemic distributed or dis-position into skin. In addition, the skin and the eye are composed of differentlayers, which can function as protective barriers and have an impact onpenetration and absorption of wavelengths from sunlight. Furthermore,binding and retention time of the compound in the different layers of the skin oreye as well as their DNA-repair mechanisms may have an impact on thephotochemical toxicity.Most of the photochemical reactions involve the generation of free-radical
oxygen species. Oxygen content and the antioxidant status may have animportant impact on the photogenotoxicity outcome.46 Due to higher oxygenpressure in vitro, higher amounts of radical oxygen species may be generated,with the consequence that a photogenotoxic effect might be overestimatedunder in vitro conditions. Therefore, more reliable test systems should enable amore thorough assessment of a photogenotoxic hazard. An in vivo Comet assay
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has been established to assess the photogenotoxic response of fluoroquinolonesin the skin of mice.34 In addition to the analysis of skin keratinocytes, theuse of cells of the retina and cornea of rats treated with model compoundssparfloxacin, dacarbazine, chlorpromazine and 8-methoxypsoralen has beenestablished.35 These investigations demonstrate that the photo-Comet assay inrodents is a reliable method to elucidate drug-induced photogenotoxicity underconditions that are relevant to the human situation.
15.4.6 Genotoxicity Testing of Chemicals
The genotoxicity testing requirements for chemicals differ from regulations forpharmaceuticals. The identification of possible genotoxic effects has long beenfundamental for toxicity testing of chemicals. Different strategies for geno-toxicity testing are applied depending on the regulated ‘‘substance class’’ (use ofsubstances, type and degree of exposure, risk-benefit considerations, etc.).While genotoxicity testing originally focused on the detection of germ cellmutagens, in current regulatory practice the emphasis is put on screening forpossible carcinogenic substances. In addition, genotoxicity testing is increas-ingly being used to clarify the contribution of genotoxicity to findings in car-cinogenicity studies.A working group sponsored by the German-speaking section of the
European Environmental Mutagen Society (GUM) proposed a simplifiedapproach to genotoxicity testing of chemicals.33 The proposed strategy consistsof basic testing using a bacterial gene mutation test plus an in vitro micro-nucleus test (stage I) and follow-up testing in vivo (stage II) in the case ofrelevant positive results observed in stage I. For the follow-up testing a singlestudy combining the analysis of micronuclei in bone marrow with the Cometassay in appropriate tissues was suggested. Negative results for both endpointsin relevant tissues would generally provide sufficient evidence to conclude thatthe test compound is nongenotoxic in vivo. Compounds recognised as in vivosomatic cell mutagens/genotoxicants in this hazard identification step wouldneed further assessment or would be considered as potential genotoxic carci-nogens and potential germ cell mutagens in the absence of additional data.33
15.5 Conclusions
The in vivo Comet assay is increasingly used to contribute to hazard identifi-cation (i.e. how likely an agent is to be genotoxic/mutagenic to humans) and todose–response assessment (i.e. the relationship between the dose of a substanceand the probability of induction of an adverse effect). Data from the in vivoComet assay data are increasingly considered by regulatory agencies in theprocess of risk assessment and may be requested under specific circumstances.The Comet assay was shown to be a reliable test system with high sensitivitythat enables detection of DNA damage in organs that cannot be investigated inthe classical assays such as the micronucleus test or the unscheduled DNA
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synthesis (UDS) test.10 One important area in which it is relevant to assess forDNA damage in specific target organs such as stomach, kidney, bladder, theComet assay at present is the most feasible method. The quality of the studyperformance is critical and recommendations have been issued by internationalexpert panels.11–13 Systematic investigations aiming at more optimised studydesigns have been issued more recently.31
A negative result in the Comet assay is considered as supportive evidence of alack of genotoxic activity of a test compound in the tissues tested. When acompound induced genotoxic effects in vitro, a negative in vivo Comet assay –generally in combination with other negative in vivo genotoxicity tests – pro-vides supporting evidence that genotoxic effects detected in vitro have norelevance for the in vivo situation. According to published experience withagrochemicals, pharmaceuticals and hair dyes, a negative in vivo Comet assaywould allow further development of a compound to proceed. However, to fulfilregulatory requirements, additional testing may be considered.A positive result indicates a genotoxic effect of the test compound in the
respective tissues of the species tested and, therefore, an indication for a muta-genic potential of the test compound. If positive in vitro data were obtained forthe compound, a positive in vivo Comet assay signal should be considered evi-dence that the in vitro signal is of biological significance in vivo. For substancesin developmental stages, a positive in vivo Comet assay generally represents amajor hurdle and will frequently result in discontinuation of further develop-ment. If further testing is considered necessary, the testing strategy needs to bedetermined on a case-by-case basis that much depends on the mode of action ofthe compound and the already existing data. Finally, the quality of the testperformance and the plausibility of the result should be critically evaluated in thecontext of existing genotoxicity data for this compound as well as available dataon absorption and disposition of the compound investigated.Isolated positive in vivo Comet assay results in the context of otherwise
negative datasets have been reported.9 Such cases should initiate a critical re-evaluation of the existing genotoxicity data and the need for additional testingshould be defined. Further testing should be performed to enable a careful riskassessment of the compound by means of the ‘‘weight of evidence’’ approach.
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