In vivo alkaline comet assay: Statistical considerations on historical negative and positive control data.

The alkaline comet assay is frequently used as in vivo follow-up test within different regulatory environments to characterize the DNA-damaging potential of different test items. The corresponding OECD Test guideline 489 highlights the importance of statistical analyses and historical control data (HCD) but does not provide detailed procedures. Therefore, the working group "Statistics" of the German-speaking Society for Environmental Mutation Research (GUM) collected HCD from five laboratories and >200 comet assay studies and performed several statistical analyses. Key results included that (I) observed large inter-laboratory effects argue against the use of absolute quality thresholds, (II) > 50% zero values on a slide are considered problematic, due to their influence on slide or animal summary statistics, (III) the type of summarizing measure for single-cell data (e.g., median, arithmetic and geometric mean) may lead to extreme differences in resulting animal tail intensities and study outcome in the HCD. These summarizing values increase the reliability of analysis results by better meeting statistical model assumptions, but at the cost of information loss. Furthermore, the relation between negative and positive control groups in the data set was always satisfactorily (or sufficiently) based on ratio, difference and quantile analyses.

Evaluation of two in vitro assays for tumorigenicity assessment of CRISPR-Cas9 genome-edited cells.

Off-target editing is one of the main safety concerns for the use of CRISPR-Cas9 genome editing in gene therapy. These unwanted modifications could lead to malignant transformation, which renders tumorigenicity assessment of gene therapy products indispensable. In this study, we established two in vitro transformation assays, the soft agar colony-forming assay (SACF) and the growth in low attachment assay (GILA) as alternative methods for tumorigenicity evaluation of genome-edited cells. Using a CRISPR-Cas9-based approach to transform immortalized MCF10A cells, we identified PTPN12, a known tumor suppressor, as a valid positive control in GILA and SACF. Next, we measured the limit of detection for both assays and proved that SACF is more sensitive than GILA (0.8% versus 3.1% transformed cells). We further validated SACF and GILA by identifying a set of positive and negative controls and by testing the suitability of another cell line (THLE-2). Moreover, in contrast to SACF and GILA, an in vivo tumorigenicity study failed to detect the known tumorigenic potential of PTPN12 deletion, demonstrating the relevance of GILA and SACF in tumorigenicity testing. In conclusion, SACF and GILA are both attractive and valuable additions to preclinical safety assessment of gene therapy products.

Gamma-H2AX immunofluorescence for the detection of tissue-specific genotoxicity in vivo.

The phosphorylation of histone H2AX in Serine 139 (gamma-H2AX) marks regions of DNA double strand breaks and contributes to the recruitment of DNA repair factors to the site of DNA damage. Gamma-H2AX is used widely as DNA damage marker in vitro, but its use for genotoxicity assessment in vivo has not been extensively investigated. Here, we developed an image analysis system for the precise quantification of the gamma-H2AX signal, which we used to monitor DNA damage in animals treated with known genotoxicants (EMS, ENU and doxorubicin). To compare this new assay to a validated standard procedure for DNA damage quantification, tissues from the same animals were also analyzed in the comet assay. An increase in the levels of gamma-H2AX was observed in most of the tissues from animals treated with doxorubicin and ENU. Interestingly, the lesions induced by doxorubicin were not easily detected by the standard comet assay, while they were clearly identified by gamma-H2AX staining. Conversely, EMS appeared strongly positive in the comet assay but only mildly in the gamma-H2AX immunofluorescence. These observations suggest that the two methods could complement each other for DNA damage analysis, where gamma-H2AX staining allows the detection of tissue-specific effects in situ. Moreover, since gamma-H2AX staining can be performed on formalin-fixed and paraffin-embedded tissue sections generated during repeated-dose toxicity studies, it does not require any further treatments or extra procedures during dissection, thus optimizing the use of resources and animals. Environ. Mol. Mutagen. 60:4-16, 2019. © 2018 Wiley Periodicals, Inc.

Inter-laboratory comparison of the in vivo comet assay including three image analysis systems.

To compare the extent of potential inter-laboratory variability and the influence of different comet image analysis systems, in vivo comet experiments were conducted using the genotoxicants ethyl methanesulfonate and methyl methanesulfonate. Tissue samples from the same animals were processed and analyzed-including independent slide evaluation by image analysis-in two laboratories with extensive experience in performing the comet assay. The analysis revealed low inter-laboratory experimental variability. Neither the use of different image analysis systems, nor the staining procedure of DNA (propidium iodide vs. SYBR® Gold), considerably impacted the results or sensitivity of the assay. In addition, relatively high stability of the staining intensity of propidium iodide-stained slides was found in slides that were refrigerated for over 3 months. In conclusion, following a thoroughly defined protocol and standardized routine procedures ensures that the comet assay is robust and generates comparable results between different laboratories.

JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for detection of genotoxic carcinogens: II. Summary of definitive validation study results.

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity.

Evaluation of methyl methanesulfonate, 2,6-diaminotoluene and 5-fluorouracil: Part of the Japanese center for the validation of alternative methods (JaCVAM) international validation study of the in vivo rat alkaline comet assay.

As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined methyl methanesulfonate, 2,6-diaminotoluene, and 5-fluorouracil under coded test conditions. Rats were treated orally with the maximum tolerated dose (MTD) and two additional descending doses of the respective compounds. In the MMS treated groups liver and stomach showed significantly elevated DNA damage at each dose level and a significant dose-response relationship. 2,6-diaminotoluene induced significantly elevated DNA damage in the liver at each dose and a statistically significant dose-response relationship whereas no DNA damage was obtained in the stomach. 5-fluorouracil did not induce DNA damage in either liver or stomach.

Critical issues with the in vivo comet assay: A report of the comet assay working group in the 6th International Workshop on Genotoxicity Testing (IWGT).

As a part of the 6th IWGT, an expert working group on the comet assay evaluated critical topics related to the use of the in vivo comet assay in regulatory genotoxicity testing. The areas covered were: identification of the domain of applicability and regulatory acceptance, identification of critical parameters of the protocol and attempts to standardize the assay, experience with combination and integration with other in vivo studies, demonstration of laboratory proficiency, sensitivity and power of the protocol used, use of different tissues, freezing of samples, and choice of appropriate measures of cytotoxicity. The standard protocol detects various types of DNA lesions but it does not detect all types of DNA damage. Modifications of the standard protocol may be used to detect additional types of specific DNA damage (e.g., cross-links, bulky adducts, oxidized bases). In addition, the working group identified critical parameters that should be carefully controlled and described in detail in every published study protocol. In vivo comet assay results are more reliable if they were obtained in laboratories that have demonstrated proficiency. This includes demonstration of adequate response to vehicle controls and an adequate response to a positive control for each tissue being examined. There was a general agreement that freezing of samples is an option but more data are needed in order to establish generally accepted protocols. With regard to tissue toxicity, the working group concluded that cytotoxicity could be a confounder of comet results. It is recommended to look at multiple parameters such as histopathological observations, organ-specific clinical chemistry as well as indicators of tissue inflammation to decide whether compound-specific toxicity might influence the result. The expert working group concluded that the alkaline in vivo comet assay is a mature test for the evaluation of genotoxicity and can be recommended to regulatory agencies for use.

Performance and data interpretation of the in vivo comet assay in pharmaceutical industry: EFPIA survey results.

In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by the ICH S2(R1) guideline. This paper summarizes a survey suggested by the Safety Working Party of European Medicines Agency (EMA), and conducted by the European Federation of Pharmaceutical Industries and Associations (EFPIA) to investigate the experience among European pharmaceutical companies by conducting the in vivo comet assay for regulatory purpose. A special focus was given on the typology of the obtained results and to identify potential difficulties encountered with the interpretation of study data. The participating companies reported a total of 147 studies (conducted in-house or outsourced) and shared the conclusion on the comet assay response for 136 studies. Most of the studies were negative (118/136). Only about 10% (14/136 studies) of the comet assays showed a positive response. None of the positive comet assay results were clearly associated with organ toxicity indicating that the positive responses are not due to cytotoxic effects of the compound in the tissue examined. The number of comet assays with an equivocal or inconclusive response was rare, respectively <1% (1/147 studies) and 2% (3/147 studies). In case additional information (e.g. repeat assay, organ toxicity, metabolism, tissue exposure) would have been available for evaluation, a final conclusion could most probably have been drawn for most or all of these studies. All (46) negative in vivo comet assays submitted alongside with a negative in vivo micronucleus assay were accepted by the regulatory authorities to mitigate a positive in vitro mammalian cell assay following the current ICH S2 guidance. The survey results demonstrate the robustness of the comet assay and the regulatory acceptance of the current ICH S2 guidance.

Characterization of vemurafenib phototoxicity in a mouse model.

Vemurafenib is a first-in-class, small molecule B-Raf kinase inhibitor for the treatment of patients with unresectable or metastatic melanoma carrying the BRAFV600E mutation, commercially available since 2011. A general phototoxic potential was identified early during development; however, based on results of an animal study in hairless rats, it was concluded that there would exist no relevant risk for humans. Surprisingly, signs of clinical photosensitivity were reported in many patients during clinical development. Therefore, it became a fundamental question to understand this discrepancy. An established mouse model (oral UV-Local Lymph Node Assay, UV-LLNA) for the assessment of in vivo photosafety was used to investigate the impact of formulations, dose levels, duration of treatment, and timing of irradiation. Moreover, a basic pharmacokinetic profile was established within the same mouse strain. We were able to demonstrate dose- and time-dependent phototoxicity of vemurafenib using commercially available tablets (stabilized amorphous material). The lowest phototoxic dose was 350 mg/kg administrated for 3 consecutive days followed by exposure to UV-visible irradiation at a UVA-normalized dose of 10 J/cm². In comparison, pure vemurafenib, which easily forms crystalline variants and is known to have poor bioavailability, was tested at 350 mg/kg, and no signs of phototoxicity could be seen. The most apparent difference between the early study in hairless rats and this study in mice was the spectral range of the irradiation light source (350-400 nm vs 320-700 nm). Because vemurafenib does not absorb sufficiently light above 350 nm, this difference can easily explain the negative earlier study result in hairless rats.

Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing.

A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.

Collaborative study on fifteen compounds in the rat-liver Comet assay integrated into 2- and 4-week repeat-dose studies.

A collaborative trial was conducted to evaluate the possibility of integrating the rat-liver Comet assay into repeat-dose toxicity studies. Fourteen laboratories from Europe, Japan and the USA tested fifteen chemicals. Two chemicals had been previously shown to induce micronuclei in an acute protocol, but were found negative in a 4-week Micronucleus (MN) Assay (benzo[a]pyrene and 1,2-dimethylhydrazine; Hamada et al., 2001); four genotoxic rat-liver carcinogens that were negative in the MN assay in bone marrow or blood (2,6-dinitrotoluene, dimethylnitrosamine, 1,2-dibromomethane, and 2-amino-3-methylimidazo[4,5-f]quinoline); three compounds used in the ongoing JaCVAM (Japanese Center for the Validation of Alternative Methods) validation study of the acute liver Comet assay (2,4-diaminotoluene, 2,6-diaminotoluene and acrylamide); three pharmaceutical-like compounds (chlordiazepoxide, pyrimethamine and gemifloxacin), and three non-genotoxic rodent liver carcinogens (methapyrilene, clofibrate and phenobarbital). Male rats received oral administrations of the test compounds, daily for two or four weeks. The top dose was meant to be the highest dose producing clinical signs or histopathological effects without causing mortality, i.e. the 28-day maximum tolerated dose. The liver Comet assay was performed according to published recommendations and following the protocol for the ongoing JaCVAM validation trial. Laboratories provided liver Comet assay data obtained at the end of the long-term (2- or 4-week) studies together with an evaluation of liver histology. Most of the test compounds were also investigated in the liver Comet assay after short-term (1-3 daily) administration to compare the sensitivity of the two study designs. MN analyses were conducted in bone marrow or peripheral blood for most of the compounds to determine whether the liver Comet assay could complement the MN assay for the detection of genotoxins after long-term treatment. Most of the liver genotoxins were positive and the three non-genotoxic carcinogens gave negative result in the liver Comet assay after long-term administration. There was a high concordance between short- and long-term Comet assay results. Most compounds when tested up to the maximum tolerated dose were correctly detected in both short- and long-term studies. Discrepant results were obtained with 2,6 diaminotoluene (negative in the short-term, but positive in the long-term study), phenobarbital (positive in the short-term, but negative in the long-term study) and gemifloxacin (positive in the short-term, but negative in the long-term study). The overall results indicate that the liver Comet assay can be integrated within repeat-dose toxicity studies and efficiently complements the MN assay in detecting genotoxins. Practical aspects of integrating genotoxicity endpoints into repeat-dose studies were evaluated, e.g. by investigating the effect of blood sampling, as typically performed during toxicity studies, on the Comet and MN assays. The bleeding protocols used here did not affect the conclusions of the Comet assay or of the MN assays in blood and bone marrow. Although bleeding generally increased reticulocyte frequencies, the sensitivity of the response in the MN assay was not altered. These findings indicate that all animals in a toxicity study (main-study animals as well as toxicokinetic (TK) satellite animals) could be used for evaluating genotoxicity. However, possible logistical issues with scheduling of the necropsies and the need to conduct electrophoresis promptly after tissue sampling suggest that the use of TK animals could be simpler. The data so far do not indicate that liver proliferation or toxicity confound the results of the liver Comet assay. As was also true for other genotoxicity assays, criteria for evaluation of Comet assay results and statistical analyses differed among laboratories. Whereas comprehensive advice on statistical analysis is available in the literature, agreement is needed on applying consistent criteria.

Repair of sparfloxacin-induced photochemical DNA damage in vivo.

The induction and subsequent repair of photochemically induced DNA damage by sparfloxacin was assessed in different tissues of juvenile Wistar rats. The animals were treated once orally with 500 mg kg(-1) of sparfloxacin and irradiated 3 hours later with 7 J cm(-2) UVA. Induction and repair of DNA damage was studied in the skin, retina and cornea using the alkaline comet assay. After a tissue-specific increase in the initial DNA damage (higher in the cornea than in skin and retina), an exponential decrease was found in the skin and retina, whereas in cornea a further increase of the DNA damage after 1 hour followed by an exponential decrease was observed. The half-lives for DNA repair were approximately 3 hours for skin and retina and 1 hour for cornea. After a recovery time of 6 hours, the majority of the induced DNA damage detectable with the comet assay had been removed. In conclusion, the data indicate that (1) photochemically induced DNA damage by sparfloxacin is efficiently removed in skin, retina and cornea, (2) repair of these DNA lesions follows an exponential decrease, (3) the induction and repair of sparfloxacin-mediated photochemical DNA damage might be tissue specific.

Detection of photogenotoxicity in skin and eye in rat with the photo comet assay.

Photosensitizing drugs increase the sensitivity of the skin and the eye toward normally harmless sunlight conditions and are known to enhance the induction of skin tumors or severe injuries to the eye. The photogenotoxicity of five common drugs (sparfloxacin, dacarbazine, chlorpromazine and 8-methoxypsoralen, promazine) was investigated in the skin as well as in the retina and cornea of Wistar rats. The compounds were administered once orally by gavage and the resulting DNA damage was analyzed in the newly developed in vivo photo comet assay. All drugs except of promazine were clearly photogenotoxic in the skin. In the cornea sparfloxacin and dacarbazine induced an increased DNA damage following irradiation. A photogenotoxic effect in the retina was observed by sparfloxacin, which is the only compound tested that absorbs wavelengths reaching the retina. The drug concentration analysis revealed that the compounds were distributed into plasma, skin and eye at concentrations, which were photogenotoxic in vitro. Additionally, histopathological analysis showed no relevant alterations or inductions of necrosis, apoptosis or inflammation in the skin or eye. In conclusion, we confirmed the photogenotoxic potential of compounds from different chemical classes in the skin. Moreover, it is the first time that photogenotoxicity has been detected in the retina and cornea in an in vivo study. Based on our results it is concluded that the photo comet assay in rat is an easy and reliable method to elucidate drug induced photogenotoxicity under conditions, which are relevant to human exposure.

The photo comet assay--a fast screening assay for the determination of photogenotoxicity in vitro.

Different classes of chemicals can induce a phototoxic effect by absorbing light energy within the wavelength range of sunlight. The assessment of photo-safety is therefore an obligatory part of the development of new drugs. Ten UV-vis (280-800nm)-absorbing compounds (ketoprofen, promazine, chlorpromazine, dacarbazine, acridine, lomefloxacin, 8-methoxypsoralen, chlorhexidine, titanium dioxide, octylmethoxycinnamate) were tested for their photogenotoxic potential in the alkaline comet assay in the presence and absence of UV-vis. In order to establish an easy and timesaving protocol for a photo comet assay screening test, the application of 96-well plates was essential. The use of mouse lymphoma L5178Y cells, a cell line growing in suspension, allowed the determination of photocytotoxicity with the Alamar Blue assay and of photogenotoxicity with the alkaline comet assay in parallel. L5178Y cells were incubated with the test compounds for 20min and irradiated with simulated sunlight in the wavelength range from 280 to 800nm. The applied UV dose was 600mJ/cm(2) UV-A and 30mJ/cm(2) UV-B. After a post-incubation of 10min, the Alamar Blue assay and the alkaline comet assay were performed. All of the compounds which are known to be photogenotoxic (8-methoxypsoralen, acridine, chlorpromazine, dacarbazine, ketoprofen, lomefloxacin) showed a positive effect under our assay conditions. Furthermore, four UV-vis absorbing chemicals which are known to be not photogenotoxic (promazine, chlorhexidine, titanium dioxide, octylmethoxycinnamate) were analysed. For none of them an increase of the DNA damage following irradiation was observed in this study. In conclusion, all of the chemical compounds tested were classified in agreement with published data. From the data presented it is concluded that the photo comet assay with L5178Y mouse lymphoma cells is a reliable model to assess photochemical genotoxicity in vitro.

Induction of gene mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue.

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.

Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations.

The alkaline Comet assay was used to investigate the in vivo genotoxicity of 17 compounds. Altogether 21 studies were conducted with these compounds. The investigations were triggered for various reasons. The main reason for performing the studies was to evaluate the in vivo relevance of in vitro genotoxicity findings with 10 compounds. Eight of these compounds showed no effects in the in vivo Comet assay while two compounds induced altered DNA migration patterns in specific organs. The remaining seven compounds were tested to follow up on neoplastic/preneoplastic or chronic toxicity changes as detected in specific target organs identified in rodent studies, to investigate the possibility of site-of-contact genotoxicity and to test the liver as a target organ for a suspected reactive metabolite. For the studies, various organs of rodents were analyzed, depending on the suspected properties of the compounds, including liver, jejunum, leukocytes, stomach mucosa, duodenum, lung and kidney. All tissues were amenable to investigation by gel electrophoresis after simple disaggregation of organs by means of mincing or, in the case of epithelial cells from the gastrointestinal tract, scraping off cells from the epithelium. In conclusion, the Comet assay was found to be a reliable and robust test to investigate in vivo genotoxicity in a variety of rodent organs. Therefore, it is concluded that in vivo Comet assay data are useful for elucidating positive in vitro genotoxicity findings and to evaluate genotoxicity in target organs of toxicity.