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Given the widespread applications in industrial and agricultural production, the health effects of rare earth elements (REEs) have garnered public attention, and the genotoxicity of REEs remains unclear. In this study, we evaluated the genetic effects of lanthanum nitrate, a typical representative of REEs, with guideline-compliant in vivo and in vitro methods. Genotoxicity assays, including the Ames test, comet assay, mice bone marrow erythrocyte micronucleus test, spermatogonial chromosomal aberration test, and sperm malformation assay were conducted to assess mutagenicity, chromosomal damage, DNA damage, and sperm malformation. In the Ames test, no statistically significant increase in bacterial reverse mutation frequencies was found as compared with the negative control. Mice exposed to lanthanum nitrate did not exhibit a statistically significant increase in bone marrow erythrocyte micronucleus frequencies, spermatogonial chromosomal aberration frequencies, or sperm malformation frequencies compared to the negative control (P > 0.05). Additionally, after a 24-h treatment with lanthanum nitrate at concentrations of 1.25, 5, and 20 μg/ml, no cytotoxicity was observed in CHL cells. Furthermore, the comet assay results indicate no significant DNA damage was observed even after exposure to high doses of lanthanum nitrate (20 μg/ml). In conclusion, our findings suggest that lanthanum nitrate does not exhibit genotoxicity., Competing Interests: Declaration of Competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: T.B.Z, A.B, P.V, C.C.V and J.J.S, declare that they are employees and equity holders at TwinStrand Biosciences, Inc. and are authors on one or more Duplex Sequencing-related patents., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Evaluation of primary DNA-damage is one way to identify potential genotoxic agents and for this purpose the Comet assay has, for the last decades, been used to monitor DNA single strand and double strand breaks in individual cells. Various attempts have been made to modify the different steps in the in vitro protocol for the Comet assay in order to improve its sensitivity. However, to the best of our knowledge, nobody has tried to replace the traditionally used NaOH-based electrophoresis solution (pH > 13), with another type of solution. In the present paper, using TK-6 cells exposed to different concentrations of H 2 O 2 or ionizing radiation, we present evidence clearly showing that a low-conductive LiOH-based electrophoresis solution at pH 12.5, and a more gentle lysis procedure, significantly improved both the speed and sensitivity of the assay. The new approach, which we call the Flash-comet, is based on a lysis buffer at pH 8.5, an unwinding time of 2.5 min in a LiOH solution without EDTA at pH 12.5, and an electrophoresis time of 1 min at 150 V (5 V/cm) using the same solution., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Ferric hexacyanoferrate(II) (Fe 4 [Fe(CN) 6 ] 3 ), i.e. Prussian blue (PB) has been used for many years to remove from the body the two toxic isotopes of cesium and thallium following irradiation. Recently, potassium cobalt hexacyanoferrate(II) (K 2 COFe(CN) 6 ), which has shown a better efficacy for decontamination, is also being considered for use to enhance the elimination of cesium isotopes. In view to its preclinical and clinical development, in vitro and in vivo GLP-compliant genotoxicity studies were carried out on this product as well as on PB for comparison. Several tests dissecting the main events leading to genotoxicity, i.e. mutagenicity and chromosomal aberrations, both structural and quantitative were implemented. In vitro, no mutagenic effect was observed in the Ames test but both compounds were positive in the mouse lymphoma assay on TK locus and induced clastogenic effects in the in vitro chromosomal aberrations test on human lymphocytes, either in absence or in presence of metabolic activation. K-Co-ferrocyanide was also assayed in vivo in the mouse bone marrow micronucleus assay and PB was assessed for DNA fragmentation in the rodent Comet assay in both glandular stomach and colon. In the in vivo micronucleus mouse bone marrow, K-Co-ferrocyanide did not display any genotoxic activity up to 2000 mg/kg/d (x2) by oral route. In opposite, PB induced a significant increase in DNA fragmentation both in the glandular stomach and in the colon of rat treated 3 times with intake ranging from 2000 to 500 mg/kg. PB should be considered as an in vivo mutagen as well as Potassium cobalt hexacyanoferrate(II) since the in vitro genotoxicity profiles of both ferrocyanides are quite similar. Their use as cesium/ thallium decontamination agents in human should be assessed following a benefit/risk approach to enable a robust decision-making., (Copyright © 2020. Published by Elsevier B.V.)

Some agrochemicals are genotoxic to several organisms. Nevertheless, few protocols are currently available for measuring the toxicogenetic effects of these compounds in target and non-target field-collected species of insects important to agriculture. Herein, we used the species Ceraeochrysa claveri (Neuroptera: Chrysopidae), a non-target predator insect, to investigate the ability of an azadirachtin-based biopesticide (Azamax™) to induce DNA damage. The alkaline version of the comet assay was standardized to evaluate genetic instability caused by the toxicant in somatic (gut) and germ (nurse cells and oocytes) cells of C. claveri. For this, C. claveri larvae were distributed into three groups (10/each) and treated with Azamax™ at 0, 0.3% or 0.5% throughout the larval stage. DNA damage (tail intensity) was measured in adult insects, four days after emerged. The data showed that both doses of Azamax™ (0.3% and 0.5%) were able to significantly (p < 0.05) increase DNA damage in somatic and germ cells of C. claveri. In conclusion, C. claveri (intestinal and ovarian cells) was a sensitive bioindicator for identifying Azamax™ genotoxic potential, whereas the comet assay was a useful tool for detecting the genotoxic hazard of the pesticide in the field-collected insect species. Given that estimation of adverse effects of pollutants on ecosystems is an essential component of environmental risk assessment, the approach used can be recommended to estimate the ecotoxicity of agricultural chemicals., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Genotoxic effects of nanomaterials (NMs) have been controversially reported in literature, and the mode of action (MoA) via DNA oxidation is cited as the main damage caused by them. Evidence of nano-silver as a crosslinker has been previously reported by the present research team in an in vivo fish genotoxicity study. Thus, aiming to confirm the evidence about NMs as crosslinker agent, the present investigation elucidated the genotoxic potential of NMs and their genotoxic MoA through in vitro assay with RTG-2 cells line (rainbow trout gonadal) by exposure to nano-silver (PVP-coated) and nano-titanium. The types and levels of DNA damage were assessed by the Comet assay (standard alkaline, hOGG1-modified alkaline, and two crosslink-modified alkaline versions). It was demonstrated that the use of the standard alkaline Comet assay alone may inaccurately predict the genotoxicity of NMs since oxidative and crosslink DNA damages were also verified in RTG-2 cells when assessed by the modified versions of the alkaline protocol. More importantly, it was confirmed that both nano-silver and nano-titanium acted as DNA-protein crosslinkers through the Comet assay version with proteinase K. As both nano-silver and nano-titanium present a great risk to aquatic life, these findings reinforce the need of genotoxicity testing strategies that encompass the assessment of different types of DNA damage, in order to ensure an accurate prediction of the genotoxic potential of NMs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In spite of its pioneer use in detecting mutational processes, Drosophila still plays an important role in those studies aiming to detect and quantify the induction of DNA damage. Here we describe two assays, one detecting primary damage (the Comet assay) and the other detecting somatic mutation and recombination effects (wing-spot test). It is important to emphasize that somatic recombination is a key event in cancer development and no assays exist at present to detect and quantify somatic recombination processes, other than the spot tests developed in Drosophila.

Due to the increasing use of gold nanoparticles (AuNPs) in different areas such as medicine, biotechnology or food sector, human exposure to them has grown significantly and its toxicity evaluation has become essential. Therefore, the purpose of this study was to compare the potential genotoxic effects of 30, 50 and 90 nm AuNPs, using in vitro comet assay with the in vivo mutagenic and recombinogenic activity (SMART Test) in Drosophila. The results indicated that in both cell lines, 30, 50 and 90 nm (1-10 μg ml -1 ) AuNPs increased DNA strand breaks following 24 h treatment. This damage was not dose and size-dependent. Moreover, a modified comet assay using endonuclease III and formamidopyrimidine-DNA glycosylase restriction enzymes showed that in both cell lines, pyrimidines and purines were oxidatively damaged by all AuNPs, being 90 nm AuNPs slightly more genotoxic. However, the data obtained with SMART showed that 30 nm AuNPs did not modify the spontaneous frequencies of spots indicating lack of mutagenic and recombinogenic activity. Therefore, further experiments must be carried out to gain a better understanding of the mechanism of action of AuNPs to ensure their safe use., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Titanium dioxide nanoparticles (TiO 2 NP) are used in the food, drug, and cosmetics industries and evaluation of their human and environmental toxicity is required. We have tested the toxicity of TiO 2 NP (anatase) with respect to developmental effects and DNA damage in Drosophila melanogaster strain Ok, using the eye-spot Somatic Mutation and Recombination Test (SMART) and the comet assay (neuroblasts). For the survival assay, TiO 2 NP were supplied to adult flies for 72 h and no adverse effects were seen. TiO 2 NP were supplied chronically for the prolificacy, SMART, and comet assays. TiO 2 NP increased fly prolificacy. With regard to genotoxicity, an increase was observed in the eye-spot SMART assay at 8 μg/mL dose, but not in the neuroblast comet assay for DNA damage., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The genotoxic potential of drugs is a serious problem, and its evaluation is one of the most critical processes of drug development. Although the comet assay of compound-exposed tissue is a frequently used genotoxicity test, its high false-positive rate is a major complication, and we consistently obtained false-positive results using the comet assay of mouse liver for nine hepatotoxic non-genotoxins (NGTXs). To identify novel genotoxin (GTX)-specific biomarkers, we screened the expression of 750 microRNAs (miRNAs) in the livers of mice treated with GTXs or NGTXs. Three miRNAs, miR-22-3p, miR-409-3p, and miR-543-3p, were significantly down-regulated in GTX-treated mouse liver. In contrast, these three miRNAs were significantly up-regulated in plasma. A discrimination model based on the expression levels of these biomarkers successfully identified GTXs and NGTXs. This novel biomarker expression-based discrimination model analysis using both liver and plasma is effective for detecting genotoxicity with high sensitivity and reliability to support drug development., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The problem of the currently used routine genotoxicity tests is relatively low predictivity of in vitro tests for in vivo genotoxicity and carcinogenicity. An important reason is considered to be inadequate expression of xenobiotic-metabolizing enzymes in indicator cell lines. The aim of our study was to generate metabolically active differentiated hepatic progenies (hDHP) from human adipose tissue-derived mesenchymal stem cells (hASC) for genotoxicity testing. hDHP, generated using a three-step hepatic differentiation procedure, expressed hepatic properties such as glycogen storage and albumin secretion. The results of the comet assay demonstrated comparable sensitivity of hASC and hDHP to detect DNA damage induced by a direct acting genotoxic agent tert-butylhydroperoxide. Exposure to model indirect acting genotoxins benzo(a)pyrene, aflatoxin B 1 , and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine did not induce DNA damage in hASC, while hDHP cells detected DNA damage induced by benzo(a)pyrene and aflatoxin B 1 , indicating their metabolic activity. The gene and protein expression analysis confirmed the presence of key enzymes involved in metabolism of the three genotoxins in hDHP cells. Moreover, the exposure of hDHP to the model pro-carcinogens altered the expression of selected metabolic genes. hDHP were further immortalized with hTERT transfection, resulting in a stable cell line that can be matured to metabolically active hDHP ready for genotoxicity testing in only 2 weeks. The advantage of these immortalized cells is their prolonged replicative life span and consequently limitless supply of hDHP cells. We conclude that hDHP cells have a great potential for the application in the routine genotoxicity testing and are therefore worth further investigations.

Nickel (Ni) compounds are classified as carcinogenic to humans but the underlying mechanisms are still poorly understood. Furthermore, effects related to nanoparticles (NPs) of Ni have not been fully elucidated. The aim of this study was to investigate genotoxicity and mutagenicity of Ni and NiO NPs and compare the effect to soluble Ni from NiCl 2 . We employed different models; i.e., exposure of (1) human bronchial epithelial cells (HBEC) followed by DNA strand break analysis (comet assay and γ-H2AX staining); (2) six different mouse embryonic stem (mES) reporter cell lines (ToxTracker) that are constructed to exhibit fluorescence upon the induction of various pathways of relevance for (geno)toxicity and cancer; and (3) mES cells followed by mutagenicity testing (Hprt assay). The results showed increased DNA strand breaks (comet assay) for the NiO NPs and at higher doses also for the Ni NPs whereas no effects were observed for Ni ions/complexes from NiCl 2 . By employing the reporter cell lines, oxidative stress was observed as the main toxic mechanism and protein unfolding occurred at cytotoxic doses for all three Ni-containing materials. Oxidative stress was also detected in the HBEC cells following NP-exposure. None of these materials induced the reporter related to direct DNA damage and stalled replication forks. A small but statistically significant increase in Hprt mutations was observed for NiO but only at one dose. We conclude that Ni and NiO NPs show more pronounced (geno)toxic effects compared to Ni ions/complexes, indicating more serious health concerns. Environ. Mol. Mutagen. 59:211-222, 2018. © 2017 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society., (© 2017 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.)

Objective: To verify the mutagenicity of 2-ethylhexyl acrylate( 2-EHA)using in vivo Pig-a gene mutation assay integrated system., Methods: The SPF-grade male SD rats( n = 30) were randomized to six treatment groups, i. e. 4 treatment groups( 100, 200, 400 and 800 mg/kg), a control group( vegetable oil) and a positive groups( Nethyl-N-nitrosourea, 10 mg/kg). All treatments were administrated by gavage for continuous 28 days. Tail vein blood specimens for Pig-a gene mutation assay were collected on days 0, 15 and 29. The number of mutant erythrocytes and reticulocytes was acquired by flow cytometer. Tail vein blood for comet assay was collected at 6 h after the final administration, followed by the bone marrow micronucleus test after animal sacrifice., Results: Later in the study, signs of mild poisoning were observed in the animals treated with 400 and 800 mg/kg BW 2-EHA. There was no significant difference among the groups in mutant cell frequency of erythrocytes and reticulocytes at all 3 timepoint in Piga gene mutation assay, and no significant difference among the groups in tail length and Olive tail moment in comet assay. There was likewise no significant difference among groups in micronucleus test., Conclusion: In present experiment conditions, 2-EHA did not show genotoxicity in Pig-a gene mutation assay, comet assay and micronucleus test, which indicated that 2-EHA probably is not mutagenic in vivo.

Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H 2 O 2 ) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H 2 O 2 , less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Development of approach or device to allow continuous multiple measurements, such as integrating cytotoxic and genotoxic analysis, is quite appealing for study of the drug's activity and mechanism of action or resistance. In this study, a single-cell-arrayed agarose chip system was developed to combine cell cultivation with subsequent in situ analysis of cytotoxicity and genotoxicity of the chemotherapeutic agent. The modified alkaline comet assay coupled with the Live/Dead assay was used to monitor the interstrand cross-links (ICLs) formation and the cytotoxic effects in different glioma cell lines. In addition, the ICL-induced double strand breaks (DSBs) was measured on the chip to reflect the level of ICLs indirectly. Compared with the traditional methods, the microarray agarose device offers higher throughput, reproducibility, and robustness, exhibiting good potential for high-content drug screening.

The goal of the current study was to evaluate different genotoxicity tools in order to assess a marine protected area (MPA) affected by former mining activities and urban settlements. A catfish (Cathorops spixii) was analyzed for genotoxic effects at the (i) molecular and at the (ii) chromosomal levels. Through factor analysis, genotoxicity was found to be linked to levels of metals bioaccumulated and PAH metabolites in the bile. Micronucleus and nuclear alteration were less vulnerable to the effects of confounding factors in mildly contaminated areas since they were more frequently associated with bioaccumulated metals than the DNA analysis. The different genotoxicity responses allowed for the identification of sources of pollution in the MPA. This approach was important for detecting environmental risks related to genotoxic contaminants in a mildly contaminated MPA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

During interlaboratory validation trials for the Pig-a gene mutation assay we assessed the genotoxicity of 4-nitroquinoline-1-oxide (4NQO) across endpoints in multiple tissues: induction of Pig-a mutant red blood cells (RBCs) and reticulocytes (RETs); micronucleated RETs (MN RETs); and DNA damage in blood and liver via the alkaline Comet assay (%tail intensity [TI]). In a previous subchronic toxicity study with 28 daily doses, biologically meaningful increases were observed only for Pig-a mutant RBCs/RETs while marginal increases in the frequency of MN RET were observed, and other clastogenic endpoints were negative. Follow up acute studies were performed using the same cumulative doses (0, 35, 70, 105, and 140 mg/kg) administered in a bolus, or split over three equal daily doses, with samples collected up to 1 month after the last dose. Both of the acute dosing regimens produced similar results, in that endpoints were either positive or negative, regardless of 1 or 3 daily doses, but the three consecutive daily dose regimen yielded more potent responses in TI (in liver and blood) and Pig-a mutant frequencies. In these acute studies the same cumulative doses of 4NQO induced positive responses in clastogenic endpoints that were negative or inconclusive using a subchronic study design. Additionally, a positive control group using combination doses of cyclophosphamide and ethyl methanesulfonate was employed to assess assay validity and potentially identify a future positive control treatment for integrated genetic toxicity studies., (© 2015 Wiley Periodicals, Inc.)

Comet assay is an invaluable tool in DNA research. It is widely used to detect DNA damage as an indicator of exposure to genotoxic stress. A canonical set of parameters and specialized software programs exist for Comet assay data quantification and analysis. None of them so far has proven its potential to employ a computer-based algorithm for assessment of the shape of the comet as an indicator of the exact mechanism by which the studied genotoxins cut in the molecule of DNA. Here, we present 14 unique measurements of the comet image based on the comet morphology. Their mathematical derivation and statistical analysis allowed precise description of the shape of the comet image which in turn discriminated the cause of genotoxic stress. This algorithm led to the development of the "CometShape" software which allowed easy discrimination among different genotoxins depending on the type of DNA damage they induce., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The food processing contaminants 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 5-hydroxymethylfurfural (HMF) and 2,5 dimethylfuran (DMF) are potentially both mutagenic and carcinogenic in vitro and/or in vivo, although data on DMF is lacking. The PHIP metabolite N-hydroxy-PhIP and HMF are bioactivated by sulfotransferases (SULTs). The substrate specificity and tissue distribution of SULTs differs between species. A single oral dose of PhIP, HMF or DMF was administered to wild-type (wt) mice and mice expressing human SULT1A1/1A2 (hSULT mice). DNA damage was studied using the in vivo alkaline single cell gel electrophoresis (SCGE) assay. No effects were detected in wt mice. In the hSULT mice, PhIP and HMF exposure increased the levels of DNA damage in the liver and kidney, respectively. DMF was not found to be genotoxic. The observation of increased DNA damage in hSULT mice compared with wt mice supports the role of human SULTs in the bioactivation of N-hydroxy-PhIP and HMF in vivo., (© 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.)

Estragole, a naturally occurring constituent of various herbs and spices, is a rodent liver carcinogen which requires bio-activation. To further understand the mechanisms underlying its carcinogenicity, genotoxicity was assessed in F344 rats using the comet, micronucleus (MN), and DNA adduct assays together with histopathological analysis. Oxidative damage was measured using human 8-oxoguanine-DNA-N-glycosylase (hOGG1) and EndonucleaseIII (EndoIII)-modified comet assays. Results with estragole were compared with the structurally related genotoxic carcinogen, safrole. Groups of seven-week-old male F344 rats received corn oil or corn oil containing 300, 600, or 1,000 mg/kg bw estragole and 125, 250, or 450 mg/kg bw safrole by gavage at 0, 24, and 45 hr and terminated at 48 hr. Estragole-induced dose-dependent increases in DNA damage following EndoIII or hOGG1 digestion and without enzyme treatment in liver, the cancer target organ. No DNA damage was detected in stomach, the non-target tissue for cancer. No elevation of MN was observed in reticulocytes sampled from peripheral blood. Comet assays, both without digestion or with either EndoIII or hOGG1 digestion, also detected DNA damage in the liver of safrole-dosed rats. No DNA damage was detected in stomach, nor was MN elevated in peripheral blood following dosing with safrole suggesting that, as far both safrole and estragole, oxidative damage may contribute to genotoxicity. Taken together, these results implicate multiple mechanisms of estragole genotoxicity. DNA damage arises from chemical-specific interaction and is also mediated by oxidative species., (© 2014 Crown copyright.)

Physical, chemical and biological agents can act in the DNA, resulting in mutation involved in cancer. Thus, genotoxic tests are required by regulatory agencies in order to evaluate potential risk of cancer. Among these tests, the comet assay (CA) and micronucleus assay (MNA) are the most commonly used. However, there are different protocols and recommendations already published. This is the first review, after the inclusion of CA in S2R1 guidance and OECD 489, which summarizes the main technical recommendations of both CA and MNA., (Copyright © 2015. Published by Elsevier Masson SAS.)

The in vivo comet assay has recently been implemented into regulatory genotoxicity testing of pharmaceuticals with inclusion into the ICH S2R1 guidance. Regulatory genotoxicity testing aims to detect DNA alterations in form of gene mutations, larger scale chromosomal damage and recombination and aneuploidy. The ICH S2R1 guideline offers two options of standard batteries of tests for the detection of these endpoints. Both options start with an AMES assay and option 1 includes an in vitro mammalian cell assay and an in vivo micronucleus assay in rodent, whereas option 2 includes an in vivo micronucleus assay in bone marrow in rodent and a second in vivo assay in a second tissue with a second endpoint. The test recommended as second in vivo test is the comet assay in rat liver. The in vivo comet assay is considered as mature enough to ensure reliable detection of relevant in vivo genotoxicants in combination with the micronucleus test in bone marrow and the AMES assay. Although lots of research papers have been published using the in vitro comet assay, the in vitro version has not been implemented into official regulatory testing guidelines. A survey of the years 1999-2014 revealed 27 in vivo comet assays submitted to BfArM with market authorisation procedures, European and national advice procedures and clinical trial applications. In three procedures, in vitro comet assays had been submitted within the genetic toxicology packages., (© The Author 2014. Published by Oxford University Press on behalf of the Mutagenesis Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Thousands of dyes are marketed daily for different purposes, including textile dyeing. However, there are several studies reporting attributing to dyes deleterious human effects such as DNA damage. Humans may be exposed to toxic dyes through either ingestion of contaminated waters or dermal contact with colored garments. With respect to dermal exposure, human skin equivalents are promising tools to assess in vitro genotoxicity of dermally applied chemicals using a three-dimensional (3D) model to mimic tissue behavior. This study investigated the sensitivity of an in-house human dermal equivalent (DE) for detecting genotoxicity of textile dyes. Two azo (reactive green 19 [RG19] and disperse red 1[DR1]) dyes and one anthraquinone (reactive blue 2 [RB2]) dye were analyzed. RG19 was genotoxic for DE in a dose-responsive manner, whereas RB2 and DR1 were nongenotoxic under the conditions tested. These findings are not in agreement with previous genotoxicological assessment of these dyes carried out using two-dimensional (2D) cell cultures, which showed that DR1 was genotoxic in human hepatoma cells (HepG2) and RG19 was nongenotoxic for normal human dermal fibroblasts (NHDF). These discrepant results probably may be due to differences between metabolic activities of each cell type (organ-specific genotoxicity, HepG2 and fibroblasts) and the test setup systems used in each study (fibroblasts cultured at 2D and three-dimensional [3D] culture systems). Genotoxicological assessment of textile dyes in context of organ-specific genotoxicity and using in vitro models that more closely resemble in vivo tissue architecture and physiology may provide more reliable estimates of genotoxic potential of these chemicals.

The Cosmetics Europe (formerly COLIPA) Genotoxicity Task Force has driven and funded three projects to help address the high rate of misleading positives in in vitro genotoxicity tests: The completed "False Positives" project optimized current mammalian cell assays and showed that the predictive capacity of the in vitro micronucleus assay was improved dramatically by selecting more relevant cells and more sensitive toxicity measures. The on-going "3D skin model" project has been developed and is now validating the use of human reconstructed skin (RS) models in combination with the micronucleus (MN) and Comet assays. These models better reflect the in use conditions of dermally applied products, such as cosmetics. Both assays have demonstrated good inter- and intra-laboratory reproducibility and are entering validation stages. The completed "Metabolism" project investigated enzyme capacities of human skin and RS models. The RS models were shown to have comparable metabolic capacity to native human skin, confirming their usefulness for testing of compounds with dermal exposure. The program has already helped to improve the initial test battery predictivity and the RS projects have provided sound support for their use as a follow-up test in the assessment of the genotoxic hazard of cosmetic ingredients in the absence of in vivo data., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Doxorubicin (DOX) is an antineoplastic drug effective against many human malignancies. DOX's clinical efficacy is greatly limited because of severe cardiotoxicity. To evaluate if DOX is genotoxic in the heart, ~7-week-old, male F344 rats were administered intravenously 1, 2, and 3 mg/kg bw DOX at 0, 24, 48, and 69 hr and the Comet assays in heart, liver, kidney, and testis and micronucleus (MN) assay in the peripheral blood (PB) erythrocytes using flow cytometry were conducted. Rats were euthanized at 72 hr and PB was removed for the MN assay and single cells were isolated from multiple tissues for the Comet assays. None of the doses of DOX induced a significant DNA damage in any of the tissues examined by the alkaline Comet assay. Contrastingly, the glycosylase enzymes-modified Comet assay showed a significant dose dependent increase in the oxidative DNA damage in the cardiac tissue (P ≤ 0.05). In the liver, only the top dose induced significant increase in the oxidative DNA damage (P ≤ 0.05). The histopathology showed no severe cardiotoxicity but non-neoplastic lesions were present in both untreated and treated samples. A severe toxicity likely occurred in the bone marrow because no viable reticulocytes could be screened for the MN assay. Gene expression profiling of the heart tissues showed a significant alteration in the expression of 11 DNA damage and repair genes. These results suggest that DOX is genotoxic in the heart and the DNA damage may be induced primarily via the production of reactive oxygen species., (Copyright © 2013 Wiley Periodicals, Inc.)

The aim of genotoxicity tests in germ cells is to assess the impact of exposure to environmental mutagens that may represent a risk for the fertility or for the offspring of exposed subject. The comet assay on mature mouse oocytes is a simple, reproductive and rapid test to study primary DNA damage in oocytes. This test is used to complete toxicology assays applied in first line to somatic cells, and could find many applications in reproductive toxicology to study impact of environmental factors on female germ cells. We describe a practical application of comet assay in reproductive biology to assess the genotoxicity of cryoprotectants used at high concentrations in oocyte vitrification protocols. This test allowed us to demonstrate that dimethylsulfoxide and ethylene glycol are non-genotoxic for the mouse oocytes and led us to hypothesize a genotoxic effect of 1,2-propanediol (PrOH) at high concentrations after having observed induction of significant DNA damage on CHO cell line and on mouse oocytes., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Aristolochic acids (AAs) are carcinogenic plant toxins that are relatively strong gene mutagens, both in vitro and in vivo, but weak inducers of micronuclei in vivo. In order to clarify the reasons for these disparate responses, we evaluated the genotoxicity of AAs in F344 rats using several assays that respond to DNA damage in bone marrow. Groups of 7- to 8-week-old male rats (n=6) were gavaged with 0, 2.75, 5.5, and 11mg/kg AAs for 28 days or with 0, 11, 22, and 30mg/kg AAs for 3 days. Day 1 being the first day of treatment, Pig-a mutant frequencies (MFs) were assayed in peripheral blood erythrocytes up to Day 56 for the 28-day treatment or Day 42 for the 3-day treatment; micronuclei were assayed in peripheral blood reticulocytes on Day 4 (both treatment protocols) and on Day 29 of the 28-day treatment protocol; and at the final sampling times (Day 59 or Day 42), the animals were sacrificed and Hprt mutant lymphocytes were measured. In a separate study, the Comet assay was performed on liver, kidney, and bone marrow of animals gavaged with 0, 11, 22, and 30mg/kg AAs for 4 days and sacrificed 3h after the last treatment. While only weak increases in micronucleated reticulocyte frequency were observed in treated animals, Pig-a MFs increased in a dose- and time-dependent manner with both treatment schedules. Lymphocyte Hprt mutant frequencies also increased dose dependently in treated animals, and the Comet assay detected elevated levels of DNA damage in all the tissues evaluated. These findings indicate that the DNA damage produced by AAs in rat bone marrow is a weak inducer of micronuclei but a relatively strong inducer of gene mutation., (Published by Elsevier B.V.)

The genotoxic potential of waters in six rivers and reservoirs from Serbia was monitored in different tissues of chub (Squalius cephalus L. 1758) with the alkaline comet assay. The comet assay, or single-cell gel electrophoresis, has a wide application as a simple and sensitive method for evaluating DNA damage in fish exposed to various xenobiotics in the aquatic environment. Three types of cells, erythrocytes, gill cells, and liver cells, were used for assessing DNA damage. Images of randomly selected cells were analyzed with a Leica fluorescence microscope and image analysis by software (Comet Assay IV Image analysis system, PI, UK). Three parameters (tail length-l, tail intensity-i, and Olive tail moment-m) were analyzed on 1,700 nuclei per cell type. The procedure for sum of ranking differences (SRD) was implemented to compare different types of cells and different parameters for estimation of DNA damage. Regarding our nine different estimations of genotoxicity: tail length, intensity, and moment in erythrocytes (rel, rei, rem), liver cells (rll, rli, rlm), and gill cells (rgl, rgi, rgm), the SRD procedure has shown that the Olive tail moment and tail intensity are (almost) equally good parameters; the SRD value was lower for the tail moment and tail intensity than for tail length in the case of all types of cells. The least reliable parameter was rel; close to the borderline case were rei, rll, and rgl (~5 % probability of random ranking).

Nanocomposite is used as a dental filling to restore the affected tooth, especially in dental caries. The dental nanocomposite (KelFil) for tooth restoration used in this study was produced by the School of Dental Sciences, Universiti Sains Malaysia, Malaysia and is incorporated with monodispersed, spherical nanosilica fillers. The aim of the study was to determine the genotoxic effect of KelFil using in vitro genotoxicity tests. The cytotoxicity and genotoxicity of KelFil was evaluated using MTT assay, comet assay and chromosome aberration tests with or without the addition of a metabolic activation system (S9 mix), using the human lung fibroblast cell line (MRC-5). Concurrent negative and positive controls were included. In the comet assay, no comet formation was found in the KelFil groups. There was a significant difference in tail moment between KelFil groups and positive control (p < 0.05). Similarly, no significant aberrations in chromosomes were noticed in KelFil groups. The mitotic indices of treatment groups and negative control were significantly different from positive controls. Hence, it can be concluded that the locally produced dental restoration nanocomposite (KelFil) is non-genotoxic under the present test conditions.

Titanium dioxide nanoparticles (TiO2-NPs) are being used increasingly for various industrial and consumer products, including cosmetics and sunscreens because of their photoactive properties. Therefore, the toxicity of TiO2-NPs needs to be thoroughly understood. In the present study, the genotoxicity of 10nm uncoated sphere TiO2-NPs with an anatase crystalline structure, which has been well characterized in a previous study, was assessed using the Salmonella reverse mutation assay (Ames test) and the single-cell gel electrophoresis (Comet) assay. For the Ames test, Salmonella strains TA102, TA100, TA1537, TA98 and TA1535 were preincubated with eight different concentrations of the TiO2-NPs for 4 h at 37 °C, ranging from 0 to 4915.2 µg per plate. No mutation induction was found. Analyses with transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDS) showed that the TiO2-NPs were not able to enter the bacterial cell. For the Comet assay, TK6 cells were treated with 0-200 µg ml(-1) TiO2-NPs for 24 h at 37 °C to detect DNA damage. Although the TK6 cells did take up TiO2-NPs, no significant induction of DNA breakage or oxidative DNA damage was observed in the treated cells using the standard alkaline Comet assay and the endonuclease III (EndoIII) and human 8-hydroxyguanine DNA-glycosylase (hOGG1)-modified Comet assay, respectively. These results suggest that TiO2-NPs are not genotoxic under the conditions of the Ames test and Comet assay., (Published 2012. This article is a US Government work and is in the public domain in the USA.)

Nowadays, there is clear progress in using the threshold concept in genetic toxicology, but its demonstration and acceptance in risk assessment is still under debate. Although it has been accepted for some non-DNA-reactive agents for which mechanisms of action were demonstrated, there is a growing weight of evidence to also support the existence of thresholded dose-responses for DNA-reactive agents. In this context, we have recently shown in human TK6 lymphoblastoid cells, that DNA-oxidizing agents [potassium bromate, bleomycin and hydrogen peroxide (via glucose oxidase)] produced non-linear dose-responses in the in vitro micronucleus test, thus allowing the determination of No-Observed-Genotoxic-Effect-Levels (NOGELs). Therefore, the aim of the present study was to focus on the analysis of thresholded dose-response curves in order to further investigate the existence of NOGELs for these same directly DNA-damaging agents, by use of other genotoxicity endpoints. Mutation frequency was determined after a 1-h treatment in the thymidine kinase (TK) gene-mutation assay. Primary DNA damage, especially oxidative DNA damage, was also assessed after 1h of treatment, followed - or not - by a 23-h recovery period, with the modified version of the comet assay (i.e. with the glycosylases Fpg and hOgg1). Overall, our analysis demonstrates that there is convincing evidence to support the existence of thresholded dose-responses for DNA-oxidizing agents. The determination of NOGELs depends on the genotoxic endpoint studied and consequently requires different genotoxicity assays performed concurrently. NOGELs could only be defined for the induction of chromosomal aberrations and gene mutations, i.e. for an effect-endpoint but not for primary DNA damage, i.e. for an exposure-endpoint. Further statistical analyses of these data are now required in order to draw conclusions on the exact level of the thresholds., (Copyright © 2011 Elsevier B.V. All rights reserved.)

As part of the Stage 3 of the Pig-a international trial, we evaluated 7,12-dimethylbenz(a)anthracene (DMBA) for induction of Pig-a gene mutation using a 28-day repeat dose study design in Sprague-Dawley rats. In the same study, chromosomal damage in peripheral blood and primary DNA damage in liver were also investigated by the micronucleus (MN) assay and the Comet assay, respectively. In agreement with previously published data (Dertinger et al., [2010]: Toxicol Sci 115:401-411), DMBA induced dose-dependent increases of CD59-negative erythrocytes/reticulocytes and micronucleated reticulocytes (MN-RETs). However, there was no significant increase in DNA damage in the liver cells when tested up to 10 mg/kg/day, which appears to be below the maximum tolerated dose. When tested up to 200 mg/kg/day in a follow-up 3 dose study, DMBA was positive in the liver Comet assay. Additionally, we evaluated diethylnitrosamine (DEN), a known mutagen/hepatocarcinogen, for induction of Pig-a mutation, MN and DNA damage in a 28-day study. DEN produced negative results in both the Pig-a mutation assay and the MN assay, but induced dose-dependent increases of DNA damage in the liver and blood Comet assay. In summary, our results demonstrated that the Pig-a mutation assay can be effectively integrated into repeat dose studies and the data are highly reproducible between different laboratories. Also, integration of multiple genotoxicity endpoints into the same study not only provides a comprehensive evaluation of the genotoxic potential of test chemicals, but also reduces the number of animals needed for testing, especially when more than one in vivo genotoxicity tests are required., (Copyright © 2011 Wiley Periodicals, Inc.)

As part of the Stage III Pig-a multilaboratory validation trial, we examined the induction of CD59-negative reticulocytes and total red blood cells (RET(CD59-) and RBC(CD59-) , respectively) in male Sprague Dawley(®) rats treated with 4-nitroquinoline-1-oxide (4NQO), for 28 consecutive days by oral gavage, at doses of 1.25, 2.50, 3.75, 5.00, and 7.50 mg kg(-1) day(-1) (the high dose group was sacrificed on Day 15 due to excessive morbidity/mortality). Animals also were evaluated for: micronucleated reticulocytes (mnRET) by flow cytometry; DNA damage in peripheral blood, liver, and stomach using the Comet assay; and chromosome aberrations (CAb) in peripheral blood lymphocytes (PBL). All endpoints were analyzed at two or more timepoints where possible. Mortality, body and organ weights, food consumption, and clinical pathology also were evaluated, and demonstrated that the maximum tolerated dose was achieved at 5.00 mg kg(-1) day(-1) . The largest increases observed for the genetic toxicology endpoints (fold-increase compared to control, where significant; all at 5.00 mg kg(-1) day(-1) on Day 29) were: RET(CD59-) (21X), RBC(CD59-) (9.0X), and mnRET (2.0X). In contrast, no significant increases were observed for the CAb or Comet response, in any tissue analyzed, at any timepoint. Because 4NQO is a well known mutagen, clastogen, and carcinogen, the lack of response for these latter endpoints was unexpected. These results emphasize the extreme care that must betaken in dose and endpoint selection when incorporating genotoxicity endpoints into routine toxicity studies as has been recommended or is under consideration by various regulatory and industrial bodies., (Copyright © 2011 Wiley-Liss, Inc.)

N-methyl-N-nitrosourea (MNU) was evaluated in the in vivo Pig-a mutation assay as part of an International Collaborative Trial to investigate laboratory reproducibility, 28-day study integration, and comparative analysis with micronucleus (MN), comet, and clinical pathology endpoints. Male Sprague Dawley rats were treated for 28 days with doses of 0, 2.5, 5, and 10 mg MNU/kg/day in two independent laboratories, GlaxoSmithKline (GSK) and Bristol Myers Squibb (BMS). Additional studies investigated the low-dose region (<2.5 mg/kg/day). Reticulocytes were evaluated for Pig-a phenotypic mutation, CD59-negative reticulocytes/erythrocytes (RETs(CD592-)/ RBCs(CD592-)) on Days 1, 4, 15, 29, 43, and 57, and for micronucleated reticulocytes (MN-RETs) on Days 4 and 29. Comet analysis was conducted for liver and whole blood, and hematology and clinical chemistry was investigated. Dose-dependent increases in the frequency of RETs(CD592-) and RBCs(CD592-) were observed by Day 15 or 29, respectively. Dose-dependent increases were observed in %MN-RET on Days 4 and 29, and in mean %tail intensity in liver and in blood. Hematology/clinical chemistry data demonstrated bone marrow toxicity. Data comparison between GSK and BMS indicated a high degree of concordance with the Pig-a mutation assay results, consistent with previous observations with MNU and N-ethyl-N-nitrosourea. These data confirm that complementary genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that can provide information on gene mutation, chromosome damage, and DNA strand breaks in a single repeat dose rodent study. Collectively, this would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints.

N-Ethyl-N-nitrosourea (ENU) was evaluated as part of the Stage III trial for the rat Pig-a gene mutation assay. Groups of six- to eight-week-old male Sprague Dawley (SD) or Fischer 344 (F344) rats were given 28 daily doses of the phosphate buffered saline vehicle, or 2.5, 5, or 10 mg/kg ENU, and evaluated for a variety of genotoxicity endpoints in peripheral blood, spleen, liver, and colon. Blood was sampled predose (Day-1) and at various time points up to Day 57. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD592-) and RET(CD592-) frequencies. Consistent with the results from a reference laboratory, RBC(CD592-) and RET(CD592-) frequencies increased in a dose and time-dependent manner, producing significant increases at all doses by Day 15, with similar frequencies seen in both rat strains. ENU also induced small but significant increases in % micronucleated RETs on Days 4 and 29. No significant increases in micronuclei were seen in the liver or colon of the ENU-treated SD rats. Hprt and Pig-a lymphocyte mutation assays conducted on splenocytes from Day 56 F344 rats detected two- to fourfold stronger responses for Hprt than Pig-a mutations. Results from the in vivo Comet assay in SD rats at Day 29 showed generally weak increases in DNA damage in all tissues evaluated. The results with ENU indicate that the Pig-a RET and RBC assays are reproducible, transferable, and complement other genotoxicity endpoints that could potentially be integrated into 28-day repeat dose rat studies.

Genotoxicity assays were conducted on rats treated with benzo[a]pyrene (BaP) as part of Stage III of a validation study on the Pig-a gene mutation assay. Assays were performed at the U.S. FDA-NCTR and Bayer-Germany. Starting on Day 1, groups of five 6- to 7-week-old male Fischer 344 (F344, used at FDA-NCTR) and Han Wistar rats (Bayer) were given 28 daily doses of 0, 37.5, 75, or 150 mg/kg BaP; blood was sampled on Days -1, 4, 15, 29, and 56. Pig-a mutant frequencies were determined on Days -1, 15, 29, and 56 in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies; percent micronucleated-RETs (%MN-RET) were measured on Days 4 and 29. RBC(CD59-) and RET(CD59-) frequencies increased in a dose- and time-dependent manner, producing significant increases by Day 29 in both rat models. The responses for RETs were stronger than those for RBCs, and the responses in F344 rats were stronger than in Han Wistar rats. BaP also produced significant increases in %MN-RET frequency at Days 4 and 29, with the responses being greater in F344 than Han Wistar rats. The overall findings were consistent with those of the reference laboratory using Han Wistar rats. Finally, mutation assays performed on splenocytes from Day 56 F344 rats indicated that BaP mutant frequencies were three to fivefold higher for the Hprt gene than the Pig-a gene. The results indicate that the Pig-a RET and RBC assays are reproducible, transferable, and show promise for integrating gene mutation into 28-day repeat-dose studies., (Copyright © 2011 Wiley-Liss, Inc.)

A workshop to reappraise the previous IWGT recommendations for photogenotoxicity testing [E. Gocke, L. Muller, P.J. Guzzie, S. Brendler-Schwaab, S. Bulera, C.F. Chignell, L.M. Henderson, A. Jacobs, H. Murli, R.D. Snyder, N. Tanaka, Considerations on photochemical genotoxicity: report of the International Workshop on Genotoxicity Test Procedures working group, Environ. Mol. Mutagen., 35 (2000) 173-184] was recently held as part of the 5th International Workshop on Genotoxicity Testing (IWGT) meeting in Basel, Switzerland (August 17-19, 2009). An Expert Panel was convened from regulatory, academic and industrial scientists (with several members serving on the original panel) and chaired by Dr Peter Kasper (BfArM, Germany). The aim of the workshop was to review progress made in photo(geno)toxicity testing over the past decade; a period which saw the introduction of several regulatory photosafety guidances in particular in Europe and the USA. Based on current regulatory guidelines a substantial proportion of compounds trigger the requirements for photosafety testing. Moreover, there has been growing concern within industry about the performance of the in vitro photosafety tests in the "real world" of compound development. Therefore, the expert group reviewed the status of the current regulatory guidance's and the impact these have had on compound development in the context of the various triggers for photosafety testing. In addition, the performance of photogenotoxicity assays (old and new) was discussed, particularly in view of reports of pseudophotoclastogencity. The Expert Panel finished with an assessment of the positioning of photogenotoxicity testing within a photosafety testing strategy. The most significant conclusion made by the Expert Panel was that photogenotoxicity testing should no longer be recommended as part of the standard photosafety testing strategy. In addition, progress was made on the refinement of triggers for photosafety testing. For example, there was support for harmonisation of methods to determine the Molar Extinction Coefficient (MEC) and a consensus agreement that there should be no requirement for testing of compounds with a MEC<1000Lmol(-1)cm(-1)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The comet assay is a sensitive and rapid method for DNA strand break detection in individual cells, and the year 2009 represents the 25th anniversary of the first description of this methodology in 1984. Over time this method has been improved, but is still not completely standardized, and variations are currently in widespread use with emphasis on applications in research and genetic toxicology. Here we review the principles of the comet assay and cite key studies that have focused on this assay in the past 25 years. In addition, we present an example of how the comet assay was used in our laboratory for studying the induction of DNA damage in human lung cancer cells after differing doses of the cytosine analog 5-aza-2'-deoxycytidine (5-aza-CdR). Finally, some insights into the potential of this assay in cancer research and work in related fields are offered.

This review considers the potential of the Comet assay (or Single Cell Gel Electrophoresis, SCGE) to evaluate the environmental impact of genotoxins in aquatic environments. It focuses on in vivo and in situ studies that have been carried out in various marine and freshwater sentinel species, published in the last 5 years. A large number of the studies reviewed report that the Comet assay is more sensitive when compared with other biomarkers commonly used in genetic ecotoxicology, such as sister chromatid exchanges or micronucleus test. Due to its high sensitivity, the Comet assay is widely influenced by laboratory procedures suggesting that standard protocols are required for both fish and mussel cells. However, there are still a wide variety of personalised Comet procedures evident in the literature reviewed, making comparison between published results often very difficult. Standardization and inter-laboratory calibration of the Comet assay as applied to aquatic species will be required if the Comet assay is to be used routinely by national bodies charged with monitoring water quality.

Retinyl palmitate (RP), an ingredient of cosmetic and medical skin-care preparations, has been reported to be photo-genotoxic/photo-clastogenic in mouse lymphoma cells (Tk locus) as well as in human Jurkat T-cells, as measured by use of the comet assay. Given that these results were obtained under exploratory conditions, we re-investigated the photo-genotoxicity of RP following a protocol consistent with current recommendations for photo-genotoxicity testing of drugs and chemicals. We tested RP in Chinese hamster ovary (CHO) cells in the dark (standard chromosome aberration test), under pre-irradiation (UVA irradiation of cells and subsequent treatment with RP) or simultaneous irradiation (irradiation of cells and RP together, standard photo-genotoxicity protocol) conditions. UVA irradiation was applied at 350 and 700 mJ/cm2 with the high UV dose targeted to produce a small increase in the incidence of structural chromosome aberrations (CA) in cells not treated with RP. RP was tested up to and above its limit of solubility in the culture medium (20-40 μg/mL). There was no overt cytotoxicity under dark or different irradiation conditions. Treatment of cells with RP in the dark, as well as treatment under pre- or simultaneous irradiation conditions failed to produce biologically significant increases in the incidence of CA, whereas the positive control substances 4-nitroquinolone and 8-methoxypsoralene produced significantly positive effects in the dark or under simultaneous irradiation, respectively. Overall, our results failed to confirm the reported positive photo-genotoxic effects, and suggest that they may have been due to the test conditions, i.e. high irradiation doses, high cytotoxicity or re-irradiation of photo-products. In conclusion, our data suggest that, under standard conditions for testing photo-genotoxicity, RP had no in vitro genotoxic or photo-genotoxic potential and is therefore unlikely to pose a local or systemic genotoxic or photo-genotoxic risk.

Cypermethrin is the most widely used Type II pyrethroid pesticide because of its high effectiveness against target species and its low mammalian toxicity reported so far. It is a fast-acting neurotoxin and is known to cause free radical-mediated tissue damage. The present study investigates the genotoxic effects of cypermethrin in multiple organs (brain, kidney, liver, spleen) and tissues (bone marrow, lymphocytes) of the mouse, using the alkaline comet assay. Male Swiss albino mice were given 12.5, 25, 50, 100, 200 mg/kg BW of cypermethrin intraperitoneally, daily for 5 consecutive days. A statistically significant (p<0.05) dose-dependent increase in DNA damage was observed in all the organs assessed, as evident from the comet-assay parameters, viz., Olive tail moment (OTM; arbitrary unit), tail DNA (%) and tail length (microm). Brain showed maximum DNA damage followed by spleen>kidney>bone marrow>liver>lymphocytes, as evident by the OTM. Our data demonstrate that cypermethrin induces systemic genotoxicity in mammals as it causes DNA damage in vital organs like brain, liver, kidney, apart from that in the hematopoietic system.

The single cell gel electrophoresis or Comet assay is one of the most popular techniques for genotoxicity assessment. The present study was undertaken to validate our previously modified version of the Comet assay for genotoxicity assessment in Drosophila melanogaster (Oregon R(+)) with four well-known mutagenic and carcinogenic alkylating agents, i.e. ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU) and cyclophosphamide (CP). Third instar larvae (74 +/- 2 h) of D.melanogaster were fed different concentrations of EMS, MMS, ENU and CP (0.05, 0.5 and 1.0 mM) mixed standard Drosophila food for 24 h. At 98 +/- 2 h, the anterior midgut from control and treated larvae were dissected out, single-cell suspensions were prepared and Comet assay was performed. Our results show a dose-dependent increase in DNA damage with all the four alkylating agents, in comparison to control. The lower concentration (0.05 mM) of the test chemicals, except MMS, did not induce any DNA damage in the gut cells of the exposed larvae. When comparison of Comet parameters was made among the chemicals, MMS was found to be the most potent genotoxicant and ENU the least. The present study validated our previous observation and shows that D.melanogaster is a sensitive and suitable model for the in vivo assessment of genotoxicity using our modified alkaline Comet assay.

It is at present considered important to include biological tests in measuring programmes of environmental samples to supplement the chemical and physical parameters that are currently used. A battery of tests is therefore necessary, also within a given "endpoint" (e.g., genotoxicity), because one single test will not give all the answers to our questions. As it is not possible to include all available tests in routine screening programmes, a selection of tests should be made. According to comparative investigations, the bacterial Ames test remains very important. When no preconcentration step is involved, other bacterial tests (e.g., the umu-C and VITOTOX tests) may be recommended. The comet assay may be used in Daphnia or human white blood cells. Further validations, comparisons, mechanistic investigations, etc. remain necessary as differences are often found between the tests that are not solely explained by differences in genetic endpoint and that therefore should further be investigated and understood.