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The current Organisation for Economic Co-Operation and Development test guideline number 487 (OECD TG No. 487) provides instruction on how to conduct the in vitro micronucleus assay. This assay is one of the gold standard approaches for measuring the mutagenicity of test items; however, it is directed at testing low molecular weight molecules and may not be appropriate for particulate materials (e.g. engineered nanoparticles [ENPs]). This study aimed to adapt the in vitro micronucleus assay for ENP testing and underpins the development of an OECD guidance document. A harmonized, nano-specific protocol was generated and evaluated by two independent laboratories. Cell lines utilized were human lymphoblastoid (TK6) cells, human liver hepatocytes (HepG2) cells, Chinese hamster lung fibroblast (V79) cells, whole blood, and buffy coat cells from healthy human volunteers. These cells were exposed to reference ENPs from the Joint Research Council (JRC): SiO2 (RLS-0102), Au5nm and Au30nm (RLS-03, RLS-010), CeO2 (NM212), and BaSO4 (NM220). Tungsten carbide-cobalt (WC/Co) was used as a trial particulate positive control. The chemical controls were positive in all cell cultures, but WC/Co was only positive in TK6 and buffy coat cells. In TK6 cells, mutagenicity was observed for SiO2- and both Au types. In HepG2 cells, Au5nm and SiO2 showed sub-two-fold increases in micronuclei. In V79 cells, whole blood, and buffy coat cells, no genotoxicity was detected with the test materials. The data confirmed that ENPs could be tested with the harmonized protocol, additionally, concordant data were observed across the two laboratories with V79 cells. WC/Co may be a suitable particulate positive control in the in vitro micronucleus assay when using TK6 and buffy coat cells. Detailed recommendations are therefore provided to adapt OECD TG No. 487 for testing ENP., (© The Author(s) 2024. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.)

This study aimed to evaluate the scientific literature on the micronucleus assay in nasal mucosa as an appropriate method for evaluating genotoxicity caused by chemical agents. According to the PRISMA guidelines, only in vivo human studies with micronucleus assays using nasal cells were considered. Reviews, case reports, editorials, letters to the editor, and articles not written in English were excluded. The following scientific databases/search engines were used: PubMed/MEDLINE, Scopus, and Web of Science. Results: This review included 13 studies. Four articles detected no statistical significance regarding the frequency of micronuclei while nine articles showed an increase in micronuclei in nasal cells. In the qualitative analysis, two articles were considered strong, eight were moderate and three were weak. The micronucleus assay using nasal mucosa cells is a sensitive and effective technique for assessing DNA damage and an appropriate method for monitoring humans continuously exposed to chemicals.

Implementation of the seventh amendment to the EU Cosmetics Directive has driven much research into suitable in vitro alternative assays to support satisfactory risk assessments. One such assay is the reconstructed skin micronucleus (RSMN) assay. First reported in 2006, further development occurred and a standard protocol was published in 2011. To evaluate and optimise the assay at Covance Laboratories, we tested nine chemicals [4-nitrophenol (4-NP), cyclohexanone (CH), 2-ethyl-1,3-hexanediol (2-EHD), methyl methansulfonate (MMS), mitomycin C (MMC), ethyl nitrosourea (ENU), benzo[a]pyrene (BaP), cyclophosphamide (CPA) and vinblastine (VIN)] using the EpiDerm™ 3D skin model (MatTek Corporation®, IVLSL, Bratislava, Slovakia) and compared the data using the standard 48-h treatment regimen and also an emerging 72-h treatment protocol. The EpiDerm™ tissue has reportedly some metabolic capacity but data using 48-h treatments has provided mixed results. Our investigations demonstrate that the two chemicals requiring metabolic activation (BaP and CPA) were negative following the 48-h protocol but were clearly positive following 72-h treatment. Furthermore, Replication Index (RI) data showed higher RI values in vehicle control treatments (indicating increased cell division) across the treatment set following 72-h treatments. A general greater magnitude of micronucleus (MN) induction was also observed following test chemical treatment. These data suggest that the 72-h treatment protocol is more suitable as a standard approach for the detection of clastogenic, aneugenic and metabolically activated chemicals in the RSMN assay. For further assay optimisation, we compare the statistical power of scoring cells from duplicate or triplicate cultures per treatment concentration and provide recommendations., (© The Author(s) 2019. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To provide a comprehensive analysis of small molecule genotoxic potential we have developed and validated an automated, high-content, high throughput, image-based in vitro Micronucleus (IVM) assay. This assay simultaneously assesses micronuclei and multiple additional cellular markers associated with genotoxicity. Acoustic dosing (≤ 2 mg) of compound is followed by a 24-h treatment and a 24-h recovery period. Confocal images are captured [Cell Voyager CV7000 (Yokogawa, Japan)] and analysed using Columbus software (PerkinElmer). As standard the assay detects micronuclei (MN), cytotoxicity and cell-cycle profiles from Hoechst phenotypes. Mode of action information is primarily determined by kinetochore labelling in MN (aneugencity) and γH2AX foci analysis (a marker of DNA damage). Applying computational approaches and implementing machine learning models alongside Bayesian classifiers allows the identification of, with 95% accuracy, the aneugenic, clastogenic and negative compounds within the data set (Matthews correlation coefficient: 0.9), reducing analysis time by 80% whilst concurrently minimising human bias. Combining high throughput screening, multiparametric image analysis and machine learning approaches has provided the opportunity to revolutionise early Genetic Toxicology assessment within AstraZeneca. By multiplexing assay endpoints and minimising data generation and analysis time this assay enables complex genotoxicity safety assessments to be made sooner aiding the development of safer drug candidates.

The Buccal Micronucleus Cytome Assay (BMCyt) has become an important biomonitoring tool for assessing cytogenetic damage in many studied populations. Each laboratory applies protocols that vary according to the method of collecting and preparing samples. Besides, Brazil is a country of great territorial extensions that received immigrants from various parts of the world with different genetic backgrounds. Therefore, the present study aimed to evaluate the inter-laboratory variation in scoring the same set of slides using the more comprehensive scoring criteria, to standardize the BMCyt protocol, to observe the basal alterations in populations of different Brazilian regions and to compare it with other places around the world. Our results showed that a valuable number of laboratories participated, ten laboratories from different regions of the country, for the validation of the BMCyt in human biomonitoring studies, resulting in the 804 healthy individuals. This was possible because we observed: a range of measures needs to be considered, such as the baseline frequency of DNA damage and cell death in non-exposed individuals; age when grouped showed an influence on DNA damage, although when evaluated by group we did not see an influence; association between smoking habit and all endpoints of the BMCyt (except karyolytic cells) was evident; the basal MN frequency, in the majority of groups, follows those around the world; and the BMCyt was confirmed as a good health status biomarker. We emphasize the need for constant discussions on the parameters of cell death due to greater difficulty among the analyzers., Competing Interests: Declaration of Competing Interest The authors declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A database of micronuclei counts for historical negative control data from rat in vivo micronuclei tests performed in 10 different laboratories was established. Data were available from over 4000 negative control rats from 10 laboratories. The mean frequency of micronucleated cells (MN)/1000 cells ranged from 0.44 to 2.22, a 5-fold range. Overall there were no major sex or strain differences in frequency, although there were some small but statistically significant differences within laboratories. There was appreciable variability between experiments compared with variability within experiments in some laboratories. No specific factor was identified which could explain this variability although it was noted that many different vehicles were used in the experiments. It is hoped that these data will help laboratories beginning studies with the rat micronucleus assay and those involved in the assessment of micronucleus assay results., (Copyright © 2019. Published by Elsevier B.V.)

Purpose: Dose assessment plays an important role in case of radiological accidents and can be performed by scoring structural changes of chromosome morphology induced in cells by ionizing radiation. The results of such a test are biased by scorer experience, therefore, simple to learn assays are recommended to be used when fast analysis of a large amount of data is needed. The aim of this study was to compare the performance of two radiobiological assays - chromosomal aberrations and micronuclei - by unexperienced scorers with the reference values generated by an expert. Materials and methods: Each participant of an EU-funded two-week radiobiology course was asked to score Chinese hamster ovary cells exposed to gamma radiation up to 4 Gy. The congruence of students' and expert's scores at each dose and the coherence of the dose-response curve parameters between the students were investigated. Results: Micronucleus test tended to be faster and easier to learn than scoring chromosomal aberrations. However, both assays carried out by inexperienced students showed reasonable dose-response curves. Conclusions: In the case of a large radiological accident involving many casualties, the unexperienced scorers would support the process of biodosimetric triage by cytogenetic biological dosimetry.

The micronucleus test in peripheral blood lymphocytes is the most widely validated technique to evaluate the DNA damage and chromosomal instability in human populations. The test is largely applied in monitoring environmental and occupational exposure to genotoxic agents. It was also proposed as a biomarker of risk/susceptibility for cancer and other degenerative diseases. The availability of "normal values" in healthy populations is a main requisite for the assay application in human biomonitoring. Age and gender-related ranges of micronucleated binucleated cells (MNBN) baseline values were established in a group of 103 healthy platelet donors (50 males and 53 females) not recently exposed to genotoxic agents and characterized for demographic, lifestyle and dietary factors. Repeatability of the test by the same scorer was evaluated. Reproducibility was estimated through analysis of repeated blood samples. High correlation between the results of the three blood samplings in two separate scoring sessions of MNBN/1000BN (R 2 values were 0.83, 0.74 and 0.68; P < 0.0001) and PI values (R 2 values were 0.69, 0.62 and 0.65; P < 0.0001) was detected. High consistency among the values obtained in three different samplings in the same individual was observed (Intraclass Correlation Coefficient (ICC) = 0.905, (95% CI = 0.868-0.933, P < 0.0001) The range of "normal" values predicted on the basis of the results of the present study appears to be sufficiently narrow to warrant application of the assay in the comparison of data obtained from groups of exposed or susceptible subjects, supporting its use in preventive programs. The large inter-individual variability predicted by the model used in the present study hampers a clinical application of the assay at individual level. The method applied in the present study represents a generally applicable model to derive "normal values" in any population, as an essential step before starting a biomonitoring study., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study investigated the effectiveness of the liver micronucleus (MN) assay using juvenile mice. Therefore, we analyzed various hepatic cytochrome P450 (CYP)- mediated activities of ethoxyresorufin O-deethylation, pentoxyresorufin O-dealkylation, tolbutamide hydroxylation, bufuralol 1'-hydroxylation, aniline hydroxylation and midazolam 4-hydroxylation by CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A, respectively, in non-treated male ICR mice aged between 3 and 8 weeks. The enzyme efficiency levels in 3- and 4-week-old mice were approximately similar to or higher than those in 8-week-old mice, except for CYP1A and CYP2E in 3- and 4-week-old mice, respectively. Since these results suggest that juvenile mice have sufficient activities for most CYP enzymes, we also conducted a liver MN assay using diethylnitrosamine (DEN), a rodent hepatocarcinogen, on male ICR mice aged between 3 and 6 weeks. A peripheral blood (PB) MN assay was performed simultaneously in 4-week-old mice. Assays incorporating DEN produced positive results in 3- and 4-week-old mice and showed a dose-dependent increase in the micronucleated hepatocyte frequencies at 4 weeks. Both the liver MN assay in 5- and 6-week-old mice and the PB MN assay had negative results when using DEN. These results suggest that 3- and 4-week-old mice have micronuclei-inducing potential in the liver to detect genotoxic compounds using the liver MN assay.

Introduction: Smoking is the most important etiologic factor of oral cancer. Exfoliative cytology is the best method for early detection of oral cancer. Toluidine blue staining is used for detection of oral premalignant and malignant lesions. The aim of this study was to enhance the accuracy of oral exfoliative cytology in evaluating dysplastic features using toluidine blue staining., Methods and Materials: This clinical trials study was performed on 60 male smokers and nonsmokers without clinically oral lesion. Oral exfoliative cytological smears were prepared before and after application of toluidine blue and stained with Papanicolaou and evaluated under light microscope. Cytological features such as cellular clumping nuclear-to-cytoplasmic ratio, cellular and nuclear pleomorphism, micronuclei, binucleation, presence of bacterial colonies, and keratin flakes were assessed and compared before and after application of toluidine blue., Results: Results showed that cellular clumping and micronuclei were significantly decreased after application of toluidine blue and conversely cellular and nuclear pleomorphisms were significantly increased. Frequency of micronuclei and binucleation were greater in smokers than nonsmokers which were insignificant. Cellular and nuclear pleomorphisms were significantly higher in smokers than nonsmokers after application of toluidine blue., Conclusion: Toluidine blue improved cellular, nuclear, and structural features of oral cytological smears and filtered false-positive or false-negative results. Thus, application of toluidine blue in combination with oral exfoliative cytology for early detection of oral cancer is recommended. Diagn. Cytopathol. 2017;45:513-519. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

The buccal micronucleus cytome (BMNcyt) assay in uncultured exfoliated epithelial cells from oral mucosa is widely applied in biomonitoring human exposures to genotoxic agents and is also proposed as a suitable test for prescreening and follow-up of precancerous oral lesions. The main limitation of the assay is the large variability observed in the baseline values of micronuclei (MNi) and other nuclear anomalies mainly related to different scoring criteria. The aim of this international collaborative study, involving laboratories with different level of experience, was to evaluate the inter- and intra-laboratory variations in the BMNcyt parameters, using recently implemented guidelines, in scoring cells from the same pooled samples obtained from healthy subjects (control group) and from cancer patients undergoing radiotherapy (treated group). The results indicate that all laboratories correctly discriminated samples from the two groups by a significant increase of micronucleus (MN) and nuclear bud (NBUD) frequencies and differentiated binucleated (BN) cells, associated with the exposure to ionizing radiation. The experience of the laboratories was shown to play an important role in the identification of the different cell types and nuclear anomalies. MN frequency in differentiated mononucleated (MONO) and BN cells showed the greatest consistency among the laboratories and low variability was also detected in the frequencies of MONO and BN cells. A larger variability was observed in classifying the different cell types, indicating the subjectivity in the interpretation of some of the scoring criteria while reproducibility of the results between scoring sessions was very good. An inter-laboratory calibration exercise is strongly recommended before starting studies with BMNcyt assay involving multiple research centers., (© The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sensitivity and/or specificity of the in vivo erythrocyte micronucleus (MN) and transgenic rodent mutation (TGR) tests to detect rodent carcinogens and non-carcinogens were investigated. The Carcinogenicity and Genotoxicity eXperience (CGX) dataset created by Kirkland et al. was used for the carcinogenicity and in vitro genotoxicity data, i.e., Ames and chromosome aberration (CA) tests. Broad literature surveys were conducted to gather in vivo MN or TGR test data to add to the CGX dataset. Genotoxicity data in vitro were also updated slightly. Data on 379 chemicals (293 carcinogens and 86 non-carcinogens) were available for the in vivo MN test; sensitivity, specificity or concordances were calculated as 41.0%, 60.5% or 45.4%, respectively. For the TGR test, data on 80 chemicals (76 carcinogens and 4 non-carcinogens) were available; sensitivity was calculated as 72.4%. Based on the recent guidance on genotoxicity testing strategies, performance (sensitivity/specificity) of the following combinations was calculated; Ames+in vivo MN (68.7%/45.3%), Ames+TGR (83.8%/not calculated (nc)), Ames+in vitro CA+in vivo MN (80.8%/21.3%), Ames+in vitro CA+TGR (89.1%/nc), Ames+in vivo MN+TGR (87.5%/nc), Ames+in vitro CA+in vivo MN+TGR (89.3%/nc). Relatively good balance in performance was shown by the Ames+in vivo MN in comparison with Ames+in vitro CA (74.3%/37.5%). Ames+TGR and Ames+in vivo MN+TGR gave even higher sensitivity, but the specificity could not be calculated (too few TGR data on non-carcinogens). This indicates that in vivo MN and TGR tests are both useful as in vivo tests to detect rodent carcinogens., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

An in vitro study of the dose responses of human peripheral blood lymphocytes was conducted with the aim of creating calibrated dose-response curves for biodosimetry measuring up to 4 Gy (0.25-4 Gy) of gamma radiation. The cytokinesis-blocked micronucleus (CBMN) assay was employed to obtain the frequencies of micronuclei (MN) per binucleated cell in blood samples from 16 healthy donors (eight males and eight females) in two age ranges of 20-34 and 35-50 years. The data were used to construct the calibration curves for men and women in two age groups, separately. An increase in micronuclei yield with the dose in a linear-quadratic way was observed in all groups. To verify the applicability of the constructed calibration curve, MN yields were measured in peripheral blood lymphocytes of two real overexposed subjects and three irradiated samples with unknown dose, and the results were compared with dose values obtained from measuring dicentric chromosomes. The comparison of the results obtained by the two techniques indicated a good agreement between dose estimates. The average baseline frequency of MN for the 130 healthy non-exposed donors (77 men and 55 women, 20-60 years old divided into four age groups) ranged from 6 to 21 micronuclei per 1000 binucleated cells. Baseline MN frequencies were higher for women and for the older age group. The results presented in this study point out that the CBMN assay is a reliable, easier and valuable alternative method for biological dosimetry.

In this study, we explored the applicability of using in vitro micronucleus (MN) data from human lymphoblastoid TK6 cells to derive in vivo genotoxicity potency information. Nineteen chemicals covering a broad spectrum of genotoxic modes of action were tested in an in vitro MN test using TK6 cells using the same study protocol. Several of these chemicals were considered to need metabolic activation, and these were administered in the presence of S9. The Benchmark dose (BMD) approach was applied using the dose-response modeling program PROAST to estimate the genotoxic potency from the in vitro data. The resulting in vitro BMDs were compared with previously derived BMDs from in vivo MN and carcinogenicity studies. A proportional correlation was observed between the BMDs from the in vitro MN and the BMDs from the in vivo MN assays. Further, a clear correlation was found between the BMDs from in vitro MN and the associated BMDs for malignant tumors. Although these results are based on only 19 compounds, they show that genotoxicity potencies estimated from in vitro tests may result in useful information regarding in vivo genotoxic potency, as well as expected cancer potency. Extension of the number of compounds and further investigation of metabolic activation (S9) and of other toxicokinetic factors would be needed to validate our initial conclusions. However, this initial work suggests that this approach could be used for in vitro to in vivo extrapolations which would support the reduction of animals used in research (3Rs: replacement, reduction, and refinement)., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.)

Every year, many countries perform a significant number of investigations based on biological radiation dose assessment to check suspected or true overexposure by irradiation of radiation workers and individuals of the general population. The scoring of dicentrics in peripheral blood lymphocytes has gradually become the "gold standard" for the biodosimetry-based assessment of accidental situations. Nevertheless, other "classical" biodosimetric methods such as micronuclei, prematurely condensed chromosomes (PCC) and FISH translocations are relevant in some exposure situations, also for surveillance of groups of populations at risk. Historical international intercomparison studies have shown discrepancies among dose-effect curves used to estimate doses from blood samples irradiated between 0 and 4Gy. Recent experimental work performed by the biological dosimetry laboratory of the French Institute for Radiation Protection and Nuclear Safety (IRSN) has shown the impact of some blood harvesting parameters on the mitotic index, and consequently on the quality of dose assessment. Therefore, it was relevant to define the best Quality Assurance (QA) and Quality Control (QC) criteria to harmonize protocols among biodosimetry laboratories. Complementary with several editions of an IAEA technical manual, ISO standards were written with the view of considering the most used chromosome aberrations assays: dicentrics and micronuclei. An important feature of these standards is to address the organization of population triage and laboratories networking that would be required in case of a large nuclear event or malicious act involving radioactive material. These ISO standards are relevant and helpful to implement a coordinated response of several biodosimetry networks in Europe, Japan, Canada, and to support European programs such as MULTIBIODOSE and RENEB. A new important ISO standard on the use of FISH translocations in retrospective dosimetry is now being drafted., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The buccal micronucleus cytome (BMCyt) assay is a minimally invasive approach for measuring DNA damage, cell proliferation, cell differentiation and cell death in exfoliated buccal cells. The main limitation for its use is the lack of knowledge about inter- and intra-laboratory variability in scoring micronuclei and other end points included in the cytome approach. In order to identify the main sources of variability across the BMCyt biomarkers, a scoring exercise was carried out between three experienced laboratories using the same set of slides and an identical set of detailed scoring criteria and associated images for the different end points. Single batches of slides were prepared from pooled samples of four groups of subjects characterised by different frequencies of cell types and micronuclei, namely Down syndrome patients, head and neck cancer patients undergoing radiotherapy and two age- and gender-matched control groups. A good agreement among the laboratories in the identification of normal differentiated cells and of micronuclei was obtained. A 3-fold and 20-fold increase in the frequency of micronucleated cells and micronuclei in differentiated cells of Down syndrome patients and in cancer patients, respectively, compared to matched controls, was a consistent result in the three laboratories. The scores of other cell types and nuclear anomalies, such as basal, binucleated, condensed chromatin and karyorrhectic cells showed significant disagreement between and within laboratories indicating that their evaluation using the current visual scoring protocol does not yield robust results for these parameters. The guidelines for BMCyt assay application could be improved by combining the anomalies associated with cell death (condensed chromatin and karyorrhectic cells) in a single category and by defining more stringent criteria in classifying basal cell, binucleated cells and buds., (© The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

At the 6th International Workshop on Genotoxicity Testing (IWGT), the liver micronucleus test working group discussed practical aspects of the in vivo rodent liver micronucleus test (LMNT). The group members focused on the three methodologies currently used, i.e., a partial hepatectomy (PH) method, a juvenile/young rat (JR) method, and a repeated-dose (RD) method in adult rodents. Since the liver is the main organ that metabolizes chemicals, the LMNT is expected to detect clastogens, especially those that need metabolic activation in the liver, and aneugens. Based on current data the three methods seem to have a high sensitivity and specificity, but more data, especially on non-genotoxic but toxic substances, would be needed to fully evaluate the test performance. The three methods can be combined with the micronucleus test (MNT) using bone marrow (BM) and/or peripheral blood (PB). The ability of the PH method to detect both clastogens and aneugens has already been established, but the methodology is technically challenging. The JR method is relatively straightforward, but animal metabolism might not be fully comparable to adult animals, and data on aneugens are limited. These two methods also have the advantage of a short testing period. The RD method is also straightforward and can be integrated into repeated-dose (e.g. 2 or 4 weeks) toxicity studies, but again data on aneugens are limited. The working group concluded that the LMNT could be used as a second in vivo test when a relevant positive result in in vitro mammalian cell genotoxicity tests is noted (especially under the condition of metabolic activation), and a negative result is observed in the in vivo BM/PB-MNT. The group members discussed LMNT protocols and reached consensus about many aspects of test procedures. However, data gaps as mentioned above remain, and further data are needed to fully establish the LMNT protocol., (Copyright © 2014 Elsevier B.V. All rights reserved.)

At the 6th International Workshop on Genotoxicity Testing, the liver micronucleus test (MNT) working group briefly discussed the MNT using tissues other than liver/erythrocytes. Many tissues other than liver/erythrocytes have been studied, primarily for research purposes. They have included the colon and intestinal epithelium, skin, spleen, lung, stomach, bladder, buccal mucosa, vagina, and fetal/neonatal tissues. These tissues were chosen because they were target sites of carcinogens, and/or relevant to a specific route of exposure. Recently, there has been particular focus on the gastrointestinal (GI) tract as it is a contact site associated with high exposure following oral gavage. Furthermore GI tumors are observed with high frequency in human populations. A collaborative study of the rat glandular stomach and colon MNT was conducted in conjunction with a collaborative study of the repeated-dose liver MNT. Based on limited data currently available, the rodent MNT using the glandular stomach and/or colon seems to detect genotoxic carcinogens with GI tract target-organ specificity. The working group concluded that the GI tract MNT would be a promising method to examine clastogenicity or aneugenicity of test chemicals in the stomach and/or colon. Further data will be needed to fully establish the methods, and to identify the sensitivity and specificity of the GI tract MNT., (Copyright © 2015 Elsevier B.V. All rights reserved.)

An Inter-Laboratory Comparison (ILC) study on Dicentric Chromosome Assay (DCA) was carried out between two Indian biodosimetry labs. Human peripheral blood samples exposed to 10 different doses of X-rays up to 5Gy were shared between the labs to generate calibration data. Validation of calibration curves was done by dose estimation of coded samples exposed to X- or gamma radiation. Reliability of the DCA data for triage application was evaluated by scoring 20, 50 and 100 metaphases in the dose range of 0.5-3.0Gy. No significant difference was observed between labs regarding the established calibration data as well as the DCA triage dose assessments. Scoring of 20 metaphases (MP) was adequate to detect radiation exposure of >2Gy whereas 50 MP were sufficient to determine exposures of 0.5Gy. Both labs performed the DCA in a reliable manner and made the first step in setting up a biodosimetry network in India., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The Vicia micronucleus assay was standardized in an international protocol, ISO 29200, "Assessment of genotoxic effects on higher plants-Vicia faba micronucleus test," for soil or soil materials (e.g., compost, sludge, sediment, waste, and fertilizing materials). The aim of this interlaboratory study on the Vicia micronucleus assay was to investigate the robustness of this in vivo assay in terms of its applicability in different countries where each participant were asked to use their own seeds and reference soil, in agreement with the ISO 29200 standard. The ISO 29200 standard protocol was adopted for this study, and seven laboratories from three countries (France, Italy, and Brazil) participated in the study. Negative and positive controls were correctly evaluated by 100 % of the participants. In the solid-phase test, the micronucleus frequency (number of micronuclei/1,000 cells) varied from 0.0 to 1.8 for the negative control (i.e., Hoagland's solution) and from 5.8 to 85.7 for the positive control (i.e., maleic hydrazide), while these values varied from 0.0 to 1.7 for the negative control and from 14.3 to 97.7 for the positive control in the case of liquid-phase test. The variability in the data obtained does not adversely affect the robustness of the protocol assessed, on the condition that the methodology described in the standard ISO 29200 is strictly respected. Thus, the Vicia micronucleus test (ISO 29200) is appropriate for complementing prokaryotic or in vitro tests cited in legislation related to risk assessment of genotoxicity potential.

Biological dosimetry using chromosome damage biomarkers is a valuable dose assessment method in cases of radiation overexposure with or without physical dosimetry data. In order to estimate dose by biodosimetry, any biological dosimetry service have to have its own dose response calibration curve. This paper reveals the results obtained after irradiation of blood samples from fourteen healthy male and female volunteers in order to establish biodosimetry in Serbia and produce dose response calibration curves for dicentrics and micronuclei. Taking into account pooled data from all the donors, the resultant fitted curve for dicentrics is: Ydic=0.0009 (±0.0003)+0.0421 (±0.0042)×D+0.0602 (±0.0022)×D(2); and for micronuclei: Ymn=0.0104 (±0.0015)+0.0824 (±0.0050)×D+0.0189 (±0.0017)×D(2). Following establishment of the dose response curve, a validation experiment was carried out with four blood samples. Applied and estimated doses were in good agreement. On this basis, the results reported here give us confidence to apply both calibration curves for future biological dosimetry requirements in Serbia., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Genetic toxicity information is critical for the safety assessment of all xenobiotics. In the absence of carcinogenicity data, genetic toxicity studies may be used to draw conclusions about the carcinogenicity potential of chemicals. However, current in vitro assays have many limitations as they produce a high rate of irrelevant positive data and possible false negative data due to the weakness of the in vitro models used. Based on the knowledge that the majority of human genotoxic carcinogens require metabolic activation to become genotoxic, it is necessary to develop in vitro cell models that mimic human liver metabolism to replace the use of liver S9 fraction, which, though helpful for predicting the potential carcinogenicity of chemicals in rodents, is questionable in humans. We therefore investigate whether the recently described human hepatoma HepaRG cells, which express the major characteristics of liver functions similarly to primary human hepatocytes, could be a suitable model for human genotoxicity assessment. We determine the performance of comet and micronucleus assays in HepaRG cells to predict in vivo genotoxins based on the list of compounds published by European Centre for the Validation of Alternative Methods (ECVAM). Twenty compounds were tested in HepaRG cells with comet and micronucleus assays over a 24-h period. The specificity, the sensitivity, and the accuracy of the two tests were determined. We found that the comet assay had higher specificity (100%) than the micronucleus (MN) test (80%), whereas the latter was far more sensitive (73%) than the former (44%), resulting nonetheless in an accuracy of 72% for the comet assay and 75% for the MN test. Taken together, our data suggest that the HepaRG cell line can be of use in genetic toxicology and that efforts to develop competent human liver cell models should be increased.

With the increasing human exposure to nanoparticles (NP), the evaluation of their genotoxic potential is of significant importance. However, relevance for NP of the routinely used in vitro genotoxicity assays is often questioned, and a nanoparticulate reference positive control would therefore constitute an important step to a better testing of NP, ensuring that test systems are really appropriate. In this study, we investigated the possibility of using tungsten carbide-cobalt (WC-Co) NP as reference positive control in in vitro genotoxicity assays, including 2 regulatory assays, the mouse lymphoma assay and the micronucleus assay, and in the Comet assay, recommended for the toxicological evaluation of nanomedicines by the French Agency of Human Health Products (Afssaps). Through these assays, we were able to study different genetic endpoints in 2 cell types commonly used in regulatory genotoxicity assays: the L5178Y mouse lymphoma cell line and primary cultures of human lymphocytes. Our results showed that the use of WC-Co NP as positive control in in vitro genotoxicity assays was conceivable, but that different parameters have to be considered, such as cell type and treatment schedule. L5178Y mouse lymphoma cells did not provide satisfactory results in the 3 performed tests. However, human lymphocytes were more sensitive to genotoxic effects induced by WC-Co NP, particularly after a 24-h treatment in the in vitro micronucleus assay and after a 4-h treatment in the in vitro Comet assay. Under such conditions, WC-Co could be used as a nanoparticulate reference positive control in these assays.

Objective: This study evaluated the role of the micronucleus (MN) in liver fine needle aspiration (FNA) cytology., Methods: Histological features of 75 cases of hepatocellular carcinoma (HCC), of which 25 were well differentiated, 37 moderately differentiated and 13 poorly differentiated, and 58 benign hepatic lesions (total, 133 cases) were correlated with MN expression observed in FNA smears reported as benign (n =40), atypical (n = 14), suspicious (n = 30) and malignant (n =49)., Results: Stepwise increases in the MN score (0.4 ± 0.6, 1.2 ± 1.3, 6.3 ± 4.2 and 14.8 ± 8.8) correlated with the degree of cytological abnormality: benign, atypia, suspicious and malignant, respectively. The mean MN scores for well-, moderately and poorly differentiated HCC were 5.4 ± 2.2, 11.5 ± 4.5 and 24.9 ± 9.1, respectively, which was significantly different between malignant and suspicious (P < 0.0001), between suspicious and atypical (P= 0.008) but not between atypical and benign. The MN scores differed significantly between all degrees of differentiation of HCC and between the HCC and benign hepatic lesions (P < 0.0001). High sensitivity, specificity and accuracy of liver FNA for diagnosing HCC (96%, 98%, and 96%, respectively) were obtained at a cutoff of three for the MN score., Conclusions: The MN score is an effective HCC biomarker and has a good potential use as an ancillary tool for diagnosing HCC using FNA cytology., (© 2012 John Wiley & Sons Ltd.)

The buccal micronucleus cytome assay is a minimally invasive cytological and interphase cytogenetic technique for measuring DNA damage and cell death biomarkers in the oral epithelium. In this report we provide an updated and more comprehensive version of the cellular and nuclear scoring criteria used in the assay accompanied with a photogallery of the various cell types and nuclear anomalies. These detailed scoring criteria complement previous published protocols of this assay and form the basis for guiding intra- and inter-laboratory slide scoring comparisons. The scoring criteria update described in this paper is the outcome of ongoing efforts of the HUMN and HUMNxl projects (www.humn.org) to standardize the application of micronucleus assay for use in human biomonitoring and to update procedures as knowledge on mechanisms and technical capability improvements., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This commentary challenges the paradigm that the cytokinesis-block micronucleus assay (CBMN assay) with cultured human lymphocytes, as it is performed currently, is a sensitive and useful tool for detecting genotoxic effects in populations exposed occupationally or environmentally to genotoxic chemicals. Based on the principle of the assay and the available data, increased micronucleus (MN) frequencies in binucleated cells (BNC) are mainly due to MN produced in vitro during the cultivation period (i.e. MN produced in vivo do not substantially contribute to the MN frequency measured in BNC). The sensitivity of the assay for the detection of induced MN in BNC after an in vivo exposure to a genotoxic chemical is limited because cytochalasin B (Cyt-B) is added relatively late during the culture period and, therefore, the BNC that are scored do not always represent cells that have completed one cell cycle only. Furthermore, this delay means that damaged cells can be eliminated by apoptosis and/or that DNA damage induced in vivo can be repaired prior to the production of a MN in the presence of Cyt-B. A comparison with the in vitro CBMN assay used for genotoxicity testing leads to the conclusion that it is highly unlikely that DNA damage induced in vivo is the cause for increased MN frequencies in BNC after occupational or environmental exposure to genotoxic chemicals. This commentary casts doubt on the usefulness of the CBMN assay as an indicator of genotoxicity in human biomonitoring and questions the relevance of many published data for hazard identification and risk assessment. Thus, it seems worthwhile to reconsider the use of the CBMN assay as presently conducted for the detection of genotoxic exposure in human biomonitoring.

An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96-well plate (Bryce SM et al. [2010]: Mutat Res 703:191-199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non-genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5- to 2-cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating ≥ 5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN-fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non-genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpoints-relative survival and quantification of ethidium monoazide-positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96-well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity., (Copyright © 2013 Wiley Periodicals, Inc.)

The aim of this paper is to compare the spontaneous and induced with cyclophosphamide micronucleus indexes in bone marrow cells of the Sprague Dawley, Lewis and Wistar rat lines. Five experimental groups were formed (10 animals of each sex and of each line, in every group). The first group was used as the negative control (intact animals), the second one was exposed to oral administration of drugs; other conditions were the same as for the other groups. The third group was treated with 2% Tween 65 and the fourth group was treated with 0.9% NaCl. Both substances were administered by oral way to 2 ml/kg during 14 days. The fifth group was treated intraperitoneally with strong mutagen cyclophosphamide in the dose of 50 mg/kg (10 ml/kg in solution), on 48th and 24th hours before euthanasia. The Sprague Dawley line (both sexes) was significantly different from the other lines. Rats of this line had lower index of spontaneous formation of micronuclei, higher index of cyclophosphamide-induced micronucle formation, percent of micronucleated erythrocytes in bone marrow and the index of cytotoxicity. The results obtained make it possible to identify the most appropriate line of rats as model animals for studies of genotoxicity. It will allow also to obtain more accurate estimates of genotoxicity of various substances.

The in vitro micronucleus assay is currently one of the most commonly used test systems for the study of genotoxic effects of chemicals. It is considered the preferred method for measuring chromosome damage as it allows the determination of both chromosomal loss and breakage. The type of chromosomal damage induced can be distinguished by using the kinetochore or pan-centromeric staining using molecular probes that label the centromeric regions of chromosomes allowing the determination of aneugenic (chromosome loss) or clastogenic (chromosome breakage) agents. In this chapter, we provide a description of the basic principles and methods of the in vitro micronucleus assay with detailed explanations of the scoring criteria for the genotoxicity and cytotoxicity end-points by manual or automated analysis.

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.)

In this study we compared the response of the Pig-a gene mutation assay to that of the lacZ transgenic rodent mutation assay, and demonstrated that multiple endpoints can be measured in a 28-day repeat dose study. Muta™Mouse were dosed daily for 28 days with benzo[a]pyrene (BaP; 0, 25, 50 and 75 mg/kg body weight/day) by oral gavage. Micronucleus (MN) frequency was determined in reticulocytes (RETs) 48 hr following the last dose. 72 h following the last dose, mice were euthanized, and tissues (glandular stomach, small intestine, bone marrow and liver) were collected for lacZ mutation and DNA adduct analysis, and blood was evaluated for Pig-a mutants. BaP-derived DNA adducts were detected in all tissues examined and significant dose-dependent increases in mutant Pig-a phenotypes (i.e., RET(CD24-) and RBC (CD24-)) and lacZ mutants were observed. We estimate that mutagenic efficiency (i.e., rate of conversion of adducts into mutations) was much lower for Pig-a compared to lacZ, and speculate that this difference is likely explained by differences in repair capacity between the gene targets, and differences in the cell populations sampled for Pig-a versus lacZ. The BaP doubling doses for both gene targets, however, were comparable, suggesting that similar mechanisms are involved in the accumulation of gene mutations. Significant dose-related increases in % MN were also observed; however, the doubling dose was considerably higher for this endpoint. The similarity in dose response kinetics of Pig-a and lacZ provides further evidence for the mutational origin of glycosylphosphatidylinositol (GPI)-anchor deficiencies detected in the Pig-a assay., (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.)

An international collaborative trial was established to systematically investigate the merits and limitations of a rat in vivo Pig-a gene mutation assay. The product of this gene is essential for anchoring CD59 to the plasma membrane, and mutations in this gene are identified by flow cytometric quantification of circulating erythrocytes without cell surface CD59 expression. Initial interlaboratory data from rats treated with several potent mutagens have been informative, but the time required for those flow cytometric analyses (∼20 min per sample) limited the number of cells that could be interrogated for the mutant phenotype. Thus, it was desirable to establish a new higher throughput scoring approach before expanding the trial to include weak mutagens or nongenotoxicants. An immunomagnetic column separation method that dramatically increases analysis rates was therefore developed (Dertinger et al. [2011]: Mutat Res 721:163-170). To evaluate this new method for use in the international collaborative trial, studies were conducted to determine the mutagenic response of male Sprague Dawley rats treated for 3 or 28 consecutive days with several doses of 1,3-propane sultone (1,3-PS). Pig-a mutant frequencies were measured over a period of several weeks and were supplemented with another indicator of genetic toxicity, peripheral blood micronucleated reticulocyte (MN-RET) counts. 1,3-PS was found to increase Pig-a mutation and MN-RET frequencies in both 3- and 28-day study designs. While the greatest induction of MN-RETs was observed in the 3-day study, the highest Pig-a responses were found with 28-days of treatment. Pig-a measurements were acquired in approximately one-third the time required in the original method, while the number of erythrocyte and reticulocyte equivalents analyzed per sample were increased by factors of 100 and 10, respectively. The data strongly support the value of using the immunomagnetic separation technique for enumerating Pig-a mutation frequencies. These results also demonstrate that the ongoing international trial will benefit from the inclusion of studies that are based on both acute and protracted repeat dosing schedules in conjunction with the acquisition of longitudinal data, at least until more data have been accumulated., (Copyright © 2011 Wiley-Liss, Inc.)

The Viciafaba root tip micronucleus test is one of the most employed plant genotoxicity assays, and has been used on various types of contaminated materials. This test has been standardized by AFNOR, the French member organization of ISO. However, this test is usually performed with a water extraction step but soil genotoxicity assessment would be more relevant when performed directly in the soil itself. In order to harmonize these protocols, an ISO standard for the V.faba micronucleus test in both liquid phase (exposure of plants to different liquid matrix, including soil water extracts) and solid phase (direct exposure of plants to the soil) would be very useful. In this context, we compared two exposure durations in the solid phase (48 h and 5 d) for the V.faba micronucleus test with two different well-known genotoxicants, maleic hydrazide and copper sulfate. We concluded that these two durations induced equivalent sensitivity: the micronucleus frequency was significantly increased with 5 μmol maleic hydrazide per kg dry soil and with 2 mmol copper sulfate per kg dry soil with both exposure durations. However, exposing roots to soil during 48 h is more practical. Moreover, organically and conventionally cultured seeds were employed to determine whether the seed provenance influenced the test sensitivity. Organic seeds were less sensitive to copper, possibly because copper-based treatments are permitted, and often applied, in organic farms. Therefore, in the absence of completely non-treated seeds, organically-cultured seeds did not appear to offer any advantages over conventional seeds., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Although the Allium cepa test has been widely used to identify potentially cytotoxic and genotoxic pollutants in aquatic environments, variable non-standardized choices have been made regarding the number of plant bulbs and roots analyzed. We propose numbers for bulbs and roots per bulb when comparing the frequencies of micronuclei, mitotic anomalies and mitotic index with this test. Roots that had been treated with aqueous solutions, such as water samples collected in August 2007 from the Paraíba do Sul River at the Brazilian cities of Tremembé and Aparecida; negative and positive controls were used for bioassays. The presence of pollutants in the river water had been presumed based on our previous cytological data and an official report by the São Paulo State Environmental Agency (Brazil) on presence of fecal contaminants (Tremembé and Aparecida) and elevated dissolved aluminium (Aparecida) in the water under study. The sampling of ten bulbs and five roots per bulb was found adequate for comparative studies to evaluate with the A. cepa test the potential damage inflicted by pollutants in aquatic environments. Furthermore, even one bulb and one root per bulb was sufficient in discerning this damage, thereby shortening the time required to attain a statistically confident comparative evaluation. However, to allow for the use of statistical programs based on the evaluation of average values, and to avoid criticism based on genetic variability, we propose that three bulbs and three roots per bulb be considered as standard sample sizes for the A. cepa test.

The OECD guideline for the in vitro mammalian cell micronucleus test (OECD 487) was recently adopted in July 22, 2010. Since its publication, it has become apparent that the guidance for testing chemicals where solubility is a limiting factor can be interpreted in a variety of ways. In this communication, we provide clarification for testing insoluble chemicals. The intent of the OECD 487 guideline is for the high dose to be the lowest precipitating concentration even if toxicity occurs above the solubility limit in tissue culture medium. Examination of precipitation can be done by the unaided eye or microscopically. Precipitation is examined at the onset or end of treatment, with the intent to identify precipitate present during treatment., (2011 Elsevier B.V. All rights reserved.)

Considering the important production of carbon nanotubes (CNTs), it is likely that some of them will contaminate the environment during each step of their life cycle. Nevertheless, there is little known about their potential ecotoxicity. Consequently, the impact of CNTs on the environment must be taken into consideration. This work evaluates the potential impact of well characterized double-walled carbon nanotubes (DWNTs) in the amphibian larvae Xenopus laevis under normalized laboratory conditions according to the International Standard micronucleus assay ISO 21427-1:2006 for 12 days of half-static exposure to 0.1-1-10 and 50 mg L(-1) of DWNTs in water. Two different endpoints were carried out: (i) toxicity (mortality and growth of larvae) and (ii) genotoxicity (induction of micronucleated erythrocytes). Moreover, intestine of larvae were analyzed using Raman spectroscopy. The DWNTs synthetized by catalytic chemical vapor deposition (CCVD) were used as produce (experiment I) and the addition of Gum Arabic (GA) was investigated to improve the stability of the aqueous suspensions (experiment II). The results show growth inhibition in larvae exposed to 10 and 50 mg L(-1) of DWNTs with or without GA. No genotoxicity was evidenced in erythrocytes of larvae exposed to DWNTs, except to 1 mg L(-1) of DWNTs with GA suggesting its potential effect in association with DWNTs at the first nonacutely toxic concentration. The Raman analysis confirmed the presence of DWNTs into the lumen of intestine but not in intestinal tissues and cells, nor in the circulating blood of exposed larvae., (Copyright © 2009 Wiley Periodicals, Inc.)

Adult rats are generally not considered as a suitable model for the peripheral blood micronucleus (PBMN) assay in regulatory consideration, owing to the splenic removal of the micronucleated cells from circulation. Although prior bleeding (PrB) increases the sensitivity of the PBMN assay in young rats, the volume of bleeding and the associated stress caused are major concerns for its possible use in genotoxicity studies. The present study was aimed to overcome these limitations in using pre-bled young rats in genotoxicity studies. The bleeding volume was reduced by the simultaneous use of erythropoietin (EPO) to increase the sensitivity of PBMN assay. Young Sprague-Dawley (SD, 26 days) rats were used in the study. The kinetics of RETs-to-ERTs ratio was determined in response to EPO (10-3000 IU/kg) or PrB (0.1-1.0 ml) at different time points (0, 6, 12, 24, 36, 48, 72 and 96 h). Injection of EPO (30 IU/kg) and PrB (0.5 ml) led to a significant increase in the MN frequency in the PBMN assay in response to cyclophosphamide and zidovudine. The effect of EPO treatment and/or PrB on cell viability and proliferation in the bone marrow (BM) was examined. The results of the present study clearly demonstrate that the simultaneous use of both EPO and PrB enhances the sensitivity of the PBMN assay in young rats due to increased cellular proliferation in the BM. This may provide a useful experimental model for the evaluation of marginally active genotoxicants.

The European Cosmetic Toiletry and Perfumery Association (COLIPA), along with contributions from the European Centre for the Validation of Alternative Methods (ECVAM), initiated a multi-lab international prevalidation project on the reconstructed skin micronucleus (RSMN) assay in EpiDerm™ for the assessment of the genotoxicity of dermally applied chemicals. The first step of this project was to standardize the protocol and transfer it to laboratories that had not performed the assay before. Here we describe in detail the protocol for the RSMN assay in EpiDerm™ and the harmonized guidelines for scoring, with an atlas of cell images. We also describe factors that can influence the performance of the assay. Use of these methods will help new laboratories to conduct the assay, thereby further increasing the database for this promising new in vitro genotoxicity test., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In the OECD Guideline 487, a total of four extended exposure treatment conditions are proposed for the in vitro micronucleus (MNvit) assay in the presence and absence of a cytokinesis block and with or without a recovery period. This guideline also states that rodent cell lines and human lymphocytes can be used as shown by many validated studies but that human cell lines such as TK6 and HepG2 are not yet validated. In this present study each extended exposure condition was characterized by investigation using TK6 cells and nine chemicals known to be able to induce micronucleus (MN) in rodent cell lines. The results revealed two concerns: six chemicals did not show significant MN induction in the 'cytokinesis block without recovery period'; two aneugens showed no dose-dependent cytotoxicity in the 'cytokinesis block with recovery period'. Further investigation revealed that 3-4 times higher spontaneous MN frequency than that in the other conditions is a possible reason for the low sensitivity, and this high spontaneous MN frequency was not observed in Chinese hamster lung cells under the identical treatment condition. With regard to the two conditions without cytokinesis block, two negative substances were evaluated and found to be negative, suggesting the validity of the TK6 test system for these conditions. Although our findings showed a few concerns for the treatment with cytokinesis block, the TK6 cells were considered to be a reliable cell line to be used for detecting potential inducers of MN in the in vitro micronucleus assay based on the overall results., (Copyright © 2010 Elsevier Inc. All rights reserved.)

At the laboratories of AstraZeneca, Alderley Park, UK the reference genotoxic agents etoposide (a topoisomerase II inhibitor), cadmium chloride (an inorganic carcinogen), colchicine (an aneugen that inhibits tubulin polymerisation), benzo[a]pyrene (a metabolism dependent reference genotoxin) and cyclophosphamide (a metabolism dependent reference genotoxin) were tested in the in vitro micronucleus assay (MNvit), using mouse lymphoma L5178Y cells, with and without cytokinesis block. Further, 2-aminoanthracene (a metabolism dependent weak clastogen) was tested in the MNvit, using TK6 cells, without cytokinesis block. This was done in support of the toxicity (cell death and cytostasis) measures recommended in the late 2007 version of the draft OECD Test Guideline 487 for the testing of chemicals. All six reference agents were positive in the MNvit without cytokinesis block at concentrations giving approximately 50% toxicity or less as defined by draft Test Guideline 487 recommended measures, relative population doublings and relative increase in cell counts. Furthermore, the five agents tested with cytokinesis block were positive in the MNvit at concentrations giving approximately 50% toxicity or less as assessed by replicative index. Accordingly, this work supports the premise that relative population doublings and relative increase in cell counts are appropriate measures of toxicity for the non-cytokinesis-blocked in vitro micronucleus assay., (Copyright © 2009. Published by Elsevier B.V.)

The known aneugens vinblastine and diethylstilboestrol (DES) were tested in the in vitro micronucleus assay, with and without cytokinesis block in Chinese hamster CHO cells, at the laboratories of Swansea University, Swansea, UK. These experiments were carried out to determine the suitability of the cell death and cytostasis measures used in the assay, as recommended in the draft OECD Test Guideline 487, 2007. Both compounds were positive in the assay without cytokinesis block at concentrations giving approximately 50% or less cell death and cytostasis, using relative population doublings and relative increase in cell counts. Moreover, both compounds were positive in the assay with cytokinesis block at concentrations giving approximately 50% cell death and cytostasis, using replicative index. Vinblastine was also positive for mitotic slippage, causing micronuclei in mononucleate cells with cytokinesis block. Relative population doublings and relative increase in cell counts were appropriate measures of cell death and cytostasis for the non-cytokinesis block in vitro micronucleus assay. In the cytokinesis blocked micronucleus assay, replicative index and cytokinesis block proliferation index were suitable cell death and cytostasis measures., (Copyright © 2009 Elsevier B.V. All rights reserved.)

Cytosine arabinoside (a nucleoside analogue that inhibits the gap-filling step of excision repair), vinblastine (an aneugen that inhibits tubulin polymerisation), 5-fluorouracil (a nucleoside analogue with a steep response profile), and 2-aminoanthracene (a metabolism-dependent reference genotoxin) were tested in the in vitro micronucleus assay with L5178Y mouse lymphoma cells, without cytokinesis block. The four chemicals were independently evaluated in two Sanofi Aventis laboratories, one of which used an image analyser to score micronuclei, while the other scored micronucleated cells manually. Very similar results were obtained in the two laboratories, highlighting the robustness of the assay. The four test chemicals induced significant increases in the incidence of micronucleated cells at concentrations that produced no more than a 55±5% reduction in survival growth, as measured with the three parameters recommended in the draft OECD Test Guideline on In Vitro Mammalian Cell Micronucleus Test (MNvit) for chemical testing, namely the relative increase in cell counts, relative population doubling, and the relative cell count. These results support the premise that the relative increase in cell counts and relative population doubling, that take into account both cell death and cytostasis, are appropriate measures of survival growth reduction in the in vitro micronucleus test conducted in the absence of cytokinesis block, as recommended in MNvit., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The following genotoxic chemicals were tested in the in vitro micronucleus assay, at Covance Laboratories, Harrogate, UK in the human lymphoblastoid cell line TK6. Cadmium chloride (an inorganic carcinogen), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation) and cyclophosphamide (an alkylating agent requiring metabolic activation) were treated with and without cytokinesis block (by addition of cytochalasin B). This work formed part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 for the in vitro micronucleus test. The toxicity measures used, capable of detecting both cytostasis and cell death, were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index or cytokinesis blocked proliferation index in the presence of cytokinesis block. All of the chemicals tested gave significant increases in the percentage of micronucleated cells with and without cytokinesis block at concentrations giving approximately 60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcomes from this series of tests support the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in the in vitro micronucleus assay., (Copyright © 2010 Elsevier B.V. All rights reserved.)