Adapting the in vitro micronucleus assay (OECD Test Guideline No. 487) for testing of manufactured nanomaterials: recommendations for best practices.

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.

Transforming early pharmaceutical assessment of genotoxicity: applying statistical learning to a high throughput, multi end point in vitro micronucleus assay.

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.

Assessment of oral cytological features in smokers and nonsmokers after application of toluidine blue.

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.

Standards in biological dosimetry: A requirement to perform an appropriate dose assessment.

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.

Soil genotoxicity assessment--results of an interlaboratory study on the Vicia micronucleus assay in the context of ISO standardization.

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.

The in vitro micronucleus assay and kinetochore staining: methodology and criteria for the accurate assessment of genotoxicity and cytotoxicity.

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.

Flow cytometric 96-well microplate-based in vitro micronucleus assay with human TK6 cells: protocol optimization and transferability assessment.

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.

Assessment of genotoxicity induced by 7,12-dimethylbenz(a)anthracene or diethylnitrosamine in the Pig-a, micronucleus and Comet assays integrated into 28-day repeat dose studies.

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.

Improvement of Vicia-micronucleus test for assessment of soil quality: a proposal for international standardization.

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.

Establishment of an x-ray standard calibration curve by conventional dicentric analysis as prerequisite for accurate radiation dose assessment.

The dicentric assay was established to carry out cytogenetic biodosimetry after suspected radiation overexposure, including a comprehensive documentation system to record the processing of the specimen, all data, results, and stored information. As an essential prerequisite for retrospective radiation dose assessment, a dose-response curve for dicentric induction by in vitro x-ray irradiation of peripheral blood samples was produced. The accelerating potential was 240 kV (maximum photon energy: 240 keV). A total of 8,377 first-division metaphases of four healthy volunteers were analyzed after exposure to doses ranging from 0.2 to 4.0 Gy at a dose rate of 1.0 Gy min. The background level of aberrations at 0-dose was determined by the analysis of 14,522 first-division metaphases obtained from unirradiated blood samples of 10 healthy volunteers. The dose-response relationship follows a linear-quadratic equation, Y = c + alphaD + betaD, with the coefficients c = 0.0005 +/- 0.0002, alpha = 0.043 +/- 0.006, and beta = 0.063 +/- 0.004. The technical competence and the quality of the calibration curve were assessed by determination of the dose prediction accuracy in an in vitro experiment simulating whole-body exposures within a range of 0.2 to 2.0 Gy. Dose estimations were derived by scoring up to 500-1,000 metaphase spreads or more (full estimation mode) and by evaluating only 50 metaphase spreads (triage mode) per subject. The triage mode was applied by performing manifold evaluations of the full estimation data in order to test the robustness of the curve for triage purposes and to assess possible variations among the estimated doses referring to a single exposure and preparation.

Comparison of different methods for an accurate assessment of cytotoxicity in the in vitro micronucleus test. I. Theoretical aspects.

A decrease in the cytokinesis-block proliferation index (CBPI) or replication index (RI) is routinely used to determine cytotoxicity of a test compound and therefore the choice of its appropriate test concentration for the in vitro micronucleus (MN) test conducted in the presence of cytochalasin B. As a number of laboratories prefer to conduct the in vitro MN test in the absence of cytochalasin B, it is important that selected test concentrations, based on cytotoxicity, should be similar to what they would have been if cytochalasin B had been used, and should be relevant of a true cytotoxicity. By using models to analyse the dynamics of the cell cultures with and without cytochalasin B we have compared different methods for evaluation of cytotoxicity, and demonstrate that relative decrease in population doubling or relative increase in cell counts are the most appropriate measures of cytotoxicity to compare with reduction in CBPI or RI.

Comparison of different methods for an accurate assessment of cytotoxicity in the in vitro micronucleus test. II: Practical aspects with toxic agents.

Appropriate measures of cytotoxicity need to be used when selecting test concentrations in in vitro genotoxicity assays. Underestimation of toxicity may lead to inappropriately toxic concentrations being selected for analysis, with the potential for generation of irrelevant positive results. As guidance for the in vitro micronucleus test is being developed, it is clearly important to compare the different measures of cytotoxicity that can be used both with and without cytokinesis blocking. Therefore, relative cell counts (RCC), relative increase in cell counts (RICC) and relative population doubling (RPD) for treatments without cytokinesis block were compared with replication index (RI) for treatments with cytokinesis block, and the corresponding induction of micronucleated cells was evaluated. A wide range of chemicals and gamma irradiation were used, and in almost all cases, RCC underestimated cytotoxicity when compared with all other measures such that RCC would have resulted in the selection of inappropriately high concentrations for micronuclei analysis. In the absence of cytokinesis block, RICC or RPD is more comparable with RI with cytokinesis block, and therefore considered more appropriate measure of survival. Furthermore, using these estimations of cytotoxicity and the limit of 50% survival, all the mutagens and aneugens tested were appropriately identified as positive in the in vitro micronucleus assay. Accordingly, it was clear that testing beyond 50% survival was not necessary to identify the potential of these agents to induce micronuclei.

Statistical analysis of in vivo rodent micronucleus assay.

The in vivo rodent micronucleus assay (MNC) is widely used as a cytogenetic assay to detect the clastogenic activity of a chemical in vivo. MNC is one of three tests in a battery recommended by the fourth International Conference on Harmonization (ICH4) of Genotoxicity Guidelines. As such it has been accepted by many regulatory authorities. However, the determination of a positive result in a genotoxicity test, including MNC, has been an issue of debate among toxicologists and biometricians. In this presentation we compare several statistical procedures that have been suggested for the analysis of MNC data and indicate which one is the most powerful. The standard protocol of MNC has at least three dose levels plus the control dose and uses at least four animals per group. For each animal, 2000 polychromatic erythrocytes (PCE) are counted. Two statistical procedures can be employed, either alone or jointly, for the analysis of the MNC dose-response curve. These are the Cochran-Armitage (C-A) trend test and the Dunnett type test. For performing Dunnett type tests, toxicologists often use negative historical control rate for the estimate of the concurrent negative control rate. Some toxicologists emphasize the reproducibility of assay results instead of the dose-response relationship for the important criterion [J. Ashby, H. Tinwell, Mutat. Res. 327 (1995) 49-55; for the rebuttal see M. Hayashi, T. Sofuni, Mutat. Res. 331 (1995) 173-174]. The following three procedures are currently employed in toxicology labs for the evaluation of MNC result. The assay response is deemed positive if it is detected by (i) the C-A trend test alone, (ii) both the C-A trend test and the Dunnett type test and (iii) either the C-A trend test or the Dunnett type test. Using Monte Carlo simulation, we first find for each procedure, sizes of tests which yield the experiment-wise type I error rate of 0.05 and show that the procedure (ii) is the most powerful against the alternatives of monotone increase. The procedure (ii) which originated from Hayashi's three-step procedure was coded in C and termed 'MNC'. The MNC software program is available in the public domain through the ftp.