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Abstract Scientific, financial, and ethical drivers have led to unprecedented interest in implementing human-relevant, mechanistic in vitro and in silico testing approaches. Further, as non-animal approaches are being developed and validated, researchers are interested in strategies that can immediately reduce the use of animals in toxicology testing. Here, we aim to outline a testing strategy for assessing genotoxicity beginning with standard in vitro methods, such as the bacterial reverse mutation test and the in vitro micronucleus test, followed by a second tier of in vitro assays including those using advanced 3D tissue models. Where regulatory agencies require in vivo testing, one demonstrated strategy is to combine genotoxicity studies traditionally conducted separately into a single test or to integrate genotoxicity studies into other toxicity studies. Standard setting organisations and regulatory agencies have encouraged such strategies, and examples of their use can be found in the scientific literature. Employing approaches outlined here will reduce animal use as well as study time and costs.

Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0-2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.

In this study, we have studied the cytotoxicity and genotoxic potency of 3 pro-oxidants; H2O2, menadione and KBrO3 in different dosing scenarios, namely acute (1-day dosing) and chronic (5-days). For this purpose, relative population doubling (RPD%) and mononucleated micronucleus (MN) test were used. TK6 cells and NH32 were employed in in vitro experiments. In the study, the total acute dose was divided into 5 days for each prooxidant chemicals by dose fractionation (1/5th per day) method. Acute dosing was compared to chronic dosing. The oxidative stress caused by the exposure of cells with pro-oxidant chemicals to the cells was determined by an optimized 2′,7′-dichlorofluorescein diacetate (DCFHDA) test method. The antioxidant levels of the cell lines were altered with buthionine sulfoxide (BSO) and N-acetyl cysteine (NAC), and the effect of antioxidant capacity on the MN formation in the cells was observed with this method. In the case of H2O2 and menadione, fractional dosing has been observed to result in lower toxicity and lower genotoxicity. But in the case of KBrO3, unlike the other 2 pro-oxidants, higher MN induction was observed with fractionated doses. DCFHDA test clearly demonstrated ROS induction with H2O2 and menadione but not with KBrO3. Unexpectedly, DCFHDA test demonstrated that KBrO3 did not cause an increase ROS levels in both acute and chronic dosing, suggesting an alternative ROS induction mechanism. It was also observed that, treatment with BSO and NAC, caused increasing and decreasing of MN fold change respectively, allowing further ROS specific mechanisms to be explored. Hence, dose fractionation expectedly caused less MN, cytotoxicity and ROS formation with H2O2 and menadione exposure, but not with KBrO3. This implies a unique mechanism of action for KBrO3 induced genotoxicity. Chronic dosing in vitro may be a valuable approach allowing better understanding of how chemicals damage DNA and pose human hazards.

In vitro genotoxicity studies are a quick and high throughput approach to assess the genotoxic potential of chemicals; however, the reliability of these tests and their relevance to in vivo effects depends on the choice of representative cell line and optimisation of assay conditions. For chemicals like urethane that require specific metabolic activation to cause genotoxicity, it is important that in vitro tests are conducted using cell lines exhibiting the activity and induction of CYP450 enzymes, including CYP2E1 enzyme that is important in the metabolism of urethane, at a concentration representing actual or perceived chemical exposure. We compared 2D MCL-5 cells and HepG2 cells with 3D HepG2 hanging drop spheroids to determine the genotoxicity of urethane using the micronucleus assay. Our 2D studies with MCL-5 did not show any statistically significant genotoxicity [99% relative population doubling (RPD)] compared to controls for concentrations and time point tested in vitro. HepG2 cells grown as 2D indicated that exposure to urethane of up to 30 mM for 23 h did not cause any genotoxic effect (102% RPD) but, at higher concentrations, genotoxicity was produced with only 89–85% RPD. Furthermore, an exposure of 20–50 mM for 23 h using 3D hanging drop spheroid assays revealed a higher MN frequency, thus exhibiting in vitro genotoxicity of urethane in metabolically active cell models. In comparison with previous studies, this study indicated that urethane genotoxicity is dose, sensitivity of cell model (2D vs. 3D) and exposure dependent.

It is well understood that poor diet and lifestyle choices can increase the risk of cancer. It is also well documented that cancer is a disease of DNA mutations, with mutations in key genes driving carcinogenesis. Measuring these mutations in a minimally invasive way may be informative as to which exposures are harmful and thus allow us to introduce primary preventative measures, in a bid to reduce cancer incidences. Here, we have measured mutations in the phosphatidylinositol glycan class A (PIG-A) gene in erythrocytes from healthy volunteers (n = 156) and from non-cancer patients attending the local endoscopy department (n = 144). The X-linked PIG-A gene encodes an enzyme involved in glycosylphosphatidylinositol (GPI) anchor synthesis. A silencing mutation in which leads to the absence of GPI anchors on the extracellular surface which can be rapidly assessed using flow cytometry. The background level of PIG-A mutant erythrocytes was 2.95 (95% CI: 2.59–3.67) mutant cells (10−6). Older age increased mutant cell frequency (P < 0.001). There was no difference in mutant cell levels between males and females (P = 0.463) or smokers and non-smokers (P = 0.186). In the endoscopy group, aspirin users had lower mutant frequencies (P = 0.001). Further information on diet and exercise was available for the endoscopy patient group alone, where those with a higher health promotion index score had lower mutant frequencies (P = 0.011). Higher dietary intake of vegetables reduced mutant cell levels (P = 0.022). Participants who exercised for at least 1 h a week appeared to have reduced mutant frequencies than those who did not exercise, although this was not statistically significant (P = 0.099). This low background level of mutant erythrocytes in a population makes this assay an attractive tool to monitor exposures such as those associated with lifestyles and diet, as demonstrated here.

This Mutagenesis special issue is on the topic of nanogenotoxicology. It unites a collection of reports that provide insight into: (i) the properties of engineered nanomaterials (ENMs) that contribute to genotoxicity, (ii) the genotoxic mechanisms associated with DNA damage observed in both in vitro and in vivo tests and (iii) the future test systems that will provide more accurate prediction of ENM genotoxicity to support regulatory hazard assessment frameworks. The contributions within therefore provide collective oversight of our current understanding, coupled to future perspectives aimed at overcoming technical hurdles and describing novel analytical methods to further advance the field.

A number of in vitro methodologies have been used to assess the genotoxicity of different nanomaterials, including titanium dioxide nanoparticles (TiO2 NPs) and silver nanoparticles (AgNPs). The in vitro micronucleus assay is one of the most commonly used test methods for genotoxicity evaluation of nanomaterials. However, due to the novel features of nanomaterials, such as high adsorption capacity and fluorescence properties, there are unexpected interactions with experimental components and detection systems. In this study, we evaluate the interference by two nanoparticles, AgNPs and TiO2 NPs, with the in vitro micronucleus assay system and possible confounding factors affecting cytotoxicity and genotoxicity assessment of the nanomaterials including cell lines with different p53 status, nanoparticle coatings and fluorescence, cytochalasin B, fetal bovine serum in cell treatment medium and different measurement methodologies for detecting micronuclei. Our results showed that micronucleus induction by AgNPs was similar when evaluated using flow cytometry or microscope, whereas the induction by TiO2 NPs was different using the two methods due to TiO2's fluorescence interference with the cytometry equipment. Cells with the mutated p53 gene were more sensitive to micronucleus induction by AgNPs than the p53 wild-type cells. The presence of serum during treatment increased the toxicity of AgNPs. The coatings of nanoparticles played an important role in the genotoxicity of AgNPs. These collective data highlight the importance of considering the unique properties of nanoparticles in assessing their genotoxicity using the in vitro micronucleus assay.

Use of imaging flow cytometry to assess induced DNA damage via the cytokinesis block micronucleus (CBMN) assay has thus far been limited to radiation dosimetry in human lymphocytes using high end, 'ImageStream X' series imaging cytometers. Its potential to enumerate chemically induced DNA damage using in vitro cell lines remains unexplored. In the present manuscript, we investigate the more affordable FlowSight® imaging cytometry platform to assess in vitro micronucleus (MN) induction in the human lymphoblastoid TK6 and metabolically competent MCL-5 cells treated with Methyl Methane Sulfonate (MMS) (0-5 µg/ml), Carbendazim (0-1.6 µg/ml), and Benzo[a]Pyrene (B[a]P) (0-6.3 µg/ml) for a period of 1.5-2 cell-cycles. Cells were fixed, and nuclei and MN were stained using the fluorescent nuclear dye DRAQ5™. Image acquisition was carried out using a 20X objective on a FlowSight® imaging cytometer (Amnis, part of Merck Millipore) equipped with a 488 nm laser. Populations of ∼20000 brightfield cell images, alongside DRAQ5™ stained nuclei/MN were rapidly collected (≤10 min). Single, in-focus cells suitable for scoring were then isolated using the IDEAS® software. An overlay of the brightfield cell outlines and the DRAQ5 nuclear fluorescence was used to facilitate scoring of mono-, bi-, tri-, and tetra-nucleated cells with or without MN events and in context of the cytoplasmic boundary of the parent cell.To establish the potential of the FlowSight® platform, and to establish 'ground truth' cell classification for the supervised machine learning based scoring algorithm that represents the next stage of our project, the captured images were scored manually. Alongside, MN frequencies were also derived using the 'gold standard' light microscopy and manual scoring. A minimum of 3000 bi-nucleated cells were assessed using both approaches. Using the benchmark dose approach, the comparability of genotoxic potency estimations for the different compounds and cell lines was assessed across the two scoring platforms as highly similar. This study therefore provides essential proof-of-concept that FlowSight® imaging cytometry is capable of reproducing the results of 'gold standard' manual scoring by light microscopy. We conclude that, with the right automated scoring algorithm, imaging flow cytometry could revolutionise the reportability and scoring throughput of the CBMN assay.

4-Nitroquinoline 1-oxide (4NQO) is used as a positive control in various genotoxicity assays because of its known mutagenic and carcinogenic properties. The chemical is converted into 4-hydroxyaminoquinoline 1-oxide and gives rise to three main DNA adducts, N-(deoxyguanosin-8-yl)-4AQO, 3-(desoxyguanosin-N (2)-yl)-4AQO and 3-(deoxyadenosin-N (6)-yl)-4AQO. This study was designed to assess the shape of the dose-response curve at low concentrations of 4NQO in three human lymphoblastoid cell lines, MCL-5, AHH-1 and TK6 as well as the mouse lymphoma L5178Y cell line in vitro. Chromosomal damage was investigated using the in vitro micronucleus assay, while further gene mutation and DNA damage studies were carried out using the hypoxanthine-guanine phosphoribosyltransferase forward mutation and comet assays. 4NQO showed little to no significant increases in micronucleus induction in the human lymphoblastoid cell lines, even up to 55±5% toxicity. A dose-response relationship could only be observed in the mouse lymphoma cell line L5178Y after 4NQO treatment, even at concentrations with no reduction in cell viability. Further significant increases in gene mutation and DNA damage induction were observed. Hence, 4NQO is a more effective point mutagen than clastogen, and its suitability as a positive control for genotoxicity testing has to be evaluated for every individual assay.

Quantum dots (QD) have unique electronic and optical properties promoting biotechnological advances. However, our understanding of the toxicological structure–activity relationships remains limited. This study aimed to determine the biological impact of varying nanomaterial surface chemistry by assessing the interaction of QD with either a negative (carboxyl), neutral (hexadecylamine; HDA) or positive (amine) polymer coating with human lymphoblastoid TK6 cells. Following QD physico-chemical characterisation, cellular uptake was quantified by optical and electron microscopy. Cytotoxicity was evaluated and genotoxicity was characterised using the micronucleus assay (gross chromosomal damage) and the HPRT forward mutation assay (point mutagenicity). Cellular damage mechanisms were also explored, focusing on oxidative stress and mitochondrial damage. Cell uptake, cytotoxicity and genotoxicity were found to be dependent on QD surface chemistry. Carboxyl-QD demonstrated the smallest agglomerate size and greatest cellular uptake, which correlated with a dose dependent increase in cytotoxicity and genotoxicity. Amine-QD induced minimal cellular damage, while HDA-QD promoted substantial induction of cell death and genotoxicity. However, HDA-QD were not internalised by the cells and the damage they caused was most likely due to free cadmium release caused by QD dissolution. Oxidative stress and induced mitochondrial reactive oxygen species were only partially associated with cytotoxicity and genotoxicity induced by the QD, hence were not the only mechanisms of importance. Colloidal stability, nanoparticle (NP) surface chemistry, cellular uptake levels and the intrinsic characteristics of the NPs are therefore critical parameters impacting genotoxicity induced by QD.

Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0–2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.