Severity of effect considerations regarding the use of mutation as a toxicological endpoint for risk assessment: A report from the 8th International Workshop on Genotoxicity Testing (IWGT).

Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose-response curve (e.g., benchmark dose) by a composite adjustment factor (AF). An "effect severity" AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health-based guidance value (HBGV) when a "severe" toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose-response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub-group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease-causing mutations by error-corrected next generation sequencing clarifies selection of ESAF values.

Within-laboratory reproducibility of Ames test results: Are repeat tests necessary?

The Ames test is required by regulatory agencies worldwide for assessing the mutagenic and carcinogenic potential of chemical compounds. This test uses several strains of bacteria to evaluate mutation induction: positive results in the assay are predictive of rodent carcinogenicity. As an initial step to understanding how well the assay may detect mutagens present as constituents of complex mixtures such as botanical extracts, a cross-sector working group examined the within-laboratory reproducibility of the Ames test using the extensive, publicly available National Toxicology Program (NTP) Ames test database comprising more than 3000 distinct test articles, most of which are individual chemicals. This study focused primarily on NTP tests conducted using the standard Organization for Economic Co-operation and Development Test Guideline 471 preincubation test protocol with 10% rat liver S9 for metabolic activation, although 30% rat S9 and 10 and 30% hamster liver S9 were also evaluated. The reproducibility of initial negative responses in all strains with and without 10% S9, was quite high, ranging from 95% to 99% with few exceptions. The within-laboratory reproducibility of initial positive responses for strains TA98 and TA100 with and without 10% rat liver S9 was ≥90%. Similar results were seen with hamster S9. As expected, the reproducibility of initial equivocal responses was lower, <50%. These results will provide context for determining the optimal design of recommended test protocols for use in screening both individual chemicals and complex mixtures, including botanicals.

Interlaboratory validation of the ToxTracker assay: An in vitro reporter assay for mechanistic genotoxicity assessment.

ToxTracker is a mammalian cell reporter assay that predicts the genotoxic properties of compounds with high accuracy. By evaluating induction of various reporter genes that play a key role in relevant cellular pathways, it provides insight into chemical mode-of-action (MoA), thereby supporting discrimination of direct-acting genotoxicants and cytotoxic chemicals. A comprehensive interlaboratory validation trial was conducted, in which the principles outlined in OECD Guidance Document 34 were followed, with the primary objectives of establishing transferability and reproducibility of the assay and confirming the ability of ToxTracker to correctly classify genotoxic and non-genotoxic compounds. Reproducibility of the assay to predict genotoxic MoA was confirmed across participating laboratories and data were evaluated in terms of concordance with in vivo genotoxicity outcomes. Seven laboratories tested a total of 64 genotoxic and non-genotoxic chemicals that together cover a broad chemical space. The within-laboratory reproducibility (WLR) was up to 98% (73%-98% across participants) and the overall between-laboratory reproducibility (BLR) was 83%. This trial confirmed the accuracy of ToxTracker to predict in vivo genotoxicants with a sensitivity of 84.4% and a specificity of 91.2%. We concluded that ToxTracker is a robust in vitro assay for the accurate prediction of in vivo genotoxicity. Considering ToxTracker's robust standalone accuracy and that it can provide important information on the MoA of chemicals, it is seen as a valuable addition to the regulatory in vitro genotoxicity battery that may even have the potential to replace certain currently used in vitro battery assays.

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

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

Toxins in Botanical Drugs and Plant-derived Food and Feed - from Science to Regulation: A Workshop Review.

In September 2022, the 3rd International Workshop on pyrrolizidine alkaloids (PAs) and related phytotoxins was held on-line, entitled 'Toxins in botanical drugs and plant-derived food and feed - from science to regulation'. The workshop focused on new findings about the occurrence, exposure, toxicity, and risk assessment of PAs. In addition, new scientific results related to the risk assessment of alkenylbenzenes, a distinct class of herbal constituents, were presented. The presence of PAs and alkenylbenzenes in plant-derived food, feed, and herbal medicines has raised health concerns with respect to their acute and chronic toxicity but mainly related to the genotoxic and carcinogenic properties of several congeners. The compounds are natural constituents of a variety of plant families and species widely used in medicinal, food, and feed products. Their individual occurrence, levels, and toxic properties, together with the broad range of congeners present in nature, represent a striking challenge to modern toxicology. This review tries to provide an overview of the current knowledge on these compounds and indicates needs and perspectives for future research.

Interpretation of in vitro concentration-response data for risk assessment and regulatory decision-making: Report from the 2022 IWGT quantitative analysis expert working group meeting.

Quantitative risk assessments of chemicals are routinely performed using in vivo data from rodents; however, there is growing recognition that non-animal approaches can be human-relevant alternatives. There is an urgent need to build confidence in non-animal alternatives given the international support to reduce the use of animals in toxicity testing where possible. In order for scientists and risk assessors to prepare for this paradigm shift in toxicity assessment, standardization and consensus on in vitro testing strategies and data interpretation will need to be established. To address this issue, an Expert Working Group (EWG) of the 8th International Workshop on Genotoxicity Testing (IWGT) evaluated the utility of quantitative in vitro genotoxicity concentration-response data for risk assessment. The EWG first evaluated available in vitro methodologies and then examined the variability and maximal response of in vitro tests to estimate biologically relevant values for the critical effect sizes considered adverse or unacceptable. Next, the EWG reviewed the approaches and computational models employed to provide human-relevant dose context to in vitro data. Lastly, the EWG evaluated risk assessment applications for which in vitro data are ready for use and applications where further work is required. The EWG concluded that in vitro genotoxicity concentration-response data can be interpreted in a risk assessment context. However, prior to routine use in regulatory settings, further research will be required to address the remaining uncertainties and limitations.

Utility of ToxTracker in animal alternative testing strategy for fragrance materials.

To determine the utility of the ToxTracker assay in animal alternative testing strategies, the genotoxic potential of four fragrance materials (2-octen-4-one, lauric aldehyde, veratraldehyde, and p-methoxy cinnamaldehyde) were tested in the ToxTracker assay. These materials have been previously evaluated in an in vitro as well as in vivo micronucleus assay, conducted as per OECD guidelines. In addition to these studies, reconstructed human skin micronucleus studies were conducted on all four materials. All four materials were positive in an in vitro micronucleus assay but were negative in both in vivo and 3D skin micronucleus assays. The ToxTracker assay, in combination with in silico methods to predict metabolism was used to identify mechanisms for the misleading positive outcomes observed in the in vitro micronucleus assays. The results show that the ToxTracker assay, in conjunction with in silico predictions, can provide the information needed to aid in the identification of an appropriate animal alternative follow-up assay, for substances with positive results in the standard in vitro test battery. Thus, the ToxTracker assay is a valuable tool to identify the genotoxic potential of fragrance materials and can aid with replacing animal-based follow-up testing with appropriate animal alternative assay(s).

Quantitative in vitro to in vivo extrapolation of genotoxicity data provides protective estimates of in vivo dose.

Genotoxicity assessment is a critical component in the development and evaluation of chemicals. Traditional genotoxicity assays (i.e., mutagenicity, clastogenicity, and aneugenicity) have been limited to dichotomous hazard classification, while other toxicity endpoints are assessed through quantitative determination of points-of-departures (PODs) for setting exposure limits. The more recent higher-throughput in vitro genotoxicity assays, many of which also provide mechanistic information, offer a powerful approach for determining defined PODs for potency ranking and risk assessment. In order to obtain relevant human dose context from the in vitro assays, in vitro to in vivo extrapolation (IVIVE) models are required to determine what dose would elicit a concentration in the body demonstrated to be genotoxic using in vitro assays. Previous work has demonstrated that application of IVIVE models to in vitro bioactivity data can provide PODs that are protective of human health, but there has been no evaluation of how these models perform with in vitro genotoxicity data. Thus, the Genetic Toxicology Technical Committee, under the Health and Environmental Sciences Institute, conducted a case study on 31 reference chemicals to evaluate the performance of IVIVE application to genotoxicity data. The results demonstrate that for most chemicals considered here (20/31), the PODs derived from in vitro data and IVIVE are health protective relative to in vivo PODs from animal studies. PODs were also protective by assay target: mutations (8/13 chemicals), micronuclei (9/12), and aneugenicity markers (4/4). It is envisioned that this novel testing strategy could enhance prioritization, rapid screening, and risk assessment of genotoxic chemicals.

The in vitro genotoxicity potency of mixtures of pyrrolizidine alkaloids can be explained by dose addition of the individual mixture components.

Plant-based 1,2-unsaturated Pyrrolizidine Alkaloids (PAs) are responsible for liver genotoxicity/carcinogenicity following metabolic activation, making them a relevant concern for safety assessment. Due to 21st century toxicology approaches, risk of PAs can be better discerned though an understanding of differing toxic potencies, but it is often mixtures of PAs that are found as contaminants in foods, for example, herbal teas and honey, food supplements and herbal medicines. Our study investigated whether genotoxicity potency of PAs dosed individually or in mixtures differed when measured using micronuclei formation in vitro in HepaRG human liver cells, which we and others have shown to be suitable for observing genotoxic potency differences across different PA structural classes. When equipotent concentrations of up to six different PAs representing a wide range of potencies in vitro were tested as mixtures, the observed genotoxic potency aligned favorably with results for single PAs. Similarly, when the BMD confidence intervals of these equipotent mixtures were compared with the confidence intervals of the individual PAs, only minimal variation was observed. These data support a conclusion that for this class of plant impurities, all acting via the same DNA-reactive mode of action, genotoxic potency can be regarded as additive when assessing the risk of mixtures of PAs.

A weight of evidence review of the genotoxicity of titanium dioxide (TiO2).

Titanium dioxide is a ubiquitous white material found in a diverse range of products from foods to sunscreens, as a pigment and thickener, amongst other uses. Titanium dioxide has been considered no longer safe for use in foods (nano and microparticles of E171) by the European Food Safety Authority (EFSA) due to concerns over genotoxicity. There are however, conflicting opinions regarding the safety of Titanium dioxide. In an attempt to clarify the situation, a comprehensive weight of evidence (WoE) assessment of the genotoxicity of titanium dioxide based on the available data was performed. A total of 192 datasets for endpoints and test systems considered the most relevant for identifying mutagenic and carcinogenic potential were reviewed and discussed for both reliability and relevance (by weight of evidence) and in the context of whether the physico-chemical properties of the particles had been characterised. The view of an independent panel of experts was that, of the 192 datasets identified, only 34 met the reliability and quality criteria for being most relevant in the evaluation of genotoxicity. Of these, 10 were positive (i.e. reported evidence that titanium dioxide was genotoxic), all of which were from studies of DNA strand breakage (comet assay) or chromosome damage (micronucleus or chromosome aberration assays). All the positive findings were associated with high cytotoxicity, oxidative stress, inflammation, apoptosis, necrosis, or combinations of these. Considering that DNA and chromosome breakage can be secondary to physiological stress, it is highly likely that the observed genotoxic effects of titanium dioxide, including those with nanoparticles, are secondary to physiological stress. Consistent with this finding, there were no positive results from the in vitro and in vivo gene mutation studies evaluated, although it should be noted that to definitively conclude a lack of mutagenicity, more robust in vitro and in vivo gene mutation studies would be useful. Existing evidence does not therefore support a direct DNA damaging mechanism for titanium dioxide (nano and other forms).

Assessing the genotoxicity and carcinogenicity of 2-chloroethanol through structure activity relationships and in vitro testing approaches.

The detection of 2-chloroethanol in foods generally follows an assumption that the pesticide ethylene oxide has been used at some stage in the supply chain. In this situation the Pesticide Residues in Food Regulation (EC) 396/2005 requires 2-chloroethanol to be assessed as if equivalent to ethylene oxide, which has been classified as a genotoxic carcinogen. This review investigated whether this is an appropriate risk assessment approach for 2-chloroethanol. This involved an assessment of existing genotoxicity and carcinogenicity data, application of Structure Activity Based Read Across for carcinogenicity assessment, biological reactivity in the ToxTracker assay and micronuclei formation in HepaRG cells. Although we identified there is an absence of a standard oral bioassay for 2-chloroethanol, carcinogenicity weight-of-evidence assessment along with data on relevant structural analogues do not show evidence for carcinogenicity for 2-chloroethanol. The absence of genotoxicity was demonstrated for 2-chloroethanol and suitable analogues. In contrast, ethylene oxide showed reactivity towards markers indicative of direct DNA damage which is consistent with what is known about its mode-of-action. These data facilitate the understanding of 2-chloroethanol and given that it is not a genotoxic carcinogen suggest it must be assessed relative to non-cancer endpoints and a health protective Reference Dose should be established on that basis.

In vivo Mammalian Alkaline Comet Assay: Method Adapted for Genotoxicity Assessment of Nanomaterials.

The in vivo Comet assay measures the generation of DNA strand breaks under conditions in which the DNA will unwind and migrate to the anode in an electrophoresis assay, producing comet-like figures. Measurements are on single cells, which allows the sampling of a diversity of cells and tissues for DNA damaging effects. The Comet assay is the most common in vivo method for genotoxicity assessment of nanomaterials (NM). The Method outlined here includes a recommended step-by-step approach, consistent with OECD 489, taking into consideration the issues impacting assessment of NM, including choice of cells or systems, handling of NM test articles, dose determination, assay methods and data assessment. This method is designed to be used along with the accompanying "Common Considerations" paper, which discusses issues common to any genotoxicity assay using NM as a test article.

Common Considerations for Genotoxicity Assessment of Nanomaterials.

Genotoxicity testing is performed to determine potential hazard of a chemical or agent for direct or indirect DNA interaction. Testing may be a surrogate for assessment of heritable genetic risk or carcinogenic risk. Testing of nanomaterials (NM) for hazard identification is generally understood to require a departure from normal testing procedures found in international standards and guidelines. A critique of the genotoxicity literature in Elespuru et al., 2018, reinforced evidence of problems with genotoxicity assessment of nanomaterials (NM) noted by many previously. A follow-up to the critique of problems (what is wrong) is a series of methods papers in this journal designed to provide practical information on what is appropriate (right) in the performance of genotoxicity assays altered for NM assessment. In this "Common Considerations" paper, general considerations are addressed, including NM characterization, sample preparation, dosing choice, exposure assessment (uptake) and data analysis that are applicable to any NM genotoxicity assessment. Recommended methods for specific assays are presented in a series of additional papers in this special issue of the journal devoted to toxicology methods for assessment of nanomaterials: the In vitro Micronucleus Assay, TK Mutagenicity assays, and the In vivo Comet Assay. In this context, NM are considered generally as insoluble particles or test articles in the nanometer size range that present difficulties in assessment using techniques described in standards such as OECD guidelines.

The BlueScreen HC assay to predict the genotoxic potential of fragrance materials.

BlueScreen HC is a mammalian cell-based assay for measuring the genotoxicity and cytotoxicity of chemical compounds and mixtures. The BlueScreen HC assay has been utilized at the Research Institute for Fragrance Materials in a safety assessment program as a screening tool to prioritize fragrance materials for higher-tier testing, as supporting evidence when using a read-across approach, and as evidence to adjust the threshold of toxicological concern. Predictive values for the BlueScreen HC assay were evaluated based on the ability of the assay to predict the outcome of in vitro and in vivo mutagenicity and chromosomal damage genotoxicity assays. A set of 371 fragrance materials was assessed in the BlueScreen HC assay along with existing or newly generated in vitro and in vivo genotoxicity data. Based on a weight-of-evidence approach, the majority of materials in the data set were deemed negative and concluded not to have the potential to be genotoxic, while only a small proportion of materials were determined to show genotoxic effects in these assays. Analysis of the data set showed a combination of high positive agreement but low negative agreement between BlueScreen HC results, in vitro regulatory genotoxicity assays, and higher-tier test results. The BlueScreen HC assay did not generate any false negatives, thereby providing robustness when utilizing it as a high-throughput screening tool to evaluate the large inventory of fragrance materials. From the perspective of protecting public health, it is desirable to have no or minimal false negatives, as a false-negative result may incorrectly indicate the lack of a genotoxicity hazard. However, the assay did have a high percentage of false-positive results, resulting in poor positive predictivity of the in vitro genotoxicity test battery outcome. Overall, the assay generated 100% negative predictivity and 3.9% positive predictivity. In addition to the data set of 371 fragrance materials, 30 natural complex substances were evaluated for BlueScreen HC, Ames, and in vitro micronucleus assay, and a good correlation in all three assays was observed. Overall, while a positive result may have to be further investigated, these findings suggest that the BlueScreen HC assay can be a valuable screening tool to detect the genotoxic potential of fragrance materials and mixtures.

AOP report: Development of an adverse outcome pathway for oxidative DNA damage leading to mutations and chromosomal aberrations.

The Genetic Toxicology Technical Committee (GTTC) of the Health and Environmental Sciences Institute (HESI) is developing adverse outcome pathways (AOPs) that describe modes of action leading to potentially heritable genomic damage. The goal was to enhance the use of mechanistic information in genotoxicity assessment by building empirical support for the relationships between relevant molecular initiating events (MIEs) and regulatory endpoints in genetic toxicology. Herein, we present an AOP network that links oxidative DNA damage to two adverse outcomes (AOs): mutations and chromosomal aberrations. We collected empirical evidence from the literature to evaluate the key event relationships between the MIE and the AOs, and assessed the weight of evidence using the modified Bradford-Hill criteria for causality. Oxidative DNA damage is constantly induced and repaired in cells given the ubiquitous presence of reactive oxygen species and free radicals. However, xenobiotic exposures may increase damage above baseline levels through a variety of mechanisms and overwhelm DNA repair and endogenous antioxidant capacity. Unrepaired oxidative DNA base damage can lead to base substitutions during replication and, along with repair intermediates, can also cause DNA strand breaks that can lead to mutations and chromosomal aberrations if not repaired adequately. This AOP network identifies knowledge gaps that could be filled by targeted studies designed to better define the quantitative relationships between key events, which could be leveraged for quantitative chemical safety assessment. We anticipate that this AOP network will provide the building blocks for additional genotoxicity-associated AOPs and aid in designing novel integrated testing approaches for genotoxicity.

Novel Insights into Pyrrolizidine Alkaloid Toxicity and Implications for Risk Assessment: Occurrence, Genotoxicity, Toxicokinetics, Risk Assessment-A Workshop Report.

This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAs' pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.

Use of the EpiDermTM 3D reconstructed skin micronucleus assay for fragrance materials.

In order to evaluate the utility of the 3D reconstructed skin micronucleus assay (3DRSMN) to assess clastogenic/aneugenic potential of the fragrance chemicals, a set of 22 fragrance materials were evaluated in 3DRSMN assay. These materials evaluated were also evaluated in an in vitro as well as in vivo micronucleus assay, conducted as per Organisation for Economic Co-operation and Development guidelines. The results of the RSMN assay were in 100% agreement with the in vivo micronucleus assay results. From this dataset, 18 materials were positive in an in vitro micronucleus assay but were negative in an in vivo micronucleus assay. All these 18 materials were also concluded to be negative in 3DRSMN assay, stressing the importance of the assay to help minimize misleading positive outcomes from the in vitro assay. Since the highest exposure for fragrances is through the dermal route, the RSMN assay fits the applicability domain for testing. Thus, RSMN assay is an important alternative to animal testing for characterization of the genotoxicity potential of fragrance materials.

In vitro and integrated in vivo strategies to reduce animal use in genotoxicity testing.

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.

A genotoxicity assessment approach for botanical materials demonstrated with Poria cocos.

Safety assessment of botanical materials often reveals genotoxicity data gaps. However, there are no harmonized regulatory genotoxicity testing approaches for botanical materials. Furthermore, literature genotoxicity testing reports often lack clear definition of the botanical materials (genus species, plant part, etc.) and/or analytical characterization. Here, upon a review of available regulatory testing batteries for botanicals, the authors conclude that an in vitro 2-test battery, consisting of the Ames test and the in vitro human lymphocyte micronucleus assay (HLM), is appropriate to assess the genotoxicity of botanical materials. This approach was then illustrated using a Poria cocos (PCS) botanical material as a case study. Before the genotoxicity testing, an analytical characterization coupled with in silico approach assured appropriate characterization of PCS and helped inform the genotoxic potential of the triterpenes that drive the genotoxicity assessment. The literature search and DEREK screening did not reveal a genotoxicity concern or a genotoxicity structural alert. PCS was then tested in OECD guideline compliant Ames and in vitro HLM and the negative results from this 2-test battery confirmed the absence of a genotoxic potential of the PCS. This fit-for-purpose approach is expected to be useful to fill genotoxicity data gaps for botanical materials.

The 3D reconstructed skin micronucleus assay using imaging flow cytometry and deep learning: A proof-of-principle investigation.

The reconstructed skin micronucleus (RSMN) assay was developed in 2006, as an in vitro alternative for genotoxicity evaluation of dermally applied chemicals or products. In the years since, significant progress has been made in the optimization of the assay, including publication of a standard protocol and extensive validation. However, the diverse morphology of skin cells makes cell preparation and scoring of micronuclei (MN) tedious and subjective, thus requiring a high level of technical expertise for evaluation. This ultimately has a negative impact on throughput and the assay would benefit by the development of an automated method which could reduce scoring subjectivity while also improving the robustness of the assay by increasing the number of cells that can be scored. Imaging flow cytometry (IFC) with the ImageStream®X Mk II can capture high-resolution transmission and fluorescent imagery of cells in suspension. This proof-of-principle study describes protocol modifications that enable such automated measurement in 3D skin cells following exposure to mitomycin C and colchicine. IFC was then used for automated image capture and the Amnis® Artificial Intelligence (AAI) software permitted identification of binucleated (BN) cells with 91% precision. On average, three times as many BN cells from control samples were evaluated using IFC compared to the standard manual analysis. When IFC MNBN cells were visually scored from within the BN cell images, their frequency compared well with manual slide scoring, showing that IFC technology can be applied to the RSMN assay. This method enables faster time to result than microscope-based scoring and the initial studies presented here demonstrate its capability for the detection of statistically significant increases in MNBN frequencies. This work therefore demonstrates the feasibility of combining IFC and AAI to automate scoring for the RSMN assay and to improve its throughput and statistical robustness.