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.

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.

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.

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.

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.

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.

A comparison of classical and 21st century genotoxicity tools: A proof of concept study of 18 chemicals comparing in vitro micronucleus, ToxTracker and genomics-based methods (TGx-DDI, whole genome clustering and connectivity mapping).

A key step in the risk assessment process of a substance is the assessment of its genotoxic potential. Irrespective of the industry involved, current approaches rely on combinations of two or three in vitro tests and while highly sensitive, their specificity is thought to be limited. A refined in vitro genotoxicity testing strategy with improved predictive capacity would be beneficial and "3R" friendly as it helps to avoid unnecessary in vivo follow-up testing. Here, we describe a proof of concept study evaluating a balanced set of compounds that have in vivo negative or positive outcomes, but variable in vitro data, to determine if we could differentiate between direct and indirect acting genotoxicants. Compounds were examined in TK6 cells using an approach in which the same sample was used to evaluate both early genomic markers (Affymetrix analysis 4 hr post treatment), and the genotoxic outcome (micronuclei [MN] after 24 hr). The resulting genomic data was then analyzed using the TGx-DDI biomarker, Connectivity mapping and whole genome clustering. Chemicals were also tested in the ToxTracker assay, which uses six different biomarker genes. None of the methods correctly differentiated all direct from indirect acting genotoxicants when used alone, however, the ToxTracker assay, TGx-DDI biomarker and whole genome approaches provided high predictive capacity when used in combination with the MN assay (1/18, 2/18, 1/18 missed calls). Ultimately, a "fit for purpose" combination will depend on the specific tools available to the end user, as well as considerations of the unique benefits of the individual assays.

Application of the adverse outcome pathway framework to genotoxic modes of action.

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114-134, 2020. © 2019 Wiley Periodicals, Inc.

Relative potency of fifteen pyrrolizidine alkaloids to induce DNA damage as measured by micronucleus induction in HepaRG human liver cells.

Plant-based 1,2-unsaturated Pyrrolizidine Alkaloids (PAs) can be found as contaminants in foods like teas, herbs and honey. PAs are responsible for liver genotoxicity/carcinogenicity following metabolic activation, making them a relevant concern for safety assessment. Current regulatory risk assessments take a precautionary approach and assume all PAs are as potent as the known most potent representatives: lasiocarpine and riddelliine. Our study investigated whether genotoxicity potency differed as a consequence of structural differences, assessing micronuclei in vitro in HepaRG cells which express metabolising enzymes at levels similar to primary human hepatocytes. Benchmark Dose (BMD) analysis was used to calculate the critical effect dose for 15 PAs representing 6 structural classes. When BMD confidence intervals were used to rank PAs, lasiocarpine was the most potent PA and plotted distinctly from all other PAs examined. PA-N-oxides were least potent, notably less potent than their corresponding parent PA's. The observed genotoxic potency compared favorably with existing in vitro data when metabolic competency was considered. Although further consideration of biokinetics will be needed to develop a robust understanding of relative potencies for a realistic risk assessment of PA mixtures, these data facilitate understanding of their genotoxic potencies and affirm that not all PAs are created equal.

Weak silica nanomaterial-induced genotoxicity can be explained by indirect DNA damage as shown by the OGG1-modified comet assay and genomic analysis.

In a previous study, 15-nm silica nanoparticles (NPs) caused small increases in DNA damage in liver as measured in the in vivo comet and micronucleus assays after intravenous administration to rats at their maximum tolerated dose, a worst-case exposure scenario. Histopathological examination supported a particle-induced, tissue damage-mediated inflammatory response. This study used a targeted approach to provide insight into the mode of action (MoA) by examining transcriptional regulation of genes in liver in a time and dose-dependent manner at 1, 2, 4, 8 and 24 h after intravenous administration of 15-nm silica NPs. DNA damage was assessed using the standard comet assay and hOGG1 glycosylase-modified comet assay that also measures oxidative DNA damage. Potassium bromate, an IARC Class 2B carcinogen that specifically operates via an oxidative stress MoA, was used as a positive control for the hOGG1 comet assay and gave a strong signal in its main target organ, the kidney, while showing less activity in liver. Treatment of rats with silica NPs at 50 mg/kg body weight (bw) caused small, statistically insignificant increases in DNA damage in liver measured by the standard comet assay, while a statistically significant increase was observed at 4 h with the hOGG1 comet assay, consistent with a MoA involving reactive oxygen species. Histopathology showed liver damage and neutrophil involvement while genomic analysis and response pattern of key genes involved in inflammation and oxidative stress supported a tissue damage-mediated inflammatory response involving the complement system for removing/phagocytising damaged cells. No changes were observed for histopathology or gene array for the low-dose (5 mg/kg bw) silica NPs. The results of this study confirm our hypothesis that the weak DNA damage observed by silica NPs occurs secondary to inflammation/immune response, indicating that a threshold can be applied in the risk assessment of these materials.