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.

Mission Alliance Community Engagement Project: Exploring the Impact of COVID-19 on Social Isolation, Loneliness, Mental Health and Wellbeing in Veterans.

During the Coronavirus disease pandemic, many U.S. veterans with posttraumatic stress disorder (PTSD) experienced increased symptomology and worsened mental health and well-being due in part to social isolation and loneliness. The Mission Alliance project explored these ramifications and prioritized critical issues expressed by U.S. veterans and stakeholders (N = 182) during virtual regional meetings (N = 32). Field notes created specifically for this project were recorded and thematically analyzed. Emerging themes included: (1) social isolation: missed opportunities, collapsed social circles, work-life balance, fostering relationships, and evolving health care delivery; (2) loneliness: deteriorated mental health, suffered with PTSD together but alone, looked out for each other, ambivalence toward technology, and strained and broken systems; (3) mental health: sense of chaos, increased demand and decreased access, aggravation, implementation of tools, innovative solutions, fear and loss, and availability of resources; (4) wellbeing: sense of purpose, holistic perspective on well-being, recognition of balance, persisting stigma, redefined pressures, freedom to direct treatment, and reconnection and disconnection. A PTSD-related patient centered outcomes research (PCOR)/comparative effectiveness research (CER) agenda was developed from these themes. Establishment of a veteran and stakeholder network is suggested to support, facilitate, and promote the PTSD-related PCOR/CER agenda. Furthermore, enhancement of opportunities for veterans with PTSD and stakeholders to partner in PCOR/CER is required to develop and conduct projects that lead to PTSD-related comprehensive care of veterans affected by traumatic events with the potential to translate findings to other populations.

In vivo genotoxicity testing strategies: Report from the 8th International Workshop on Genotoxicity Testing (IWGT).

The in vivo working group (WG) considered three topics: acceptable maximum doses for negative erythrocyte micronucleus (MN) tests, validation status of MN assays in non-hematopoietic tissues, and nuisance factors in the comet assay. The WG reached agreement on many issues, including: negative erythrocyte MN studies should be acceptable if dosing is conducted to Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 474 recommendations and if sufficient bone marrow exposure is demonstrated; consensus on the evidence required to demonstrate "sufficient" exposure was not reached. The liver MN test using six-week-old rats is sufficiently validated to develop an OECD TG, but the impact of animal age warrants additional study. Ki-67 is a reliable marker for cellular proliferation in hepatocytes. The gastrointestinal tract MN test is useful for detecting poorly absorbed or rapidly degraded aneugens, and for genotoxic metabolites formed in the colon. Although current validation data are insufficient to support the development of an OECD TG, the methodologies are sufficient to consider as an appendix to OECD TG474. Comparison of comet assay results to laboratory historical control data (HCD) should not be used in data evaluation, unless the HCD distribution is demonstrated to be stable and the predominant source of HCD variation is due to animal, not study, factors. No universally acceptable negative control limit for any tissue was identified. Methodological differences in comet studies can result in variable data interpretations; more data are required before best practice recommendations can be made. Hedgehogs alone are unreliable indicators of cytotoxicity and additional investigations into cytotoxicity markers are required.

International Workshops on Genotoxicity Testing (IWGT): Origins, achievements and ambitions.

Over the past thirty years, the International Workshops on Genotoxicity Testing (IWGT) became one of the leading groups in the field of regulatory genotoxicology, not only due to the diversity of participants with respect to geography and professional affiliation, but also due to the unique setup of recurring IWGT meetings every four years. The hallmarks of the IWGT process have been diligent initial planning approaches of the working groups, collection of data so as to stimulate data-driven discussions and debate, and striving to reach consensus recommendations. The scientific quality of the Working Groups (WGs) has been exceptional due to the selection of highly regarded experts on each topic. As a result, the IWGT working group reports have become important documents. The deliberations and publications have provided guidance on test systems and testing protocols that have influenced the development or revision of test guidelines of the Organisation for Economic Co-operation and Development (OECD), guidance by the International Council for Harmonisation (ICH), and strategic testing or data analysis approaches in general. This article summarizes the history of the IWGT, identifies some of its major achievements, and provides an outlook for the future.

Valuing Negative Affect Weakens Affect-Health Linkages: Similarities and Differences Across Affect Valuation Measures.

Negative affect (NA) has been robustly linked to poorer psychological health, including greater depressive symptoms, personal burnout, and perceived stress. These associations, known as affect-health links, have been postulated by our research team to vary with different levels of negative affect valuation (NAV), such that people who evaluate NA states as more pleasant, helpful, appropriate, and/or meaningful may show weaker affect-health links. Another affect valuation construct is ideal NA, which is the degree to which people ideally want to experience NA states (i.e., desirability of affective states). The current study extends previous research by examining these two different measures of affect valuation (NAV and ideal NA) and comparing the extent to which they moderate affect-health links for psychological health and functioning. Participants from the Health and Daily Experiences (HEADE) study (N = 162 comprising of 56 younger adults and 106 older adults) completed questionnaires in a laboratory setting and ecological momentary assessments of NA 6 times a day for 7 consecutive days (i.e., trait NA). The results demonstrated that the two affect valuation constructs were distinct and showed different patterns of buffering effects. NAV attenuated the association between trait NA and depressive symptoms, personal burnout, and intolerance of uncertainty. Ideal NA attenuated affect-health links for depressive symptoms and perceived stress. These findings point to the importance of sharpening the distinctions between various affect valuation constructs to elucidate their unique contributions to attenuating affect-health links.

Impact of sampling time on the detection of mutations in rapidly proliferating tissues using transgenic rodent gene mutation models: A review.

The OECD Test Guideline 488 (TG 488) for the Transgenic Rodent Gene Mutation Assay has undergone several revisions to update the recommended design for studying mutations in somatic tissues and male germ cells. The recently revised TG recommends a single sampling time of 28 days following 28 days of exposure (i.e., 28 + 28 days) for all tissues, irrespective of proliferation rates. An alternative design (i.e., 28 + 3 days) is appropriate when germ cell data is not required, nor considered. While the 28 + 28 days design is clearly preferable for slowly proliferating somatic tissues and germ cells, there is still uncertainty about the impact of extending the sampling time to 28 days for rapidly somatic tissues. Here, we searched the available literature for evidence supporting the applicability and utility of the 28 + 28 days design for rapidly proliferating tissues. A total of 79 tests were identified. When directly comparing results from both designs in the same study, there was no evidence that the 28 + 28 days regimen resulted in a qualitatively different outcome from the 28 + 3 days design. Studies with a diverse range of agents that employed only a 28 + 28 days protocol provide further evidence that this design is appropriate for rapidly proliferating tissues. Benchmark dose analyses demonstrate high quantitative concordance between the 28 + 3 and 28 + 28 days designs for rapidly proliferating tissues. Accordingly, our review confirms that the 28 + 28 days design is appropriate to assess mutagenicity in both slowly and rapidly proliferating somatic tissues, and germ cells, and provides further support for the recommended design in the recently adopted TG 488.

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).

A comparison of the lowest effective concentration in culture media for detection of chromosomal damage in vitro and in blood or plasma for detection of micronuclei in vivo.

It is often assumed that genotoxic substances will be detected more easily by using in vitro rather than in vivo genotoxicity tests since higher concentrations, more cytotoxicity and static exposures can be achieved. However, there is a paucity of data demonstrating whether genotoxic substances are detected at lower concentrations in cell culture in vitro than can be reached in the blood of animals treated in vivo. To investigate this issue, we compared the lowest concentration required for induction of chromosomal damage in vitro (lowest observed effective concentration, or LOEC) with the concentration of the test substance in blood at the lowest dose required for biologically relevant induction of micronuclei in vivo (lowest observed effective dose, or LOED). In total, 83 substances were found for which the LOED could be identified or estimated, where concentrations in blood and micronucleus data were available via the same route of administration in the same species, and in vitro chromosomal damage data were available. 39.8 % of substances were positive in vivo at blood concentrations that were lower than the LOEC in vitro, 22.9 % were positive at similar concentrations, and 37.3 % of substances were positive in vivo at higher concentrations. Distribution analysis showed a very wide scatter of > 6 orders of magnitude across these 3 categories. When mode of action was evaluated, the distribution of clastogens and aneugens across the 3 categories was very similar. Thus, the ability to detect induction of micronuclei in bone marrow in vivo regardless of the mechanism for micronucleus induction, is clearly not solely determined by the concentration of test substance which induced chromosomal damage in vitro.

A comprehensive weight of evidence assessment of published acetaminophen genotoxicity data: Implications for its carcinogenic hazard potential.

In 2019, the California Office of Environmental Health Hazard Assessment initiated a review of the carcinogenic hazard potential of acetaminophen, including an assessment of its genotoxicity. The objective of this analysis was to inform this review process with a weight-of-evidence assessment of more than 65 acetaminophen genetic toxicology studies that are of widely varying quality and conformance to accepted standards and relevance to humans. In these studies, acetaminophen showed no evidence of induction of point or gene mutations in bacterial and mammalian cell systems or in in vivo studies. In reliable, well-controlled test systems, clastogenic effects were only observed in unstable, p53-deficient cell systems or at toxic and/or excessively high concentrations that adversely affect cellular processes (e.g., mitochondrial respiration) and cause cytotoxicity. Across the studies, there was no clear evidence that acetaminophen causes DNA damage in the absence of toxicity. In well-controlled clinical studies, there was no meaningful evidence of chromosomal damage. Based on this weight-of-evidence assessment, acetaminophen overwhelmingly produces negative results (i.e., is not a genotoxic hazard) in reliable, robust high-weight studies. Its mode of action produces cytotoxic effects before it can induce the stable, genetic damage that would be indicative of a genotoxic or carcinogenic hazard.

A weight of evidence assessment of the genotoxicity of 2,6-xylidine based on existing and new data, with relevance to safety of lidocaine exposure.

Lidocaine has not been associated with cancer in humans despite 8 decades of therapeutic use. Its metabolite, 2,6-xylidine, is a rat carcinogen, believed to induce genotoxicity via N-hydroxylation and DNA adduct formation, a non-threshold mechanism of action. To better understand this dichotomy, we review literature pertaining to metabolic activation and genotoxicity of 2,6-xylidine, identifying that it appears resistant to N-hydroxylation and instead metabolises almost exclusively to DMAP (an aminophenol). At high exposures (sufficient to saturate phase 2 metabolism), this may undergo metabolic threshold-dependent activation to a quinone-imine with potential to redox cycle producing ROS, inducing cytotoxicity and genotoxicity. A new rat study found no evidence of genotoxicity in vivo based on micronuclei in bone marrow, comets in nasal tissue or female liver, despite high level exposure to 2,6-xylidine (including metabolites). In male liver, weak dose-related comet increases, within the historical control range, were associated with metabolic overload and acute systemic toxicity. Benchmark dose analysis confirmed a non-linear dose response. The weight of evidence indicates 2,6-xylidine is a non-direct acting (metabolic threshold-dependent) genotoxin, and is not genotoxic in vivo in rats in the absence of acute systemic toxic effects, which occur at levels 35 × beyond lidocaine-related exposure in humans.

A weight of evidence assessment of the genotoxic potential of 4-methylimidazole as a possible mode of action for the formation of lung tumors in exposed mice.

4-Methylimidazole (4-MeI) is a byproduct formed during the cooking of foods containing carbohydrates and amino acids, including the production of flavors and coloring substances, e.g., class III and IV caramel colors, used in many food products with extensive human exposure. Two-year rodent bioassays via oral exposure conducted by the National Toxicology Program reported evidence of carcinogenicity only in B6C3F1 mice (increased alveolar/bronchial neoplasms). In 2011, the International Agency for Research on Cancer classified 4-MeI as Group 2B, "possibly carcinogenic to humans". An expert panel was commissioned to assess the genotoxic potential of 4-MeI and the plausibility of a genotoxic mode of action in the formation of lung tumors in mice when exposed to high doses of 4-MeI. The panel defined and used a weight-of-evidence (WOE) approach that included thorough evaluation of studies assessing the genotoxic potential of 4-MeI. The panelists categorized each study, consisting of study weight, degree of technical performance, study reliability, and contribution to the overall WOE. Based on the reviewed studies' weighted contribution, the panel unanimously concluded that the WOE supports no clear evidence of in vivo genotoxicity of 4-MeI and no association for a genotoxic mode of action in the formation of mouse lung tumors.

EURL ECVAM Genotoxicity and Carcinogenicity Database of Substances Eliciting Negative Results in the Ames Test: Construction of the Database.

The bacterial reverse mutation test (Ames test) is the most commonly used genotoxicity test; it is a primary component of the chemical safety assessment data required by regulatory agencies worldwide. Within the current accepted in vitro genotoxicity test battery, it is considered capable of revealing DNA reactivity, and identifying substances that can produce gene mutations via different mechanisms. The previously published consolidated EURL ECVAM Genotoxicity and Carcinogenicity Database, which includes substances that elicited a positive response in the Ames test, constitutes a collection of data that serves as a reference for a number of regulatory activities in the area of genotoxicity testing. Consequently, we considered it important to expand the database to include substances that fail to elicit a positive response in the Ames test, i.e., Ames negative substances. Here, we describe a curated collection of 211 Ames negative substances, with a summary of complementary data available for other genotoxicity endpoints in vitro and in vivo, plus available carcinogenicity data. A descriptive analysis of the data is presented. This includes a representation of the chemical space formed by the Ames-negative database with respect to other substances (e.g. REACH registered substances, approved drugs, pesticides, etc.) and a description of the organic functional groups found in the database. We also provide some suggestions on further analyses that could be made.

Use of in vitro 3D tissue models in genotoxicity testing: Strategic fit, validation status and way forward. Report of the working group from the 7th International Workshop on Genotoxicity Testing (IWGT).

Use of three-dimensional (3D) tissue equivalents in toxicology has been increasing over the last decade as novel preclinical test systems and as alternatives to animal testing. In the area of genetic toxicology, progress has been made with establishing robust protocols for skin, airway (lung) and liver tissue equivalents. In light of these advancements, a "Use of 3D Tissues in Genotoxicity Testing" working group (WG) met at the 7th IWGT meeting in Tokyo in November 2017 to discuss progress with these models and how they may fit into a genotoxicity testing strategy. The workshop demonstrated that skin models have reached an advanced state of validation following over 10 years of development, while liver and airway model-based genotoxicity assays show promise but are at an early stage of development. Further effort in liver and airway model-based assays is needed to address the lack of coverage of the three main endpoints of genotoxicity (mutagenicity, clastogenicity and aneugenicity), and information on metabolic competence. The IWGT WG believes that the 3D skin comet and micronucleus assays are now sufficiently validated to undergo an independent peer review of the validation study, followed by development of individual OECD Test Guidelines.

In vivo genotoxicity testing strategies: Report from the 7th International workshop on genotoxicity testing (IWGT).

The working group reached complete or majority agreement on many issues. Results from TGR and in vivo comet assays for 91 chemicals showed they have similar ability to detect in vivo genotoxicity per se with bacterial mutagens and Ames-positive carcinogens. TGR and comet assay results were not significantly different when compared with IARC Group 1, 2 A, and unclassified carcinogens. There were significantly more comet assay positive responses for Group 2B chemicals, and for IARC classified and unclassified carcinogens combined, which may be expected since mutation is a sub-set of genotoxicity. A liver comet assay combined with the bone marrow/blood micronucleus (MNviv) test would detect in vivo genotoxins that do not exhibit tissue-specific or site-of-contact effects, and is appropriate for routine in vivo genotoxicity testing. Generally for orally administered substances, a comet assay at only one site-of-contact GI tract tissue (stomach or duodenum/jejunum) is required. In MNviv tests, evidence of target tissue exposure can be obtained in a number of different ways, as recommended by ICH S2(R1) and EFSA (Hardy et al., 2017). Except for special cases the i.p. route is inappropriate for in vivo testing; for risk evaluations more weight should be given to data from a physiologically relevant administration route. The liver MN test is sufficiently validated for the development of an OECD guideline. However, the impact of dosing animals >6 weeks of age needs to be evaluated. The GI tract MN test shows promise but needs more validation for an OECD guideline. The Pig-a assay detects systemically available mutagens and is a valuable follow-up to in vitro positive results. A new freeze-thaw protocol provides more flexibility. Mutant reticulocyte and erythrocyte frequencies should both be determined. Preliminary data are available for the Pig-a assay in male rat germ cells which require validation including germ cell DNA mutation origin.

A comparison of transgenic rodent mutation and in vivo comet assay responses for 91 chemicals.

A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset. There were fewer datasets from rats than mice, particularly for the TGR assay, and therefore, results from both species were combined for further analysis. TGR and comet responses were compared in liver and bone marrow (the most commonly studied tissues), and in stomach and colon evaluated either separately or in combination with other GI tract segments. Overall positive, negative, or equivocal test results were assessed for each chemical across the tissues examined in the TGR and comet assays using two approaches: 1) overall calls based on weight of evidence (WoE) and expert judgement, and 2) curation of the data based on a priori acceptability criteria prior to deriving final tissue specific calls. Since the database contains a high prevalence of positive results, overall agreement between the assays was determined using statistics adjusted for prevalence (using AC1 and PABAK). These coefficients showed fair or moderate to good agreement for liver and the GI tract (predominantly stomach and colon data) using WoE, reduced agreement for stomach and colon evaluated separately using data curation, and poor or no agreement for bone marrow using both the WoE and data curation approaches. Confidence in these results is higher for liver than for the other tissues, for which there were less data. Our analysis finds that comet and TGR generally identify the same compounds (mainly potent mutagens) as genotoxic in liver, stomach and colon, but not in bone marrow. However, the current database content precluded drawing assay concordance conclusions for weak mutagens and non-DNA reactive chemicals.

A review of the genotoxic potential of 1,4-naphthoquinone.

1,4-Naphthoquinone (1,4-NQ; CAS RN 130-15-4), a derivative of naphthalene, is a commonly used pre-cursor in industrial processes. Since the early 1980's 1,4-NQ has been tested in a number of genotoxicity assays, both in vitro and in vivo. There is strong evidence that 1,4-NQ does not induce gene mutations in bacteria or mammalian cells in vitro with predominantly negative Ames tests and negative Hprt and tk mutation studies. However, there is clear evidence of a clastogenic response in vitro from positive micronucleus, sister chromatid exchange and chromosome aberration assays. 1,4-NQ-treated mice and hamsters were, however, negative for micronucleus or chromosomal aberration induction in GLP-compliant studies with clear evidence of target tissue exposure, suggesting an in vitro only effect. Evidence indicates that the mechanism of in vitro clastogenicity is predominantly via ROS generation, and since in vitro mammalian cell tests systems have poor anti-oxidant defence mechanisms, they are particularly sensitive to oxidative DNA damage. On the other hand, healthy mammalian tissues have more efficient anti-oxidant defence mechanisms, and therefore it is not surprising that 1,4-NQ is not genotoxic in vivo.

An updated review of the genotoxicity of respirable crystalline silica.

Human exposure to (certain forms of) crystalline silica (CS) potentially results in adverse effects on human health. Since 1997 IARC has classified CS as a Group 1 carcinogen [1], which was confirmed in a later review in 2012 [2]. The genotoxic potential and mode of genotoxic action of CS was not conclusive in either of the IARC reviews, although a proposal for mode of actions was made in an extensive review of the genotoxicity of CS by Borm, Tran and Donaldson in 2011 [3]. The present study identified 141 new papers from search strings related to genotoxicity of respirable CS (RCS) since 2011 and, of these, 17 relevant publications with genotoxicity data were included in this detailed review.Studies on in vitro genotoxic endpoints primarily included micronucleus (MN) frequency and % fragmented DNA as measured in the comet assay, and were mostly negative, apart from two studies using primary or cultured macrophages. In vivo studies confirmed the role of persistent inflammation due to quartz surface toxicity leading to anti-oxidant responses in mice and rats, but DNA damage was only seen in rats. The role of surface characteristics was strengthened by in vitro and in vivo studies using aluminium or hydrophobic treatment to quench the silanol groups on the CS surface.In conclusion, the different modes of action of RCS-induced genotoxicity have been evaluated in a series of independent, adequate studies since 2011. Earlier conclusions on the role of inflammation driven by quartz surface in genotoxic and carcinogenic effects after inhalation are confirmed and findings support a practical threshold. Whereas classic in vitro genotoxicity studies confirm an earlier no-observed effect level (NOEL) in cell cultures of 60-70 µg/cm2, transformation frequency in SHE cells suggests a lower threshold around 5 µg/cm2. Both levels are only achieved in vivo at doses (2-4 mg) beyond in vivo doses (> 200 µg) that cause persistent inflammation and tissue remodelling in the rat lung.