An orthogonal methods assessment of topical drug concentrations in skin and the impact for risk assessment in the viable epidermis.

Systemic toxicity assessments for oral or parenteral drugs often utilize the concentration of drug in plasma to enable safety margin calculations for human risk assessment. For topical drugs, there is no standard method for measuring drug concentrations in the stratum basale of the viable epidermis. This is particularly important since the superficial part of the epidermis, the stratum corneum (SC), is nonviable and where most of a topically applied drug remains, never penetrating deeper into the skin. We investigated the relative concentrations of a prototype kinase inhibitor using punch biopsy, laser capture microdissection, and imaging mass spectrometry methods in the SC, stratum basale, and dermis of minipig skin following topical application as a cream formulation. The results highlight the value of laser capture microdissection and mass spectrometry imaging in quantifying the large difference in drug concentration across the skin and even within the epidermis, and supports use of these methods for threshold-based toxicity risk assessments in specific anatomic locations of the skin, like of the stratum basale.

Synthesis, molecular editing, and biological assessment of the potent cytotoxin leiodermatolide.

It was by way of total synthesis that the issues concerning the stereostructure of leiodermatolide (1) have recently been solved; with the target now being unambiguously defined, the mission of synthesis changes as to secure a meaningful supply of this exceedingly scarce natural product derived from a deep-sea sponge. To this end, a scalable route of 19 steps (longest linear sequence) has been developed, which features a catalytic asymmetric propargylation of a highly enolizable ß-keto-lactone, a ring closing alkyne metathesis and a modified Stille coupling as the key transformations. Deliberate digression from this robust blueprint brought a first set of analogues into reach, which allowed the lead qualities of 1 to be assessed. The acquired biodata show that 1 is a potent cytotoxin in human tumor cell proliferation assays, distinguished by GI50 values in the ≤3 nM range even for cell lines expressing the Pgp efflux transporter. Studies with human U2OS cells revealed that 1 causes mitotic arrest, micronucleus induction, centrosome amplification and tubulin disruption, even though no evidence for direct tubulin binding has been found in cell-free assays; moreover, the compound does not seem to act through kinase inhibition. Indirect evidence points at centrosome declustering as a possible mechanism of action, which provides a potentially rewarding outlook in that centrosome declustering agents hold promise of being inherently selective for malignant over healthy human tissue.

Comparative analysis of micronucleus induction and DNA damage biomarkers in TK6 and A375 cells using flow cytometry.

Previously, we introduced an alternative adherent A375 cell line for clastogenicity and aneugenicity testing using a high content imaging platform. To further characterize the performance of A375 cells, we investigated the sensitivity and specificity of A375 and TK6 cells by directly comparing micronucleus (MN) induction, cytotoxicity (relative cell counts, viability, and apoptosis), clastogenicity (γH2AX), and aneuploidy markers (pH 3, MPM-2, and polyploidy) using flow cytometric methods. We evaluated 14 compounds across different mechanisms (non-genotoxic apoptosis inducers, clastogens, and aneugens with either tubulin binding or aurora kinase inhibiting phenotypes) at 4-h and 24-h post treatment. Both aneugens and clastogens tested positive for micronucleus induction in both cell lines. Apoptosis continued to be a confounding factor for flow cytometry-based micronuclei assessment in TK6 cells as evidenced by positive responses by the three cytotoxicants. Conversely, A375 cells were not affected by apoptosis-related false positive signals and did not produce a positive response in the in vitro micronucleus assay. Benchmark dose response (BMD) analysis showed that the induction of micronuclei and biomarkers occurred at similar concentrations in both cell lines for clastogens and aneugens. By showing that A375 cells have similar sensitivity to TK6 cells but a greater specificity, these results provide additional support for A375 cells to be used as an alternative adherent cell line for in vitro genetic toxicology assessment.

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.

Comparison of the transgenic rodent mutation assay, error corrected next generation duplex sequencing, and the alkaline comet assay to detect dose-related mutations following exposure to N-nitrosodiethylamine.

N-Nitrosodiethylamine (NDEA), a well-studied N-nitrosamine, was tested in rats to compare the dose-response relationship of three genotoxicity endpoints. Mutant / mutation frequencies were determined using the transgenic rodent (TGR) gene mutation assay and error corrected next generation sequencing (ecNGS) (i.e., duplex sequencing (DS)), and genetic damage was detected by the alkaline comet assay. Big Blue® (cII Locus) animals (n = 6 per dose group) were administered doses of 0.001, 0.01, 0.1, 1, 3 mg/kg/day NDEA by oral gavage. Samples were collected for cII mutation and DS analyses following 28-days of exposure and 3 days recovery. In a separate study, male Sprague-Dawley (SD) rats (n = 6 per dose group) were administered the same doses by oral gavage for two consecutive days and then samples collected for the alkaline comet assay. A dose-related increase in mutant / mutation frequencies of the liver but not duodenum was observed using the TGR assay and DS with DS resulting in a slightly more sensitive response, with a lower benchmark dose (BMD). In addition, a dose-related increase in percent tail DNA was observed in the liver using the alkaline comet assay. Therefore, DS and comet assays showed good utility for hazard identification and dose-response analysis of a representative N-nitrosamine comparable to the TGR gene mutation assay.

Development and validation of an in vitro micronucleus assay platform in TK6 cells.

The Organization for Economic Co-operation and Development (OECD) has recently adopted Test Guideline 487 (TG487) for conducting the in vitro micronucleus (MNvit) assay. The purpose of this study is to evaluate and validate treatment conditions for the use of p53 competent TK6 human lymphoblastoid cells in a TG487 compliant MNvit assay. The ten reference compounds suggested in TG487 (mitomycin C, cytosine arabinoside, cyclophosphamide, benzo-a-pyrene, vinblastine sulphate, colchicine, sodium chloride, nalidixic acid and di(2-ethylhexyl)phthalate and pyrene) and noscapine hydrochloride were chosen for this study. In order to optimize the micronucleus response after treatment with some positive substances, we extended the recovery time after pulse treatment from 2 cell cycles recommended in TG487 to 3 cell cycles for untreated cells (40h). Each compound was tested in at least one of four exposure conditions: a 4h exposure followed by a 40h recovery, a 4h exposure followed by a 24h recovery, a 4h exposure in the presence of an exogenous metabolic activation system followed by a 40h recovery period, and a 27h continuous direct treatment. Results show that the direct acting clastogens, clastogens requiring metabolic activation and aneugens caused a robust increase in micronuclei in at least one test condition whereas the negative compounds did not induce micronuclei. The negative control cultures exhibited reproducibly low and consistent micronucleus frequencies ranging from 0.4 to 1.8% (0.8±0.3% average and standard deviation). Furthermore, extending the recovery period from 24h to 40h produced a 2-fold higher micronucleus frequency after a 4h pulse treatment with mitomycin C. In summary, the protocol described in this study in TK6 cells produced the expected result with model compounds and should be suitable for performing the MNvit assay in accordance with guideline TG487.

Development challenges for carcinogenicity risk assessments of topical drugs.

The nonclinical safety package to support development and approval of drugs intended to be administered by topical application generally follows International Council for Harmonisation multidisciplinary 3 (ICH M3) and topic specific safety (ICH S) guidances. However, some aspects of topical drug development may require case-by-case determination of nonclinical safety strategies. The necessity to conduct a dermal rodent carcinogenicity study is one such example that is not considered an obligate component of a nonclinical safety data package for drug approval. While absence of systemic exposure, as stated in ICH M3, is a primary reason to forego a dermal carcinogenicity assessment, there may also be other factors for consideration in determining the need for a life-time carcinogencity study by dermal route to aid in the overall human cancer risk assessment. We therefore reviewed nonclinical carcinogencity data packages from drugs approved by the FDA or PMDA over a ~25 year time period to evaluate outcomes of oral versus topical carcinogencity studies and to understand their utility for informing the overall human risk assessment. We also discuss various other properties of topical small molecules that could impact the decisions to conduct a dermal life-time rodent carcinogenicity study. Collectively, the need to conduct 2-year dermal carcinogenicity studies in rodents should be determined case-by-case and consider scientific factors such existing systemic toxicity and carcinogenicity study data, anticipated drug exposures in skin, skin evaluation from the chronic minipig toxicity study, and genetic toxicity profile.

A new imaging platform (iScreen) allows for the concurrent assessment of micronucleus induction and genotoxic mode of action in human A375 cells.

Genotoxicity testing guidelines require the assessment of the clastogenic and aneugenic potential of compounds. While in vitro micronucleus assays detect both types of endpoints, it requires labor-intensive microscopic scoring and does not discriminate between the two modes of actions. Here, we present a novel high-content imaging platform in A375 human cells that addresses the need for rapid scoring while providing additional mechanistic information. We evaluated the new platform with 12 compounds, three compounds from each mechanistic class (clastogen, aneugen tubulin binder, aneugen aurora inhibitor, and nongenotoxicant) following 4- and 24-h compound treatments. The approach we developed is first discriminating between genotoxicant and nongenotoxicant using an image analysis algorithm to quantify micronucleus induction below a 60% cytotoxicity cutoff. Then it uses centromere protein A (CENPA) staining for the genotoxic compounds to discriminate between aneugens and clastogens. Lastly, we use phosphorylated histone H2AX Ser139 (γH2AX) staining to confirm clastogenicity and changes in phosphorylated histone 3 Ser10 (pH 3) and increases in polyploidy in mitotic cells to discriminate between aneugens that bind tubulin from those that affect aurora kinases. All compounds were correctly classified, and we showed by using benchmark dose-response analysis that the imaging platform in A375 cells is at least as sensitive as the MicroFlow® assay in TK6 cells for genotoxicant but appears to be more specific for the nongenotoxicants. A detailed comparison of the cell lines and a more comprehensive validation with a much larger compound set, predictive and dose-response modeling will be presented in the future.

Defining EMS and ENU dose-response relationships using the Pig-a mutation assay in rats.

In recent years, experimental evidence has accumulated that supports the existence of sublinear dose-response relationships at low doses of DNA reactive mutagens. However, creating the in vivo data necessary to allow for a more detailed dose-response modeling with the currently available tools might not always be practical. The purpose of the current work was to evaluate the utility of the Pig-a gene mutation assay to rapidly identify dose-response relationships for direct acting genotoxicants. The induction of mutations in the peripheral blood of rats was evaluated following 28 days of exposure down to low doses of the direct acting alkylating agents ethyl methane sulfonate (EMS) and ethylnitrosourea (ENU). Using statistical modeling based on the 28-day studies, a threshold for mutation induction for EMS was estimated to be 21.9mg/kg, whereas for the more potent ENU, the threshold was estimated to be 0.88mg/kg. Comparing mutation frequencies from acute and sub-chronic dosing indicated less than additive dose-response relationships, further confirming the possibility of a threshold dose-response relationship for both compounds. In conclusion, the work presented provides evidence that the Pig-a assay might be a practical alternative to other in vivo mutation assays when assessing dose-response relationships for direct acting mutagens and that an experimental approach using fractionated dosing could be used to substantiate a biological mechanism responsible for the observation of a sublinear dose-response relationship.

Workshop summary: Top concentration for in vitro mammalian cell genotoxicity assays; and report from working group on toxicity measures and top concentration for in vitro cytogenetics assays (chromosome aberrations and micronucleus).

The selection of maximum concentrations for in vitro mammalian cell genotoxicity assays was reviewed at the 5th International Workshop on Genotoxicity Testing (IWGT), 2009. Currently, the top concentration recommended when toxicity is not limiting is 10mM or 5mg/ml, whichever is lower. The discussion was whether to reduce the limit, and if so whether the 1mM limit proposed for human pharmaceuticals was appropriate for testing other chemicals. The consensus was that there was reason to consider reducing the 10mM limit, and many, but not all, attendees favored a reduction to 1mM. Several proposals are described here for the concentration limit. The in vitro cytogenetics expert working group also discussed appropriate measures and level of cytotoxicity. Data were reviewed from a multi-laboratory trial of the in vitro micronucleus (MN) assay with multiple cell types and several types of toxicity measurements. The group agreed on a preference for toxicity measures that take cell proliferation after the beginning of treatment into account (relative increase in cell counts, relative population doubling, cytokinesis block proliferation index or replicative index), and that this applies both to in vitro MN assays and to in vitro chromosome aberration assays. Since relative cell counts (RCC) underestimate toxicity, many group members favored making a recommendation against the use of RCC as a toxicity measure for concentration selection. All 14 chemicals assayed for MN induction in the multi-laboratory trial were detected without exceeding 50% toxicity by any measure, but some were positive only at concentrations with toxicity quite close to 50%. The expert working group agreed to accept the cytotoxicity range recommended by OECD guideline 487 (55±5% toxicity at the top concentration scored). This also reinforces the original intent of the guidance for the in vitro chromosome aberration assay, where ">50%" was intended to target the range close to 50% toxicity.

Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing.

A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.

Experiments in the EpiDerm 3D Skin In Vitro Model and Minipigs In Vivo Indicate Comparatively Lower In Vivo Skin Sensitivity of Topically Applied Aneugenic Compounds.

Risk management of in vitro aneugens for topically applied compounds is not clearly defined because there is no validated methodology to accurately measure compound concentration in proliferating stratum basale keratinocytes of the skin. Here, we experimentally tested several known aneugens in the EpiDerm reconstructed human skin in vitro micronucleus assay and compared the results to flow cytometric mechanistic biomarkers (phospho-H3; MPM2, DNA content). We then evaluated similar biomarkers (Ki-67, nuclear area) using immunohistochemistry in skin sections of minipigs following topical exposure the potent aneugens, colchicine, and hesperadin. Data from the EpiDerm model showed positive micronucleus responses for all aneugens tested following topical or direct media dosing with similar sensitivity when adjusted for applied dose. Quantitative benchmark dose-response analysis exhibited increases in the mitotic index biomarkers phospho-H3 and MPM2 for tubulin binders and polyploidy for aurora kinase inhibitors are at least as sensitive as the micronucleus endpoint. By comparison, the aneugens tested did not induce histopathological changes, increases in Ki-67 immunolabeling or nuclear area in skin sections from the in vivo minipig study at doses in significant excess of those eliciting a response in vitro. Results indicate the EpiDerm in vitro micronucleus assay is suitable for the hazard identification of aneugens. The lack of response in the minipig studies indicates that the barrier function of the minipig skin, which is comparable to human skin, protects from the effects of aneugens in vivo. These results provide a basis for conducting additional studies in the future to further refine this understanding.

Evaluation of a modified CD71 MicroFlow method for the flow cytometric analysis of micronuclei in rat bone marrow erythrocytes.

The aim of this study was to evaluate a modified flow cytometric method for the quantification of micronuclei in rat bone marrow reticulocytes. The method identified uses the erythrocyte pure fraction from cellulose filtered bone marrow with slight modifications to the widely published MicroFlow(®) method developed by Litron Laboratories, Rochester, NY for the detection of micronuclei in peripheral blood. A number of experiments were conducted to compare the micronucleus induction measured by flow cytometry with traditional microscopic analysis in male rats treated daily for 2 days with appropriate vehicle controls or various doses of cyclophosphamide (CP), mitomycin C (MMC), vinblastine sulfate (VBS), 1,2-dimethylhydrazine (DMH), etoposide (ETO), colchicine (COL), or 4-nitroquinoline-1-oxide (4NQO). In addition, for a subset of chemical we compared the induction of micronuclei in bone marrow and peripheral blood. The results from this study showed a very good correlation of micronucleus frequencies in bone marrow between microscopic analysis and the flow cytometry as well as between blood and bone marrow. In general, micronucleus frequencies of test compound treated animals and inter-animal variability were slightly lower by flow cytometric analysis compared to manual slide analysis. The data presented in this study support the use of the CD71 flow method for the analysis of micronuclei in rat bone marrow and also suggest that peripheral blood may be equally as sensitive as bone marrow in detecting a micronucleus response in short term studies.

Evaluation of phenolphthalein, diazepam and quinacrine dihydrochloride in the in vitro mammalian cell micronucleus test in Chinese hamster ovary (CHO) and TK6 cells.

The in vitro micronucleus assay has been extensively used as an in vitro screening tool to identify test articles that might have aneugenic or clastogenic potential. Currently, the Organization for Economic Co-operation and Development (OECD) is working towards a final version of the guideline for the conduct of the in vitro mammalian cell micronucleus Test (MNvit). A few questions regarding appropriate cytotoxicity measurements and cytotoxicity limits to use remain to be answered. In order to resolve the remaining questions, we compared the induction of micronuclei at the top dose (50-60% cytotoxicity) determined by either Relative Cell Counts (RCC), Relative Increase in Cell Counts (RICC), Relative Population Doublings (RPD), or Cytokinesis-Blocked Proliferating Index (CBPI) using weak and strong inducers of micronuclei in both the presence and absence of cytochalasin B (CYB) in Chinese hamster ovary (CHO) and human lymphoblastoid TK6 cells. In order to assess extensive dose-response relationships, we selected expected weak (diazepam, phenolphthalein, quinacrine dihydrochloride dihydrate) and strong (cytosine arabinoside, mitomycin C, vinblastine sulphate) inducers of micronuclei with a variety of different mechanisms of action for testing. The results clearly demonstrated that all six compounds produced positive responses using either cytotoxicity measurement. The outcome from these studies further supports the cytotoxicity measurements and cytotoxicity limits proposed in the draft OECD guideline.

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.

Chemically induced aneuploidy in germ cells. Part II of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

As part of the 7th International Workshops on Genotoxicity Testing held in Tokyo, Japan in November 2017, a workgroup of experts reviewed and assessed the risk of aneugens for human health. The present manuscript is one of three manuscripts from the workgroup and reports on the unanimous consensus reached on the evidence for aneugens affecting germ cells, their mechanisms of action and role in hereditary diseases. There are 24 chemicals with strong or sufficient evidence for germ cell aneugenicity providing robust support for the ability of chemicals to induce germ cell aneuploidy. Interference with microtubule dynamics or inhibition of topoisomerase II function are clear characteristics of germ cell aneugens. Although there are mechanisms of chromosome segregation that are unique to germ cells, there is currently no evidence for germ cell-specific aneugens. However, the available data are heavily skewed toward chemicals that are aneugenic in somatic cells. Development of high-throughput screening assays in suitable animal models for exploring additional targets for aneuploidy induction, such as meiosis-specific proteins, and to prioritize chemicals for the potential to be germ cell aneugens is encouraged. Evidence in animal models support that: oocytes are more sensitive than spermatocytes and somatic cells to aneugens; exposure to aneugens leads to aneuploid conceptuses; and, the frequencies of aneuploidy are similar in germ cells and zygotes. Although aneuploidy in germ cells is a significant cause of infertility and pregnancy loss in humans, there is currently limited evidence that aneugens induce hereditary diseases in human populations because the great majority of aneuploid conceptuses die in utero. Overall, the present work underscores the importance of protecting the human population from exposure to chemicals that can induce aneuploidy in germ cells that, in contrast to carcinogenicity, is directly linked to an adverse outcome.

High information content assays for genetic toxicology testing: A report of the International Workshops on Genotoxicity Testing (IWGT).

We live in an era of 'big data', where the volume, velocity, and variety of the data being generated is increasingly influencing the way toxicological sciences are practiced. With this in mind, a workgroup was formed for the 2017 International Workshops on Genotoxicity Testing (IWGT) to consider the use of high information content data in genetic toxicology assessments. Presentations were given on adductomics, global transcriptional profiling, error-reduced single-molecule sequencing, and cellular phenotype-based assays, which were identified as methodologies that are relevant to present-day genetic toxicology assessments. Presenters and workgroup members discussed the state of the science for these methodologies, their potential use in genetic toxicology, current limitations, and the future work necessary to advance their utility and application. The session culminated with audience-assisted SWOT (strength, weakness, opportunities, and threats) analyses. The summary report described herein is structured similarly. A major conclusion of the workgroup is that while conventional regulatory genetic toxicology testing has served the public well over the last several decades, it does not provide the throughput that has become necessary in modern times, and it does not generate the mechanistic information that risk assessments ideally take into consideration. The high information content assay platforms that were discussed in this session, as well as others under development, have the potential to address aspect(s) of these issues and to meet new expectations in the field of genetic toxicology.

Targets and mechanisms of chemically induced aneuploidy. Part 1 of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

An aneuploidy workgroup was established as part of the 7th International Workshops on Genotoxicity Testing. The workgroup conducted a review of the scientific literature on the biological mechanisms of aneuploidy in mammalian cells and methods used to detect chemical aneugens. In addition, the current regulatory framework was discussed, with the objective to arrive at consensus statements on the ramifications of exposure to chemical aneugens for human health risk assessment. As part of these efforts, the workgroup explored the use of adverse outcome pathways (AOPs) to document mechanisms of chemically induced aneuploidy in mammalian somatic cells. The group worked on two molecular initiating events (MIEs), tubulin binding and binding to the catalytic domain of aurora kinase B, which result in several adverse outcomes, including aneuploidy. The workgroup agreed that the AOP framework provides a useful approach to link evidence for MIEs with aneuploidy on a cellular level. The evidence linking chemically induced aneuploidy with carcinogenicity and hereditary disease was also reviewed and is presented in two companion papers. In addition, the group came to the consensus that the current regulatory test batteries, while not ideal, are sufficient for the identification of aneugens and human risk assessment. While it is obvious that there are many different MIEs that could lead to the induction of aneuploidy, the most commonly observed mechanisms involving chemical aneugens are related to tubulin binding and, to a lesser extent, inhibition of mitotic kinases. The comprehensive review presented here should help with the identification and risk management of aneugenic agents.

Role of aneuploidy in the carcinogenic process: Part 3 of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

Aneuploidy is regarded as a hallmark of cancer, however, its role is complex with both pro- and anti-carcinogenic effects evident. In this IWGT review, we consider the role of aneuploidy in cancer biology; cancer risk associated with constitutive aneuploidy; rodent carcinogenesis with known chemical aneugens; and chemotherapy-related malignant neoplasms. Aneuploidy is seen at various stages in carcinogenesis. However, the relationship between induced aneuploidy occurring after exposure and clonal aneuploidy present in tumours is not clear. Recent evidence indicates that the induction of chromosomal instability (CIN), may be more important than aneuploidy per se, in the carcinogenic process. Down Syndrome, trisomy 21, is associated with altered hematopoiesis in utero which, in combination with subsequent mutations, results in an increased risk for acute megakaryoblastic and lymphoblastic leukemias. In contrast, there is reduced cancer risk for most solid tumours in Down Syndrome. Mouse models with high levels of aneuploidy are also associated with increased cancer risk for particular tumours with long latencies, but paradoxically other types of tumour often show decreased incidence. The aneugens reviewed that induce cancer in humans and animals all possess other carcinogenic properties, such as mutagenicity, clastogenicity, cytotoxicity, organ toxicities, hormonal and epigenetic changes which likely account for, or interact with aneuploidy, to cause carcinogenesis. Although the role that aneuploidy plays in carcinogenesis has not been fully established, in many cases, it may not play a primary causative role. Tubulin-disrupting aneugens that do not possess other properties linked to carcinogenesis, were not carcinogenic in rodents. Similarly, in humans, for the tubulin-disrupting aneugens colchicine and albendazole, there is no reported association with increased cancer risk. There is a need for further mechanistic studies on agents that induce aneuploidy, particularly by mechanisms other than tubulin disruption and to determine the role of aneuploidy in pre-neoplastic events and in early and late stage neoplasia.

Analysis of historical negative control group data from the in vitro micronucleus assay using human lymphocytes.

A database of the micronuclei counts was built up for historical negative control data from human lymphocyte in vitro micronuclei tests (MnVit) carried out in 8 laboratories with experience of the method. The mean incidence of micronucleated cells (mnt)/1000 cells ranged from 2.2/1000 to 15.9/1000. There were no large differences in incidence between the presence or absence of S9 mix or between different treatment lengths. There was also little evidence that different solvents affected the numbers of micronuclei appreciably. A number of laboratories did show significant inter-experiment variability, indicating that there remained unidentified factors affecting frequencies. Donor variance may be one such factor. Inter-individual variability may explain some of these differences. The approximate 7.5-fold difference in mnt/1000 scores in a relatively small group of experienced laboratories illustrates the potential complications that can arise if a metric like a fold increase was considered the only biologically important finding. Although there is inherent variability between experiments, it was evident that within a laboratory the overall laboratory mean remains constant over time. It is believed that these findings will provide help to laboratories conducting studies using human lymphocytes in the MnVit and to those involved in the assessment of MnVit results.