Assessing the genotoxicity of N-nitrosodiethylamine with three in vivo endpoints in male Big Blue® transgenic and wild-type C57BL/6N mice.

The detection of N-nitrosamines in drug products has raised global regulatory interest in recent years due to the carcinogenic potential of some nitrosamines in animals and a need to identify a testing strategy has emerged. Ideally, methods used would allow for the use of quantitative analysis of dose-response data from in vivo genotoxicity assays to determine a compound-specific acceptable intake for novel nitrosamines without sufficient carcinogenicity data. In a previous study we compared the dose-response relationships of N-nitrosodiethylamine (NDEA) in three in vivo genotoxicity endpoints in rats. Here we report a comparison of NDEA's genotoxicity profile in mice. Big Blue® mice were administered NDEA at doses of 0.001, 0.01, 0.1, 1 and 3 mg/kg/day by oral gavage for 28 days followed by 3 days of expression. Statistically significant increases in the NDEA induced mutations were detected by both the transgenic rodent mutation assay (TGR) using the cII endpoint and by duplex sequencing in the liver but not bone marrow of mice. In addition, administration of NDEA for two consecutive days in male C57BL/6N mice caused elevated DNA damage levels in the liver as measured by % tail DNA in comet assay. The benchmark dose (BMD) analysis shows a BMDL50 of 0.03, 0.04 and 0.72 mg/kg/day for TGR, duplex sequencing and comet endpoints, respectively. Overall, this study demonstrated a similar genotoxicity profile of NDEA between mice and rats and provides a reference that can be used to compare the potential potency of other novel nitrosamines for the induction of gene mutations.

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.

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.

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.

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.

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.

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.

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.

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.

Analysis of negative historical control group data from the in vitro micronucleus assay using TK6 cells.

The recent revisions of the Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines emphasize the importance of historical negative controls both for data quality and interpretation. The goal of a HESI Genetic Toxicology Technical Committee (GTTC) workgroup was to collect data from participating laboratories and to conduct a statistical analysis to understand and publish the range of values that are normally seen in experienced laboratories using TK6 cells to conduct the in vitro micronucleus assay. Data from negative control samples from in vitro micronucleus assays using TK6 cells from 13 laboratories were collected using a standard collection form. Although in some cases statistically significant differences can be seen within laboratories for different test conditions, they were very small. The mean incidence of micronucleated cells/1000 cells ranged from 3.2/1000 to 13.8/1000. These almost four-fold differences in micronucleus levels cannot be explained by differences in scoring method, presence or absence of exogenous metabolic activation (S9), length of treatment, presence or absence of cytochalasin B or different solvents used as vehicles. The range of means from the four laboratories using flow cytometry methods (3.7-fold: 3.5-12.9 micronucleated cells/1000 cells) was similar to that from the nine laboratories using other scoring methods (4.3-fold: 3.2-13.8 micronucleated cells/1000 cells). No laboratory could be identified as an outlier or as showing unacceptably high variability. Quality Control (QC) methods applied to analyse the intra-laboratory variability showed that there was evidence of inter-experimental variability greater than would be expected by chance (i.e. over-dispersion). However, in general, this was low. This study demonstrates the value of QC methods in helping to analyse the reproducibility of results, building up a 'normal' range of values, and as an aid to identify variability within a laboratory in order to implement processes to maintain and improve uniformity.

Interlaboratory evaluation of a multiplexed high information content in vitro genotoxicity assay.

We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow® DNA Damage Kit-p53, γH2AX, Phospho-Histone H3. For these experiments, seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and nongenotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hr. At 4 and 24 hr, cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all interlaboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or nongenotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals' predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay. Environ. Mol. Mutagen. 58:146-161, 2017. © 2017 Wiley Periodicals, Inc.

IWGT report on quantitative approaches to genotoxicity risk assessment I. Methods and metrics for defining exposure-response relationships and points of departure (PoDs).

This report summarizes the discussion, conclusions, and points of consensus of the IWGT Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (QWG) based on a meeting in Foz do Iguaçu, Brazil October 31-November 2, 2013. Topics addressed included (1) the need for quantitative dose-response analysis, (2) methods to analyze exposure-response relationships & derive point of departure (PoD) metrics, (3) points of departure (PoD) and mechanistic threshold considerations, (4) approaches to define exposure-related risks, (5) empirical relationships between genetic damage (mutation) and cancer, and (6) extrapolations across test systems and species. This report discusses the first three of these topics and a companion report discusses the latter three. The working group critically examined methods for determining point of departure metrics (PoDs) that could be used to estimate low-dose risk of genetic damage and from which extrapolation to acceptable exposure levels could be made using appropriate mode of action information and uncertainty factors. These included benchmark doses (BMDs) derived from fitting families of exponential models, the No Observed Genotoxic Effect Level (NOGEL), and "threshold" or breakpoint dose (BPD) levels derived from bilinear models when mechanistic data supported this approach. The QWG recognizes that scientific evidence suggests that thresholds below which genotoxic effects do not occur likely exist for both DNA-reactive and DNA-nonreactive substances, but notes that small increments of the spontaneous level cannot be unequivocally excluded either by experimental measurement or by mathematical modeling. Therefore, rather than debating the theoretical possibility of such low-dose effects, emphasis should be placed on determination of PoDs from which acceptable exposure levels can be determined by extrapolation using available mechanistic information and appropriate uncertainty factors. This approach places the focus on minimization of the genotoxic risk, which protects against the risk of the development of diseases resulting from the genetic damage. Based on analysis of the strengths and weaknesses of each method, the QWG concluded that the order of preference of PoD metrics is the statistical lower bound on the BMD > the NOGEL > a statistical lower bound on the BPD. A companion report discusses the use of these metrics in genotoxicity risk assessment, including scaling and uncertainty factors to be considered when extrapolating below the PoD and/or across test systems and to the human.

IWGT report on quantitative approaches to genotoxicity risk assessment II. Use of point-of-departure (PoD) metrics in defining acceptable exposure limits and assessing human risk.

This is the second of two reports from the International Workshops on Genotoxicity Testing (IWGT) Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (the QWG). The first report summarized the discussions and recommendations of the QWG related to the need for quantitative dose-response analysis of genetic toxicology data, the existence and appropriate evaluation of threshold responses, and methods to analyze exposure-response relationships and derive points of departure (PoDs) from which acceptable exposure levels could be determined. This report summarizes the QWG discussions and recommendations regarding appropriate approaches to evaluate exposure-related risks of genotoxic damage, including extrapolation below identified PoDs and across test systems and species. Recommendations include the selection of appropriate genetic endpoints and target tissues, uncertainty factors and extrapolation methods to be considered, the importance and use of information on mode of action, toxicokinetics, metabolism, and exposure biomarkers when using quantitative exposure-response data to determine acceptable exposure levels in human populations or to assess the risk associated with known or anticipated exposures. The empirical relationship between genetic damage (mutation and chromosomal aberration) and cancer in animal models was also examined. It was concluded that there is a general correlation between cancer induction and mutagenic and/or clastogenic damage for agents thought to act via a genotoxic mechanism, but that the correlation is limited due to an inadequate number of cases in which mutation and cancer can be compared at a sufficient number of doses in the same target tissues of the same species and strain exposed under directly comparable routes and experimental protocols.

Histone markers identify the mode of action for compounds positive in the TK6 micronucleus assay.

The in vitro micronucleus assay with TK6 cells is frequently used as part of the genotoxicity testing battery for pharmaceuticals. Consequently, follow-up testing strategies are needed for positive compounds to determine their mode of action, which would then allow for deployment of appropriate in vivo follow-up strategies. We have chosen 3 micronucleus positive compounds, the clastogen etoposide, the aneugen noscapine and the cytotoxicant tunicamycin to evaluate different approaches to determine their aneugenic or clastogenic properties. Each of the three compounds were evaluated following 4 and 24h of continuous treatment by flow cytometry for micronucleus induction, the aneugenicity markers phosphorylated-histone 3 (p-H3) and polyploidy, the clastogenicity marker γH2AX and the apoptosis marker cleaved caspase 3. They were further evaluated by Western blot for mono-ubiquitinated and γH2AX. Results show that the clastogen etoposide produced a dose related increase in γH2AX and mono-ubiquitinated H2AX and a dose related decrease in p-H3 positive mitotic cells. Conversely, the aneugen produced increases in p-H3 and polyploidy with no significant increases seen in mono-ubiquitinated H2AX or γH2AX. Lastly, the cytotoxicant tunicamycin induced neither an increase in p-H3 nor γH2AX. All three compounds produced dose-related increases in cleaved caspase 3. The results from this study provide evidence that adding clastogenicity and aneugenicity markers to the in vitro micronucleus assay in TK6 cells could help to identify the mode of action of positive compounds. The combination of endpoints suggested here needs to be further evaluated by a broader set of test compounds.

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.