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.

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.

Recommendations for the evaluation of complex genetic toxicity data sets when assessing carcinogenic risks to humans.

Genotoxicity testing plays an important role in the assessment of carcinogenic and heritable risks. In many cases, experts charged with assessing genotoxicity test results need to evaluate widely varying numbers and types of bioassays of differing quality, conducted in a variety of cells and species using a wide range of protocols. The recommendations in this article were initially prepared as general guidelines to assist experts involved in the 2016 Joint Food and Agricultural Organization and World Health Organization Meeting on Pesticide Residues (JMPR) in their evaluation of the human health risks associated with exposure to pesticide residues in the diet. A weight of evidence approach is recommended in which studies are evaluated based on quality, reproducibility and consistency, significance of the genetic alteration, phylogenetic relevance to humans, type (in vivo vs. in vitro), and relevance of the route of administration. Using the recommended approach, the most weight will generally be given to high quality in vivo studies of gene and chromosome mutations (including aberrations) in humans or mammals exposed to the chemical through a physiologically relevant route such as oral or dermal administration or by inhalation. The guidelines are intended to give reviewers flexibility in evaluating all relevant scientific information, and allow them to use their best scientific judgment in reaching conclusions about the significance of the genotoxicity results. The use of these guidelines and the associated weighting considerations should facilitate the evaluation of complex and sometimes contradictory data sets, and provide more consistency in evaluations across risk assessments. Environ. Mol. Mutagen. 58:380-385, 2017. © 2017 Wiley Periodicals, Inc.

The Importance of Hand Exposures to Absorbed Dosage of Hand Harvesters.

Fruit harvesters' primary pesticide exposure results from direct dermal and clothing contact with foliar residues. The transfer of pesticide residues from foliage to strawberry harvesters' hands and their subsequent dissipation under normal occupational conditions in the field was examined. The effectiveness of latex gloves as sampling dosimeters was evaluated and compared with bare-handed harvester exposures. After application of malathion and fenpropathrin insecticides on strawberry fields, resulting harvester exposures using four independent methods were studied. Between d 4 (preharvest interval, PHI) and d 7 after pesticide application, (1) dislodgeable foliar residues, (2) pesticide residues accumulating on the gloves, and (3) end-of-shift harvester hand-wash residues decreased by 90, 75, and 85%, respectively. In contrast, the 7-d decline in excreted urine metabolites was only 43% for gloved harvesters and 29% for barehanded ones. In addition, gloved harvesters displayed 23% lower biomonitored exposures than barehanded ones, demonstrating that latex gloves are an effective protective barrier against surface residues. Since malathion and its metabolites are readily excreted, data indicated that there were likely other sources of excreted malathion breakdown products present on foliar surfaces after dissipation of malathion itself.

Latex Rubber Gloves as a Sampling Dosimeter Using a Novel Surrogate Sampling Device.

Pesticide exposure during harvesting of crops occurs primarily to the workers' hands. When harvesters wear latex rubber gloves for personal safety and hygiene harvesting reasons, gloves accumulate pesticide residues. Hence, characterization of the gloves' properties may be useful for pesticide exposure assessments. Controlled field studies were conducted using latex rubber gloves to define the factors that influence the transfer of pesticides to the glove and that would affect their use as a residue monitoring device. A novel sampling device called the Brinkman Contact Transfer Unit (BCTU) was constructed to study the glove characteristics and residue transfer and accumulation under controlled conditions on turf. The effectiveness of latex rubber gloves as sampling dosimeters was evaluated by measuring the transferable pesticide residues as a function of time. The validation of latex rubber gloves as a residue sampling dosimeter was performed by comparing pesticide transfer and dissipation from the gloves, with the turf transferable residues sampled using the validated California (CA) Roller, a standard measure of residue transfer. The observed correlation (Pearson's correlation coefficient R(2)) between the two methods was .84 for malathion and .96 for fenpropathrin, indicating that the BCTU is a useful, reliable surrogate tool for studying available residue transfer to latex rubber gloves under experimental conditions. Perhaps more importantly, these data demonstrate that latex gloves worn by workers may be useful quantifiable matrices for measuring pesticide exposure.

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.

Lymphohematopoietic cancers induced by chemicals and other agents and their implications for risk evaluation: An overview.

Lymphohematopoietic neoplasia are one of the most common types of cancer induced by therapeutic and environmental agents. Of the more than 100 human carcinogens identified by the International Agency for Research on Cancer, approximately 25% induce leukemias or lymphomas. The objective of this review is to provide an introduction into the origins and mechanisms underlying lymphohematopoietic cancers induced by xenobiotics in humans with an emphasis on acute myeloid leukemia, and discuss the implications of this information for risk assessment. Among the agents causing lymphohematopoietic cancers, a number of patterns were observed. Most physical and chemical leukemia-inducing agents such as the therapeutic alkylating agents, topoisomerase II inhibitors, and ionizing radiation induce mainly acute myeloid leukemia through DNA-damaging mechanisms that result in either gene or chromosomal mutations. In contrast, biological agents and a few immunosuppressive chemicals induce primarily lymphoid neoplasms through mechanisms that involve alterations in immune response. Among the environmental agents examined, benzene was clearly associated with acute myeloid leukemia in humans, with increasing but still limited evidence for an association with lymphoid neoplasms. Ethylene oxide and 1,3-butadiene were linked primarily to lymphoid cancers. Although the association between formaldehyde and leukemia remains controversial, several recent evaluations have indicated a potential link between formaldehyde and acute myeloid leukemia. The four environmental agents examined in detail were all genotoxic, inducing gene mutations, chromosomal alterations, and/or micronuclei in vivo. Although it is clear that rapid progress has been made in recent years in our understanding of leukemogenesis, many questions remain for future research regarding chemically induced leukemias and lymphomas, including the mechanisms by which the environmental agents reviewed here induce these diseases and the risks associated with exposures to such agents.

Concordance of transcriptional and apical benchmark dose levels for conazole-induced liver effects in mice.

The ability to anchor chemical class-based gene expression changes to phenotypic lesions and to describe these changes as a function of dose and time informs mode-of-action determinations and improves quantitative risk assessments. Previous global expression profiling identified a 330-probe cluster differentially expressed and commonly responsive to 3 hepatotumorigenic conazoles (cyproconazole, epoxiconazole, and propiconazole) at 30 days. Extended to 2 more conazoles (triadimefon and myclobutanil), the present assessment encompasses 4 tumorigenic and 1 nontumorigenic conazole. Transcriptional benchmark dose levels (BMDL(T)) were estimated for a subset of the cluster with dose-responsive behavior and a ≥ 5-fold increase or decrease in signal intensity at the highest dose. These genes primarily encompassed CAR/RXR activation, P450 metabolism, liver hypertrophy- glutathione depletion, LPS/IL-1-mediated inhibition of RXR, and NRF2-mediated oxidative stress pathways. Median BMDL(T) estimates from the subset were concordant (within a factor of 2.4) with apical benchmark doses (BMDL(A)) for increased liver weight at 30 days for the 5 conazoles. The 30-day median BMDL(T) estimates were within one-half order of magnitude of the chronic BMDLA for hepatocellular tumors. Potency differences seen in the dose-responsive transcription of certain phase II metabolism, bile acid detoxification, and lipid oxidation genes mirrored each conazole's tumorigenic potency. The 30-day BMDL(T) corresponded to tumorigenic potency on a milligram per kilogram day basis with cyproconazole > epoxiconazole > propiconazole > triadimefon > myclobutanil (nontumorigenic). These results support the utility of measuring short-term gene expression changes to inform quantitative risk assessments from long-term exposures.

The use of genetically modified mice in cancer risk assessment: challenges and limitations.

The use of genetically modified (GM) mice to assess carcinogenicity is playing an increasingly important role in the safety evaluation of chemicals. While progress has been made in developing and evaluating mouse models such as the Trp53⁺/⁻, Tg.AC and the rasH2, the suitability of these models as replacements for the conventional rodent cancer bioassay and for assessing human health risks remains uncertain. The objective of this research was to evaluate the use of accelerated cancer bioassays with GM mice for assessing the potential health risks associated with exposure to carcinogenic agents. We compared the published results from the GM bioassays to those obtained in the National Toxicology Program's conventional chronic mouse bioassay for their potential use in risk assessment. Our analysis indicates that the GM models are less efficient in detecting carcinogenic agents but more consistent in identifying non-carcinogenic agents. We identified several issues of concern related to the design of the accelerated bioassays (e.g., sample size, study duration, genetic stability and reproducibility) as well as pathway-dependency of effects, and different carcinogenic mechanisms operable in GM and non-GM mice. The use of the GM models for dose-response assessment is particularly problematic as these models are, at times, much more or less sensitive than the conventional rodent cancer bioassays. Thus, the existing GM mouse models may be useful for hazard identification, but will be of limited use for dose-response assessment. Hence, caution should be exercised when using GM mouse models to assess the carcinogenic risks of chemicals.

A comparative study of the cytotoxic and genotoxic effects of ICRF-154 and bimolane, two catalytic inhibitors of topoisomerase II.

ICRF-154 and bimolane have been used for the treatment of cancer, psoriasis, and uveitis in humans. Previous reports have revealed that the two drugs are topoisomerase II catalytic inhibitors, and patients treated with these agents have developed unique types of secondary leukemia. A study published in 1984 by Camerman and colleagues proposed that the therapeutic effects of bimolane could be due to ICRF-154, an impurity present within the bimolane samples that may also be responsible for the toxic effects attributed to bimolane. To date, this hypothesis has not been evaluated. In addition, little is known about the potential cytotoxic and genotoxic effects of ICRF-154. In this study, a combination of in vitro tests in human TK6 lymphoblastoid cells has been used to characterize the cytotoxic and genotoxic effects of ICRF-154 and bimolane as well as to compare the results for the two chemicals. ICRF-154 and bimolane were both cytotoxic, exhibiting very similar effects in three measures of cytotoxicity and cell proliferation. In the cytokinesis-block micronucleus assay with CREST-antibody staining, the two agents similarly induced chromosome breakage and, to a lesser extent, chromosome loss. Intriguingly, both drugs resulted in the formation of binucleated cells, perhaps as a consequence of an interference with cytokinesis. To further investigate their aneugenic effects, flow cytometry and fluorescence in situ hybridization analyses revealed that both compounds also produced similar levels of non-disjunction and polyploidy. In each of the cellular and cytogenetic assays employed, the responses of the ICRF-154-treated cells were very similar to those observed with the bimolane, and generally occurred at equimolar test concentrations. Our results, combined with those from previous studies, strongly suggest that bimolane degrades to ICRF-154, and that ICRF-154 is most likely the chemical species responsible for the cytotoxic, genotoxic, and leukemogenic effects exerted by bimolane.

Dose-response analysis of bromate-induced DNA damage and mutagenicity is consistent with low-dose linear, nonthreshold processes.

Mutagenic agents have long been inferred to act through low-dose linear, nonthreshold processes. However, there is debate about this assumption, with various studies interpreting datasets as showing thresholds for DNA damage and mutation. We have applied rigorous statistical analyses to investigate the shape of dose-response relationships for a series of in vitro and in vivo genotoxicity studies using potassium bromate (KBrO(3) ), a water ozonation byproduct that is bioactivated to a reactive species causing oxidative damage to DNA. We analyzed studies of KBrO(3) genotoxicity where no-effect/threshold levels were reported as well as other representative datasets. In all cases, the data were consistent with low-dose linear models. In the majority of cases, the data were fit either by a linear (straight line) model or a model which was linear at low doses and showed a saturation-like downward curvature at high doses. Other datasets with apparent upward curvature were still adequately represented by models that were linear at low dose. Sensitivity analysis of datasets showing upward curvature revealed that both low-dose linear and nonlinear models provide adequate fits. Additionally, a simple biochemical model of selected key processes in bromate-induced DNA damage was developed and illustrated a situation where response for early primary events suggested an apparent threshold while downstream events were linear. Overall, the statistical analyses of DNA damage and mutations induced by KBrO(3) are consistent with a low-dose linear response and do not provide convincing evidence for the presence of a threshold.

Factors influencing mutagenic mode of action determinations of regulatory and advisory agencies.

The determination of whether a chemical induces cancer through a mutagenic or genotoxic mechanism frequently plays an important role in evaluating the risks associated with low dose exposure. Although various approaches are employed for making mode of action decisions, a systematic investigation to identify the major factors that influence these determinations has not been performed. To accomplish this, over 40 chemical risk assessments conducted by U.S. or international regulatory agencies and organizations were reviewed to identify components that had played a significant role, either directly or indirectly, in the decision-making process. The major factors identified included the chemical properties of the agent, its metabolites and degradation products; its metabolism and toxicokinetics; genotoxic effects seen in vivo, particularly in the target organ; structural or metabolic similarities to known mutagenic or nonmutagenic chemicals; characteristics of the tumors induced in the animal bioassays; and the origin of the observed effects. The quality of the data, the specific genotoxic endpoint and its sensitivity to assay conditions and toxicity were also important considerations. In all cases, the authoritative groups used a weight-of-evidence approach and, in most cases where evaluations were conducted by more than one authoritative body, similar conclusions were reached. In summary, a critical evaluation of the data as well as expert judgment is necessary in reaching mechanism of action conclusions. These determinations should be made within the broader context of evaluating the chemical's overall toxicity and carcinogenicity.

Bimolane induces multiple types of chromosomal aberrations in human lymphocytes in vitro.

Bimolane has been commonly used in China for the treatment of psoriasis and various types of cancer. Patients treated with bimolane have been reported to have an increased risk of developing therapy-related leukemias. Although bimolane has been identified as a human leukemia-inducing agent, little is known about its genotoxic effects, and a systematic study of the types of chromosomal alterations induced by this compound has not been performed. In this study, a combination of immunochemical, molecular and conventional cytogenetic techniques has been used to study the chromosomal alterations induced by bimolane in cultured human lymphocytes. Immunochemical staining with the CREST antibody indicated that bimolane induces micronuclei (MN) originating primarily from chromosome breakage. Interestingly fluorescence in situ hybridization (FISH) with differentially labeled chromosomes 1 and 9 centromeric probes indicated that bimolane also caused non-disjunction and polyploidy. Consistent with this, an expedited analysis of Giemsa-stained metaphase chromosomes in bimolane-treated lymphocytes revealed a high frequency of polyploidy/hyperdiploidy as well as dicentric chromosomes, and premature centromeric division (PCD). In addition, bimolane was also found to produce binucleated cells, possibly through an interference with normal functioning of intermediate filaments. As a follow-up to these studies, three different types of commercially available bimolane formulations obtained from different Chinese manufacturers were also evaluated. The effects seen with the formulated bimolane were similar to those seen with the synthesized compound. Our studies indicate that bimolane effectively induces a variety of cellular and chromosomal changes in cultured lymphocytes and that similar alterations occurring in bone marrow stem cells could contribute to the development of the secondary cancers seen in bimolane-treated patients.

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.

Topoisomerase II inhibition by the bioactivated benzene metabolite hydroquinone involves multiple mechanisms.

While benzene is widely recognized as a human and animal carcinogen, the key mechanisms underlying its carcinogenic effects remain unknown. Inhibition of topoisomerase II (topoII) by benzene and its metabolites represents a potential mechanism by which benzene could induce its chromosome-altering and leukemogenic effects. Previous work from our laboratory and others has demonstrated that bioactive benzene metabolites are capable of inhibiting topoII in isolated enzyme and cell culture systems. Similarly, a decrease in topoII activity has been seen in the bone marrow of mice administered benzene in vivo. The objective of these studies was to further investigate the mechanisms by which the bioactivated benzene metabolite, hydroquinone (BAHQ), inhibits topoII in vitro, and to identify the point(s) in the enzyme's catalytic cycle where inhibition occurs. Our experiments indicate that BAHQ inhibits topoII at the DNA binding stage as well as in the closed clamp stage in the catalytic cycle, thereby interfering with either the binding to, or the release of, DNA from the enzyme. While increases in the cleavable complex were also seen with BAHQ treatment, our results suggest that this is related to a shift in equilibrium due to an accumulation of the topoII enzyme at the closed clamp stage rather than a major inhibitory effect on the religation step. An increase in cleavable complex formation as well as the inhibition of enzymatic activity at the closed clamp and other stages of the catalytic cycle in bone marrow cells would likely result in DNA breakage, the formation of chromosomal aberrations, and could potentially result in leukemia-associated chromosomal translocations, similar to those seen in leukemias induced by the bisdioxopiperazine type of catalytic topoII inhibitors.

Reduction of use of animals in regulatory genotoxicity testing: Identification and implementation opportunities-Report from an ECVAM workshop.

In vivo genetic toxicology tests measure direct DNA damage or the formation of gene or chromosomal mutations, and are used to predict the mutagenic and carcinogenic potential of compounds for regulatory purposes and/or to follow-up positive results from in vitro testing. These tests are widely used and consume large numbers of animals, with a foreseeable marked increase as a result of the EU chemicals legislation (REACH), which may require follow-up of any positive outcome in the in vitro standard battery with appropriate in vivo tests, regardless of the tonnage level of the chemical. A 2-day workshop with genotoxicity experts from academia, regulatory agencies and industry was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) in Ranco, Italy from 24 to 25 June 2008. The objectives of the workshop were to discuss how to reduce the number of animals in standard genotoxicity tests, whether the application of smarter test strategies can lead to lower animal numbers, and how the possibilities for reduction can be promoted and implemented. The workshop agreed that there are many reduction options available that are scientifically credible and therefore ready for use. Most of these are compliant with regulatory guidelines, i.e. the use of one sex only, one administration and two sampling times versus two or three administrations and one sampling time for micronucleus (MN), chromosomal aberration (CA) and Comet assays; and the integration of the MN endpoint into repeat-dose toxicity studies. The omission of a concurrent positive control in routine CA and MN tests has been proven to be scientifically acceptable, although the OECD guidelines still require this; also the combination of acute MN and Comet assay studies are compliant with guidelines, except for sampling times. Based on the data presented at the workshop, the participants concluded that these options have not been sufficiently utilized to date. Key factors for this seem to be the uncertainty regarding regulatory compliance/acceptance, lack of awareness, and an in many cases unjustified uncertainty regarding the scientific acceptance of reduction options. The workshop therefore encourages the use and promotion of these options as well as the dissemination of data related to reduction opportunities by the scientific community in order to boost the acceptance level of these approaches. Furthermore, experimental proof is needed and under way to demonstrate the credibility of additional options for reduction of the number of animals, such as the integration of the Comet assay into repeat-dose toxicity studies.

Mutagenicity testing for chemical risk assessment: update of the WHO/IPCS Harmonized Scheme.

Since the publication of the International Programme on Chemical Safety (IPCS) Harmonized Scheme for Mutagenicity Testing, there have been a number of publications addressing test strategies for mutagenicity. Safety assessments of substances with regard to genotoxicity are generally based on a combination of tests to assess effects on three major end points of genetic damage associated with human disease: gene mutation, clastogenicity and aneuploidy. It is now clear from the results of international collaborative studies and the large databases that are currently available for the assays evaluated that no single assay can detect all genotoxic substances. The World Health Organization therefore decided to update the IPCS Harmonized Scheme for Mutagenicity Testing as part of the IPCS project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals. The approach presented in this paper focuses on the identification of mutagens and genotoxic carcinogens. Selection of appropriate in vitro and in vivo tests as well as a strategy for germ cell testing are described.

Improving prediction of chemical carcinogenicity by considering multiple mechanisms and applying toxicogenomic approaches.

While scientific knowledge of the potential health significance of chemical exposures has grown, experimental methods for predicting the carcinogenicity of environmental agents have not been substantially updated in the last two decades. Current methodologies focus first on identifying genotoxicants under the premise that agents capable of directly damaging DNA are most likely to be carcinogenic to humans. Emphasis on the distinction between genotoxic and non-genotoxic carcinogens is also motivated by assumed implications for the dose-response curve; it is purported that genotoxicants would lack a threshold in the low dose region, in contrast to non-genotoxic agents. However, for the vast majority of carcinogens, little if any empirical data exist to clarify the nature of the cancer dose-response relationship at low doses in the exposed human population. Recent advances in scientific understanding of cancer biology-and increased appreciation of the multiple impacts of carcinogens on this disease process-support the view that environmental chemicals can act through multiple toxicity pathways, modes and/or mechanisms of action to induce cancer and other adverse health outcomes. Moreover, the relationship between dose and a particular outcome in an individual could take multiple forms depending on genetic background, target tissue, internal dose and other factors besides mechanisms or modes of action; inter-individual variability and susceptibility in response are, in turn, key determinants of the population dose-response curve. New bioanalytical approaches (e.g., transcriptomics, proteomics, and metabolomics) applied in human, animal and in vitro studies could better characterize a wider array of hazard traits and improve the ability to predict the potential carcinogenicity of chemicals.