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.

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.

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.

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.

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.

Formaldehyde exposure and leukemia: a new meta-analysis and potential mechanisms.

Formaldehyde is an economically important chemical, to which more than 2 million U.S. workers are occupationally exposed. Substantially more people are exposed to formaldehyde environmentally, as it is generated by automobile engines, is a component of tobacco smoke and is released from household products, including furniture, particleboard, plywood, and carpeting. The International Agency for Research on Cancer (IARC) recently classified formaldehyde as a human carcinogen that causes nasopharyngeal cancer and also concluded that there is "strong but not sufficient evidence for a causal association between leukemia and occupational exposure to formaldehyde". Here, we review the epidemiological studies published to date on formaldehyde-exposed workers and professionals in relation to lymphohematopoietic malignances. In a new meta-analysis of these studies, focusing on occupations known to have high formaldehyde exposure, we show that summary relative risks (RRs) were elevated in 15 studies of leukemia (RR=1.54; confidence interval (CI), 1.18-2.00) with the highest relative risks seen in the six studies of myeloid leukemia (RR=1.90; 95% CI, 1.31-2.76). The biological plausibility of this observed association is discussed and potential mechanisms proposed. We hypothesize that formaldehyde may act on bone marrow directly or, alternatively, may cause leukemia by damaging the hematopoietic stem or early progenitor cells that are located in the circulating blood or nasal passages, which then travel to the bone marrow and become leukemic stem cells. To test these hypotheses, we recommend that future studies apply biomarkers validated for other chemical leukemogens to the study of formaldehyde.

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.

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.

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.

Evaluating genotoxicity data to identify a mode of action and its application in estimating cancer risk at low doses: A case study involving carbon tetrachloride.

In the new USEPA cancer risk assessment guidelines, mode of action (MoA) information, combined with a determination of whether or not a chemical is mutagenic, plays an important role in determining whether a linear or nonlinear approach should be used to estimate cancer risks at low doses. In this article, carbon tetrachloride (CT) is used as an example to illustrate how mixed genotoxicity data can be evaluated and used to identify a likely MoA. CT is essentially negative in inducing gene mutations in Salmonella, but is consistently positive in inducing recombination and aneuploidy in fungi. Negative or equivocal results were seen in most in vitro and in vivo studies in mammals, including mutation studies in transgenic mice. However, DNA adducts, primarily those derived from oxidation- and lipid-peroxidation-derived products as well as DNA double-strand breaks and micronucleated cells, have been seen repeatedly in the liver of CT-treated animals. On the basis of the weight of evidence, CT should not be considered a directly mutagenic agent. Mutagenic as well as other genotoxic effects, as they occur, will most likely be generated through indirect mechanisms resulting from oxidative and lipid peroxidative damage and/or damage occurring during necrosis or apoptosis. As key events in this process are expected to occur in a nonlinear fashion, the expected relationship between CT dose and carcinogenic response in the liver is likely to be nonlinear with a steep dose response. This conclusion is consistent with rodent cancer bioassay results in which steep nonlinear dose responses have been seen.

Differential cell cycle-specificity for chromosomal damage induced by merbarone and etoposide in V79 cells.

Merbarone, a topoisomerase II (topo II) inhibitor which, in contrast to etoposide, does not stabilize topo II-DNA cleavable complexes, was previously shown to be a potent clastogen in vitro and in vivo. To investigate the possible mechanisms, we compared the cell cycle-specificity of the clastogenic effects of merbarone and etoposide in V79 cells. Using flow cytometry and BrdU labeling techniques, etoposide was shown to cause a rapid and persistent G2 delay while merbarone was shown to cause a prolonged S-phase followed by a G2 delay. To identify the stages which are susceptible to DNA damage, we performed the micronucleus (MN) assay with synchronized cells or utilized a combination of BrdU pulse labeling and the cytokinesis-blocked MN assay with non-synchronized cells. Treatment of M phase cells with either agent did not result in increased MN formation. Etoposide but not merbarone caused a significant increase in MN when cells were treated during G2 phase. When treated during S-phase, both chemicals induced highly significant increases in MN. However, the relative proportion of MN induced by merbarone was substantially higher than that induced by etoposide. Both chemicals also caused significant increases in MN in cells that were treated during G1 phase. To confirm the observations in the MN assay, first division metaphases were evaluated in the chromosome aberration assay. The chromosomes of cells treated with merbarone and etoposide showed increased frequencies of both chromatid- and chromosome-type of aberrations. Our findings indicate that while etoposide causes DNA damage more evenly throughout the G1, S and G2 phases of the cell cycle, an outcome which may be closely associated with topo II-mediated DNA strand cleavage, merbarone induces DNA breakage primarily during S-phase, an effect which is likely due to the stalling of replication forks by inhibition of topo II activity.

Non-disjunction events induced by albendazole in human cells.

Albendazole (ABZ), a benzimidazole carbamate used for the treatment of several human helminthiases has high affinity for tubulin, which results in an inhibition of microtubule polymerization, blocking several vital processes in the parasites, such as motility and nutrient uptake. The ability of ABZ to act as mitotic spindle poison leads to a potential risk for aneuploidy induction in exposed human beings. ABZ, as well as albendazole sulphoxide (ABZSO), its main metabolite, induce micronuclei in human cells in a dose-dependent manner. Despite recognition that ABZ and ABZSO increase micronucleus frequency, their potential as inducers of non-disjunction in human cells, an event considered more frequent than chromosome loss, and one of the main mechanisms involved in aneuploidy induction, has not been evaluated. In the present work, we investigated the ability of ABZ and ABZSO to induce non-disjunction in cultured human lymphocytes. Non-disjunction was scored by chromosome-specific FISH using a classical or alpha satellite probe for chromosomes 1 and 7, respectively. Significant increase in non-disjunction events that involved either chromosome were observed in cells treated with ABZ or ABZSO. Both ABZ and ABZSO induced non-disjunction at lower concentrations than those at which MN were observed.

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.

Childhood acute lymphocytic leukemia and perspectives on risk assessment of early-life stage exposures.

Recognition that children are a potentially susceptible subpopulation has led to the development of child-specific sensitivity factors. Establishing reliable sensitivity factors in support of risk assessment of early-life stage exposures can be aided by evaluating studies that enhance our understanding both of the biological basis of disease processes and the potential role of environmental exposures in disease etiology. For these reasons, we evaluated childhood acute lymphocytic leukemia (ALL) studies from the point of view of mechanism and etiology. ALL is the most common form of childhood cancer proposed to result from a prenatal primary event and a postnatal second event. This multi-stage model is supported by the observation that chromosomal translocations/fusion genes (e.g., TEL-AML1) involved in producing ALL are detected at birth (prenatal event), and a postnatal event (e.g., TEL deletion) is required for disease manifestation. It appears that a proportion of ALL cases are the result of environmental exposures, in which case preconceptional, prenatal, and postnatal stages are likely to be critical exposure windows. To this end, we recognized postnatal infection-related risk factors as potential candidates associated with the ALL second event. Additionally, we discuss use of ALL-associated fusion genes and genetic polymorphisms, together or separately, as indicators of ALL susceptibility and increased risk. The possibility of using fusion genes alone as biomarkers of response is also discussed because they can serve as predictors of key events in the development of a mode of action (a sequence of key events, starting with interaction of an agent with a cell, ultimately resulting in cancer formation) for particular environmental exposures. Furthermore, we discuss use of an initiated animal model for ALL, namely transgenic mice with TEL-AML1 expression, for exploring mechanisms by which different classes of environmental exposures could be involved in inducing the postnatal step in ALL formation.

Induction of cell proliferation, micronuclei and hyperdiploidy/polyploidy in the mammary cells of DDT- and DMBA-treated pubertal rats.

The environmental estrogen, dichlorodiphenyltrichloroethane (DDT), and its metabolites have been implicated in the development of breast cancer through mechanisms that remain to be elucidated. It has been hypothesized that exposure to DDT and its metabolites, during critical periods of development, can contribute to an elevated risk for breast cancer in adults. In the present study, we have investigated the effect of o,p'-DDT on mammary gland cell proliferation and chromosomal alterations, in a rat mammary cancer model (commonly used to study human cancer), to gain insights into its potential role in the development of breast cancer. Twenty-one-day-old female Sprague-Dawley (SD) rats were administered o,p'-DDT, 7,12-dimethylbenz[a]anthracene (DMBA), genistein, DDT+DMBA, or DDT+DMBA+genistein, over a 14-day period. To determine changes in chromosome number and structure, we used the micronucleus assay as well as multicolor fluorescence in situ hybridization (FISH) region-specific DNA probes for rat chromosomes 4 and 19. Cell proliferation was evaluated using 5-bromo-2'-deoxyuridine (BrdU). Significant increases in BrdU-incorporated cells were seen in the rats treated with DDT+DMBA. Although micronucleus frequencies were somewhat elevated in several of the treatment groups, significant increases were not seen in any of them. Significant increases in numerical chromosomal aberrations were detected in all of the DDT- and DMBA-treated groups. Genistein significantly reduced BrdU incorporation and polyploidy in the DDT+DMBA-treated rats. These initial studies indicate that DDT and DMBA can induce cellular and chromosomal alterations in the rat mammary gland, which is consistent with the hypothesis that these agents can induce early events in mammary carcinogenesis.

Topoisomerase II inhibition by myeloperoxidase-activated hydroquinone: a potential mechanism underlying the genotoxic and carcinogenic effects of benzene.

Benzene is an established human and animal carcinogen. While many of the key mechanisms underlying its carcinogenic effects remain unknown, there is increasing evidence that chromosomal alterations play an important role in the development of the induced leukemias. Inhibition of enzymes involved in DNA replication and maintenance such as topoisomerases by benzene metabolites represents a potential mechanism by which benzene may induce its chromosome-altering effects. Previous work from our laboratory and others has demonstrated that bioactivated benzene metabolites are capable of inhibiting topoisomerase II (topo II) in isolated enzyme and cell systems as well as in mice administered benzene in vivo. The current studies were designed to build upon this hypothesis, and show that in the presence of human myeloperoxidase and H2O2, hydroquinone can be activated to a potent topo II inhibitor. In the absence of dithiothreitol, partial inhibition can be seen at hydroquinone concentrations as low as 50 nM. The potential role of topo II inhibition in the development of benzene-induced leukemia is also discussed in the context of other known leukemia-inducing agents. Current evidence indicates that multiple mechanisms are likely to contribute to benzene-induced leukemias, and that inhibition of topo II could represent an important step in the development of certain leukemia subtypes.

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.

Catalytic inhibitors of topoisomerase II are DNA-damaging agents: induction of chromosomal damage by merbarone and ICRF-187.

Merbarone is a catalytic inhibitor of topoisomerase II (topo II) that has been proposed to act primarily by blocking topo II-mediated DNA cleavage without stabilizing DNA-topo II-cleavable complexes. In this study merbarone was used as a model compound to investigate the genotoxic effects of catalytic inhibitors of topo II. The clastogenic properties of merbarone were evaluated using in vitro and in vivo micronucleus (MN) assays combined with CREST staining. For the in vitro MN assay, ICRF-187, a different type of catalytic inhibitor, and etoposide, a topo II poison, were used for comparison. Treatment of TK6 cells with all three of these drugs resulted in highly significant dose-related increases in kinetochore-lacking MN and, to a lesser extent, kinetochore-containing MN. In addition, a good correlation between p53 accumulation and MN formation was seen in the drug-treated cells. A mouse MN assay was performed to confirm that similar DNA-damaging effects would occur in vivo. Bone marrow smears from merbarone-treated B6C3F1 mice showed a dose-related increase in micronucleated polychromatic erythrocytes with a mean of 26 MN per 1000 cells being seen at the 60 mg/kg dose. Almost all MN lacked a kinetochore signal, indicating that merbarone was predominantly clastogenic under these conditions in vivo. The present study clearly shows that merbarone is genotoxic both in vitro and in vivo, and demonstrates the inaccuracy of earlier statements that merbarone and other catalytic inhibitors block the enzymatic activity of topo II without damaging DNA.