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Genotoxicity of styrene monomer was evaluated in male Fischer 344 rats using the alkaline comet assay for DNA damage, micronucleus assay for cytogenetic damage and the Pig‐a assay for gene mutations. In a dose range finding (DRF) study, styrene was administered by oral gavage in corn oil for 28 consecutive days at 0, 100, 500, and 1000 mg/kg/day. The bioavailability of styrene was confirmed in the DRF by measuring its plasma levels at approximately 7‐ or 15‐min following dosing. The 1000 mg/kg/day group exceeded the maximum tolerated dose based on body weight and organ weight changes and signs of central nervous system depression. Based on these findings, doses of 0, 100, 250, and 500 mg/kg/day (for 28 or 29 days) were selected for the genotoxicity assays. Animals were sacrificed 3–4 h after treatment on Day 28 or 29 for assessing various genotoxicity endpoints. Pig‐a mutant frequencies and micronucleus frequencies were determined in peripheral blood erythrocytes. The comet assay was conducted in the glandular stomach, duodenum, liver, lung, and kidney. These studies were conducted in accordance with the relevant OECD test guidelines. Oral administration of styrene did not lead to genotoxicity in any of the investigated endpoints. The adequacy of the experimental conditions was assured by including animals treated by oral gavage with the positive control chemicals ethyl nitrosourea and ethyl methane sulfonate. Results from these studies supplement to the growing body of evidence suggesting the lack of in vivo genotoxic potential for styrene. [ABSTRACT FROM AUTHOR]

Male B6C3F1 mice were administered styrene monomer by oral gavage for 29 consecutive days at dose levels of 0, 75, 150, or 300 mg/kg/day. The highest dose level represented the maximum tolerated dose based on findings in a 28‐day dose range‐finding study, in which the bioavailability of orally administered styrene was also confirmed. The positive control group received ethyl nitrosourea (ENU; 51.7 mg/kg/day) on Study Days 1–3 and ethyl methanesulfonate (EMS; 150 mg/kg/day) on Study Days 27–29 by oral gavage. Approximately 3 h following the final dose, blood was collected to assess erythrocyte Pig‐a mutant and micronucleus frequencies. DNA strand breakage was assessed in glandular stomach, duodenum, kidney, liver, and lung tissues using the alkaline comet assay. The %tail DNA for stomach, liver, lung, and kidney in the comet assay among the styrene‐treated groups was neither significantly different from the respective vehicle controls nor was there any dose‐related increasing trend in any of the tissues; results for duodenum were interpreted to be inconclusive because of technical issues. The Pig‐a and micronucleus frequencies among styrene‐treated groups also did not show significant increases relative to the vehicle controls and there was also no evidence for a dose‐related increasing trend. Thus, orally administered styrene did not induce DNA damage, mutagenesis, or clastogenesis/aneugenesis in these Organization of Economic Co‐operation and Development test guideline‐compliant genotoxicity studies. Data from these studies can contribute to the overall assessment of genotoxic hazard and risk posed to humans potentially exposed to styrene. [ABSTRACT FROM AUTHOR]

3‐Chloroallyl alcohol (3‐CAA) can be found in the environment following the application of plant protection products. 3‐CAA is formed in groundwater following the injection of 1,3‐dichloropropene, a fumigant used to control nematodes. 3‐CAA is also formed, in leafy crops, as a glycoside conjugate following application of the herbicide, clethodim. Human exposure may occur from groundwater used as drinking water or through dietary consumption. To characterize 3‐CAA's potential to cause genotoxicity in mammals, in vitro and in vivo studies were conducted. 3‐CAA was negative in an Ames test and positive in a mouse lymphoma forward mutation assay. 3‐CAA was negative in an acute in vivo CD‐1 mouse bone marrow micronucleus assay when administered up to a dose level of 125 mg/kg/day for two consecutive days. In a combined gene mutation assay and erythrocyte micronucleus assay, using transgenic Big Blue® Fischer 344 rats, 3‐CAA was administered via drinking water at targeted dose levels of 0, 10, 30, and 100 mg/kg/day for 29 days. Peripheral blood samples, collected at the end of treatment, were analyzed for micronucleus induction in reticulocytes using flow cytometry. Liver and bone marrow samples, collected 2 days after the termination of the treatment, were analyzed for the induction of mutations at the cII locus. 3‐CAA did not induce an increase in mutant frequency or micronuclei under the experimental conditions. In conclusion, the mutagenic response observed in the in vitro mouse lymphoma assay is not confirmed in the whole animal. 3‐CAA is not considered to pose a mutagenic risk. [ABSTRACT FROM AUTHOR]

Disruption of microtubule stability in mammalian cells may lead to genotoxicity and carcinogenesis. The ability to screen for microtubule destabilization or stabilization is therefore a useful and efficient approach to aid in the design of molecules that are safe for human health. In this study, we developed a high-throughput 384-well assay combining immunocytochemistry with high-content imaging to assess microtubule disruption in the metabolically competent human liver cell line: HepaRG. To enhance analysis throughput, we implemented a supervised machine learning approach using a curated training library of 180 compounds. A majority voting ensemble of eight machine learning classifiers was employed for predicting microtubule disruptions. Our prediction model achieved over 99.0% accuracy and a 98.4% F1 score, which reflects the balance between precision and recall for in-sample validation and 93.5% accuracy and a 94.3% F1 score for out-of-sample validation. This automated image-based testing can provide a simple, high-throughput screening method for early stage discovery compounds to reduce the potential risk of genotoxicity for crop protection product development.

Aneuploidy is characterized by the presence of an abnormal number of chromosomes and is a common hallmark of cancer. However, exposure to aneugenic compounds does not necessarily lead to cancer. Aneugenic compounds are mainly identified using the in vitro micronucleus assay but this assay cannot standardly discriminate between aneugens and clastogens and cannot be used to identify the exact mode-of-action (MOA) of aneugens; tubulin stabilization, tubulin destabilization, or inhibition of mitotic kinases. To improve the classification of aneugenic substances and determine their MOA, we developed and validated the TubulinTracker assay that uses a green fluorescent protein-tagged tubulin reporter cell line to study microtubule stability using flow cytometry. Combining the assay with a DNA stain also enables cell cycle analysis. Substances whose exposure resulted in an accumulation of cells in G2/M phase, combined with increased or decreased tubulin levels, were classified as tubulin poisons. All known tubulin poisons included were classified correctly. Moreover, we correctly classified compounds, including aneugens that did not affect microtubule levels. However, the MOA of aneugens not affecting tubulin stability, such as Aurora kinase inhibitors, could not be identified. Here, we show that the TubulinTracker assay can be used to classify microtubule stabilizing and destabilizing compounds in living cells. This insight into the MOA of aneugenic agents is important, eg, to support a weight-of-evidence approach for risk assessment, and the classification as an aneugen as opposed to a clastogen or mutagen, has a big impact on the assessment. [ABSTRACT FROM AUTHOR]

The EFSA Scientific Committee was asked to provide guidance on the most appropriate in vivo tests to follow up on positive in vitro results for aneugenicity, and on the approach to risk assessment for substances that are aneugenic but not clastogenic nor causing gene mutations. The Scientific Committee confirmed that the preferred approach is to perform an in vivo mammalian erythrocyte micronucleus test with a relevant route of administration. If this is positive, it demonstrates that the substance is aneugenic in vivo. A negative result with evidence that the bone marrow is exposed to the test substance supports a conclusion that aneugenic activity is not expressed in vivo. If there is no evidence of exposure to the bone marrow, a negative result is viewed as inconclusive and further studies are required. The liver micronucleus assay, even though not yet fully validated, can provide supporting information for substances that are aneugenic following metabolic activation. The gastrointestinal micronucleus test, conversely, to be further developed, may help to assess aneugenic potential at the initial site of contact for substances that are aneugenic in vitro without metabolic activation. Based on the evidence in relation to mechanisms of aneugenicity, the Scientific Committee concluded that, in principle, health-based guidance values can be established for substances that are aneugenic but not clastogenic nor causing gene mutations, provided that a comprehensive toxicological database is available. For situations in which the toxicological database is not sufficient to establish health-based guidance values, some approaches to risk assessment are proposed. The Scientific Committee recommends further development of the gastrointestinal micronucleus test, and research to improve the understanding of aneugenicity to support risk assessment. [ABSTRACT FROM AUTHOR]

Abstract The EFSA Scientific Committee was asked to provide guidance on the most appropriate in vivo tests to follow up on positive in vitro results for aneugenicity, and on the approach to risk assessment for substances that are aneugenic but not clastogenic nor causing gene mutations. The Scientific Committee confirmed that the preferred approach is to perform an in vivo mammalian erythrocyte micronucleus test with a relevant route of administration. If this is positive, it demonstrates that the substance is aneugenic in vivo. A negative result with evidence that the bone marrow is exposed to the test substance supports a conclusion that aneugenic activity is not expressed in vivo. If there is no evidence of exposure to the bone marrow, a negative result is viewed as inconclusive and further studies are required. The liver micronucleus assay, even though not yet fully validated, can provide supporting information for substances that are aneugenic following metabolic activation. The gastrointestinal micronucleus test, conversely, to be further developed, may help to assess aneugenic potential at the initial site of contact for substances that are aneugenic in vitro without metabolic activation. Based on the evidence in relation to mechanisms of aneugenicity, the Scientific Committee concluded that, in principle, health‐based guidance values can be established for substances that are aneugenic but not clastogenic nor causing gene mutations, provided that a comprehensive toxicological database is available. For situations in which the toxicological database is not sufficient to establish health‐based guidance values, some approaches to risk assessment are proposed. The Scientific Committee recommends further development of the gastrointestinal micronucleus test, and research to improve the understanding of aneugenicity to support risk assessment.

Aflatoxin B1 (AFB1) is the most toxic mycotoxin and a proven human carcinogen that requires metabolic activation, known by cytochrome P450 (CYP) 1A2 and 3A4. Previous evidence showed that AFB1 is activated by human recombinant CYP1A1 expressed in budding yeast. Yet, the toxicity, in particular the genotoxicity of the reactive metabolites formed from AFB1 remains unclear. Humans could be exposed to both AFB1 and benzo(a)pyrene (BaP) simultaneously, thus we were interested in their combined genotoxic effects subsequent to metabolic activation by CYP1A1. In this study, molecular docking of AFB1 to human CYP1A1 indicated that AFB1 is valid as a substrate. In the incubations with AFB1 in human CYP1A1-expressed microsomes, AFM1 as a marking metabolite of AFB1 was detected. Moreover, AFB1 induced micronucleus formation in a Chinese hamster V79-derived cell line and in a human lung epithelial BEAS-2B cell line, both expressing recombinant human CYP1A1, V79-hCYP1A1 and 2B-hCYP1A1 cells, respectively. Immunofluorescence of centromere protein B stained micronuclei was dominant in AFB1-treated BEAS-2B cells exposed to AFB1, suggesting an aneugenic effect. Moreover, AFB1 elevated the levels of ROS, 8-OHdG, AFB1-DNA adduct, and DNA breaks in 2B-hCYP1A1 cells, compared with those in the parental BEAS-2B cells. Meanwhile, AFB1 increased CYP1A1, RAD51, and γ-H2AX protein levels in 2B-hCYP1A1 cells, which were attenuated by the CYP1A1 inhibitor bergamottin. Co-exposure of AFB1 with BaP increased 8-OHdG, RAD51, and γ-H2AX levels (indicating DNA damage). In conclusion, AFB1 could be activated by human CYP1A1 for potent aneugenicity, which may be further enhanced by co-exposure to BaP. • AFB1 could be activated by human CYP1A1 for potent aneugenicity. • AFM1 is formed by AFB1 incubated in human CYP1A1-expressed microsomes. • AFB1 and BaP coexposure enhanced the genotoxic effects associated with human CYP1A1 activity. [ABSTRACT FROM AUTHOR]

Abstract The European Commission requested EFSA to provide scientific advice on the translocation potential by Pseudomonas chlororaphis MA342 in plants after seed treatment of cereals and peas and, if applicable, for a revision of the assessment of the risk to humans by its metabolite 2,3‐deepoxy‐2,3‐didehydro‐rhizoxin (DDR) and this based on the evidence available in the dossier for renewal of the approval. The information from other P. chlororaphis strains than MA342 was taken into account with care, because the studies available in the dossier did not confirm the identity of the strain MA342 as belonging to the species P. chlororaphis. It has been concluded that there is a potential for translocation of P. chlororaphis MA342 to edible plant parts following seed treatment till an estimated concentration up to about 105 cfu/g and some exposure can be assumed by consumption of fresh commodities. Also, production of the metabolite DDR in the plant cannot be excluded. Regarding levels of DDR in the raw agricultural commodities, exposure estimates based on the limit of quantification (LOQ) for DDR in cereals cannot be further refined while there is no information on the levels of DDR in peas in the dossier. As regards genotoxicity, DDR induced chromosomal damage; however, it was not possible to conclude whether it is through an aneugenic or clastogenic mechanism. Hence, it is not possible to draw a reliable conclusion that DDR is producing an aneugenic effect nor to determine a threshold dose for aneugenicity. Thus, it is not possible to revise the human risk assessment as regards exposure to DDR. The concerns identified in the EFSA conclusion of 2017 remain.

The European Commission requested EFSA to provide scientific advice on the translocation potential by Pseudomonas chlororaphis MA342 in plants after seed treatment of cereals and peas and, if applicable, for a revision of the assessment of the risk to humans by its metabolite 2,3‐deepoxy‐2,3‐didehydro‐rhizoxin (DDR) and this based on the evidence available in the dossier for renewal of the approval. The information from other P. chlororaphis strains than MA342 was taken into account with care, because the studies available in the dossier did not confirm the identity of the strain MA342 as belonging to the species P. chlororaphis. It has been concluded that there is a potential for translocation of P. chlororaphis MA342 to edible plant parts following seed treatment till an estimated concentration up to about 105 cfu/g and some exposure can be assumed by consumption of fresh commodities. Also, production of the metabolite DDR in the plant cannot be excluded. Regarding levels of DDR in the raw agricultural commodities, exposure estimates based on the limit of quantification (LOQ) for DDR in cereals cannot be further refined while there is no information on the levels of DDR in peas in the dossier. As regards genotoxicity, DDR induced chromosomal damage; however, it was not possible to conclude whether it is through an aneugenic or clastogenic mechanism. Hence, it is not possible to draw a reliable conclusion that DDR is producing an aneugenic effect nor to determine a threshold dose for aneugenicity. Thus, it is not possible to revise the human risk assessment as regards exposure to DDR. The concerns identified in the EFSA conclusion of 2017 remain. [ABSTRACT FROM AUTHOR]

Nicotine's genotoxic potential has been extensively studied in vitro. While the results of mammalian cell‐based studies have inferred that it can potentially damage chromosomes, in general and with few exceptions, adverse DNA effects have been observed primarily at supraphysiological concentrations in nonregulatory assays that provide little information on its mode‐of‐action (MoA). In this study, a modern‐day regulatory genotoxicity assessment was conducted using a flow cytometry‐based in vitro micronucleus (MN) assay, Good Laboratory Practice study conditions, Chinese hamster ovary cells of known provenance, and acceptance/evaluation criteria from the current OECD Test Guideline 487. Nicotine concentrations up to 3.95 mM had no effect on background levels of DNA damage; however, concentrations above the point‐of‐departure range of 3.94–4.54 mM induced increases in MN and hypodiploid nuclei, indicating a possible aneugenicity hazard. Follow‐up experiments designed to elucidate nicotine's MoA revealed cellular vacuolization, accompanying distortions in microtubules, inhibition of tubulin polymerization, centromere‐positive DNA, and multinucleate cells at MN‐inducing concentrations. Vacuoles likely originated from acidic cellular compartments (e.g., lysosomes). Remarkably, genotoxicity was suppressed by chemicals that raised the luminal pH of these organelles. Other endpoints (e.g., changes in phosphorylated histones) measured in the study cast doubt on the biological relevance of this apparent genotoxicity. In addition, three major nicotine metabolites, including cotinine, had no MN effects but nornicotine induced a nicotine‐like profile. It is possible that nicotine's lysosomotropic properties drive the genotoxicity observed in vitro; however, the potency and mechanistic insights revealed here indicate that it is likely of minimal physiological relevance for nicotine consumers. Environ. Mol. Mutagen. 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA), an autoimmune disorder in which the immune system attacks healthy cells, is associated with elevated risk of lymphoma. Rituximab, a treatment for non-Hodgkin's lymphoma, has been approved as a treatment for RA. We studied the effects of rituximab on chromosomal stability in collagen-induced arthritis DBA/1J animal models. Micronucleus levels were increased in the mouse models, mainly due to chromosome loss, as detected by fluorescence in situ hybridization; rituximab-treated arthritic mice had significantly less micronucleus formation. Serum 8-hydroxydeoxyguanosine, a DNA oxidative stress marker, was increased in the mice models but reduced following rituximab administration. • Increased levels of chromosomal instability in arthritic male DBA/1J mouse model. • Repeated treatment with rituximab did not exhibit significant mutagenic effects. • Rituximab reduced chromosomal instability in arthritic mice. • Rituximab reduced DNA oxidative stress in arthritic mice. [ABSTRACT FROM AUTHOR]

The potential of molybdenum substances to cause genotoxic effects has been studied previously. However, a review of existing in vitro data, including an assessment of relevance and reliability, has shown that inconsistent results have been observed in the past. To resolve the inconsistencies, new studies were performed with the highly soluble sodium molybdate dihydrate according to OECD test guidelines. In a bacterial reverse mutation assay sodium molybdate dihydrate did not induce reverse mutations in five strains of Salmonella typhimurium . No mutagenic or clastogenic effect was observed at the tk locus of L5178Y mouse lymphoma cells. In a micronucleus test in cultured human peripheral blood lymphocytes no clastogenic or aneugenic effects were seen. These results can be read across to other inorganic molybdenum substances, that all release the molybdate ion [MoO 4 ] 2− under physiological conditions as the only toxicologically relevant species. In summary, a weight of evidence assessment of all available in vitro data shows no evidence of genotoxicity of molybdenum substances. [ABSTRACT FROM AUTHOR]

Coffee wastewater (CWW) is an effluent produced through wet processing of coffee containing high concentration of organic matter, nutrients, salts and also agrochemicals. It is released directly into the argillaceous soil or into decantation tanks for later disposal into soils, by fertigation, subsurface infiltration or superficial draining. However, this practice is not followed by the monitoring the toxicity potential of this effluent. In this sense, the present work aimed to evaluate the phytotoxic, cytogenotoxic and mutagenic potential of CWW on seed germination, root elongation and cell cycle alterations in the plant model Lactuca sativa L. The effluent (CWW) collected was diluted in distilled water into six concentrations solutions (1.25%, 1.66%, 2.5%, 5.0%, 10%, 20%). A solution of raw CWW (100%) was also applied. Distilled water was used as negative control), and the DNA alkylating agent, metilmetano sulfonate (4×10 −4 M) as positive control. Physico-chemical parameters of the CWW was accessed and it was found that the effluent contained total phenols and inorganic matter in amounts within the limits established by the National Environment Council (CONAMA). Nevertheless, the biologicals assays performed demonstrated the phytotoxicity and cytogenotoxicty of CWW. Seed germination was totally inhibited after exposure of raw CWW. In addition, a decrease in seed germination speed as well as in root growth dose-dependently manner was noticed. Moreover, nuclear and chromosomal alterations were observed in the cell cycle, mostly arising from aneugenic action. [ABSTRACT FROM AUTHOR]

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 24 h 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. [ABSTRACT FROM AUTHOR]

The in vivo bone marrow (BM) micronucleus assay is one of the three tests in the standard test battery to assess the genotoxic potential of a pharmaceutical candidate. In some cases, depending on results of in vitro studies, the route of administration or the degree of systemic exposure, in vivo assessment of genotoxicity in the BM alone may not be sufficient. Based on the potential for high gut exposures to orally administered compounds with low systemic exposures as well as the potential susceptibility of rapidly dividing cells of the intestinal tissues, we have developed a modified technique for evaluating micronuclei formation in both the duodenum and colon of rats based on earlier publications. Adult male Sprague Dawley rats were treated once daily for 2 days with either vehicle control or with the test articles acetyl salicylic acid (ASA), carbendazim (CAR), cyclophosphamide (CP), dimethylhydrazine (DMH), mitomycin C (MMC) or vinblastine sulfate (VIN). The duodenum, colon, and BM were harvested, processed, and analyzed for micronucleus induction. Results from these studies demonstrated differences in the susceptibility for different test compounds in the three tissues tested. When MMC and VIN were dosed by different routes at the same dose levels both compounds produced positive results in all three tissues by intraperitoneal injection but not oral administration. These studies suggest that overall the GI micronucleus assay might be a useful tool for clastogenic and aneugenic compounds that are expected to produce high sustained concentrations in the gastrointestinal tract with little systemic exposure. Environ. Mol. Mutagen., 2011. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

1-Phenylethanol is one of the major primary phase-I metabolites of ethylbenzene. In principle it may yield an electrophilic intermediate by phase-II metabolism. Because of the extensive use of ethylbenzene as a solvent, 2-year carcinogenicity inhalation studies were carried out leading to renal hyperplasia and tubular neoplasms both in male and female rats and alveolar/bronchiolar neoplasms in male mice and hepatocellular neoplasms in female mice. Whereas the mechanism underlying the increased renal tumor incidences in rats has been clarified, the mechanism of tumor formation (genotoxic or nongenotoxic mode of action) in the lung and liver of mice is still unclear. The genotoxicity data available to date for 1-phenylethanol include in vitro studies using either bacteria (Salmonella reverse mutation assay, E. coli Pol A+/Pol A− test) or mammalian cells (mouse lymphoma assay, chromosome aberration test and sister chromatid exchanges using CHO cells). These experiments, however, did not always follow current standard procedures and some of the data obtained are compromised and not always convincing. The present database thus does not allow a definitive assessment of the in vitro genotoxic potential of 1-phenylethanol. The in vitro database suggests that clastogenicity may be the most relevant genetic end point, and therefore an in vivo micronucleus assay in mouse bone marrow was carried out. The animals were given 1-phenylethanol in single oral doses up to the maximum tolerated dose of 750 mg/kg body weight. Bone marrow was sampled 24 and 48 h after treatment. Under the experimental conditions used, there was no evidence of increased micronuclei frequencies at any dose or sampling time. These findings indicate that 1-phenylethanol is not clastogenic in vivo. This information, together with other negative or inconclusive genotoxicity data available so far, suggests a nongenotoxic mode of action responsible for the lung and liver tumors observed in mice following 2 years of inhalation exposure to ethylbenzene. [ABSTRACT FROM AUTHOR]

Hormonally active chemicals in the human diet, such as man-made estrogenic chemicals or plant-derived compounds (phytoestrogens), have become a matter of public concern. A significant part of human exposure to phytoestrogens is attributable to soy isoflavones. Besides their estrogenic properties, soy isoflavones also exert genotoxic actions. In this paper, the micronucleus (MN) assay in V79 cells was used to study chromosomal genotoxicity. Genistein caused a clear dose-related induction of MN within the range of 5–25 μM; MN rates were declining at higher genistein concentrations. This was probably due to cytotoxicity of genistein since reduced neutral red uptake and MTT formation with an IC50 of about 75 μM occurred. Daidzein induced a comparatively shallow increase in the number of MN between 25 and 100 μM. In contrast, the daidzein metabolite equol caused an increase in the number of MN up to 25 μM with no further increase at higher concentrations. Additional staining with anti-kinetochore (CREST) antibodies served to determine if the micronuclei contain whole chromosomes or acentric fragments. Genistein induced mostly CREST(-) micronuclei, i.e. MN with chromosomal fragments, thus indicative of a clastogenic mode of action. MN induced by high concentrations of daidzein were partly CREST(+) and CREST(-), whilst equol induced mostly CREST(+) micronuclei indicative of an aneugenic action. These results point to a differential genotoxicity of phytoestrogens. [Copyright &y& Elsevier]

• The safety of low- and no- calorie sweeteners is frequently revisited. • Genotoxicity potential of five common LNCS was surveyed and summarized. • Peer-review literature and HTS data were integrated with authoritative assessments. • Emphasis was placed on assays most frequently considered for hazard identification. • Findings support authoritative conclusions regarding lack of genotoxic potential. Low- and no-calorie sweeteners (LNCS) are food additives that have been widely consumed for many decades. Their safety has been well established by authoritative bodies globally and is re-evaluated periodically. The objective herein was to survey and summarize the genotoxicity potential of five commonly utilized LNCS: acesulfame potassium (Ace-K), aspartame, saccharin, steviol glycosides and sucralose. Data from peer-reviewed literature and the ToxCast/Tox21 database were evaluated and integrated with the most recent weight-of-evidence evaluations from authoritative sources. Emphasis was placed on assays most frequently considered for hazard identification and risk assessment: mutation, clastogenicity and/or aneugenicity, and indirect DNA damage, such as changes in DNA repair mechanisms or gene expression data. These five sweeteners have been collectively evaluated in hundreds of in vivo or in vitro studies that employ numerous testing models, many of which have been conducted according to specific testing guidelines. The weight-of-evidence demonstrates overall negative findings across assay types for each sweetener when considering the totality of study design, reliability and reporting quality, as well as the lack of carcinogenic responses (or lack of responses relevant to humans) in animal cancer bioassays as well as observational studies in humans. This conclusion is consistent with the opinions of authoritative sources that have consistently determined that these sweeteners lack mutagenic and genotoxic potential. [ABSTRACT FROM AUTHOR]

The purpose of this review is to evaluate the literature on the genotoxicity of cumene (CAS # 98−82-8) and to assess the role of mutagenicity, if any, in the mode of action for cumene-induced rodent tumors. The studies reviewed included microbial mutagenicity, DNA damage/ repair, cytogenetic effects, and gene mutations. In reviewing these studies, attention was paid to their conformance to applicable OECD test guidelines which are considered as internationally recognized standards for performing these assays. Cumene was not a bacterial mutagen and did not induce Hprt mutations in CHO cell cultures. In the primary rat hepatocyte cultures, cumene induced unscheduled DNA synthesis in one study but this response could not be reproduced in an independent study using a similar protocol. In a study that is not fully compliant to the current OECD guideline, no increase in chromosomal aberrations was observed in CHO cells treated with cumene. The weight of the evidence (WoE) from multiple in vivo studies indicates that cumene is not a clastogen or aneugen. The weak positive response in an in vivo comet assay in the rat liver and mouse lung tissues is of questionable significance due to several study deficiencies. The genotoxicity profile of cumene does not match that of a classic DNA-reactive molecule and the available data does not support a conclusion that cumene is an in vivo mutagen. As such, mutagenicity does not appear to be an early key event in cumene-induced rodent tumors and alternate hypothesized non-mutagenic modes-of-action are presented. Further data are necessary to rule in or rule out a particular MoA. [ABSTRACT FROM AUTHOR]

• Increased levels of micronuclei formation in EAE-mouse model of multiple sclerosis. • The majority of increased micronuclei were due to aneugenic effect. • PD98059 decreased chromosomal instability and oxidative stress in EAE mice. Multiple sclerosis (MS), a disease in which the immune system attacks nerve cells, has been associated with both genetic and environmental risk factors. We observed increased micronucleus (MN) formation in SJL/J mouse experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Most of these MN were due to chromosomal loss. Increased activation of MAP kinases, which leads to disruption of the mitotic spindle and improper segregation of chromosomes, is associated with MS. MAP kinase inhibitors, such as PD98059, may therefore be beneficial for MS. In the EAE model, PD98059 treatment reduced adverse effects, including MN formation, lipid peroxidation, and GSH oxidation. Interventions that mitigate chromosomal instability may have therapeutic value in MS. [ABSTRACT FROM AUTHOR]

• Vorinostat was a genotoxic and epigenotoxic at the equivalent human doses. • It induced chromosomal aberrations, oxidative damages, apoptosis & hypomethylation. • Vorinostat downregulated the expression of genes involved in DNA repair pathway. • It's medical use should be weighed against the hazards of carcinogenicity. Vorinostat was approved as the first histone deacetylase inhibitor for the management of cutaneous T cell lymphoma. However, it's in vivo genetic and epigenetic effects on non-cancerous cells remain poorly understood. As genetic and epigenetic changes play a critical role in the pathogenesis of carcinogenesis, we investigated whether vorinostat induces genetic and epigenetic alterations in mouse bone marrow cells. Bone marrow cells were isolated 24 h following the last oral administration of vorinostat at the doses of 25, 50, or 100 mg/kg/day for five days (approximately equal to the recommended human doses). The cells were then used to assess clastogenicity and aneugenicity by the micronucleus test complemented by fluorescence in situ hybridization assay; DNA strand breaks, oxidative DNA strand breaks, and DNA methylation by the modified comet assay; apoptosis by annexin V/PI staining analysis and the occurrence of the hypodiploid DNA content; and DNA damage/repair gene expression by polymerase chain reaction (PCR) Array. The expression of the mRNA transcripts were also confirmed by real-time PCR and western blot analysis. Vorinostat caused structural chromosomal damage, numerical chromosomal abnormalities, DNA strand breaks, oxidative DNA strand breaks, DNA hypomethylation, and programed cell death in a dose-dependent manner. Furthermore, the expression of numerous genes implicated in DNA damage/repair were altered after vorinostat treatment. Accordingly, the genetic/epigenetic mechanism(s) of action of vorinostat may play a role in its carcinogenicity and support the continued study and development of new compounds with lower toxicity. [ABSTRACT FROM AUTHOR]