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Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety of a feed additive consisting of vitamin B 2 /riboflavin produced with Eremothecium ashbyi CCTCCM 2019833 intended for use as a nutritional additive (functional group: vitamins, pro-vitamins and chemically well-defined substances having similar effects) for all animal species. The characterisation, safety and efficacy of the additive have been already assessed previously; however, the FEEDAP Panel could not conclude on the safety of the additive for the target species, consumers and the users due to lack of reliable toxicological data. In the present assessment, the applicant submitted new genotoxicity and repeated dose oral toxicity studies. After the assessment of the data newly submitted, the FEEDAP Panel concluded that the use of the feed additive in animal nutrition under the conditions of use proposed is of no safety concern for the target species and the consumer. The additive is not a skin/eye irritant nor a skin sensitiser, but it is considered a respiratory sensitiser., (© 2024 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

Green agronomy promotes the implementation of natural and naturally derived substances in crop protection. In the present study, we evaluated the endocrine-disrupting potential of the allelopathic herbicide tembotrione in Wistar rats by studying the hormone status of offspring from the treated dams. Three doses of tembotrione (0.0004, 0.0007, and 4.0 mg/kg b.w./day) have been administered to dams during gestation and/or lactation. In the serum of newborn, weaning, and pubertal female and male offspring, 17β-estradiol and testosterone were determined using enzyme-linked immunosorbent assay. A decrease in 17β-estradiol and testosterone was observed in female and male weaning and pubertal offspring exposed to all doses of tembotrione during gestation and lactation. In weaning offspring exposed only during lactation, 17β-estradiol dropped significantly after exposure to the two lower doses and testosterone after exposure to the lowest dose of tembotrione. The greatest effect was observed at the lowest dose of tembotrione. In newborns, we observed increased 17β-estradiol after exposure to two lower doses of tembotrione and significantly increased testosterone after exposure to the lowest dose. The highest dose of tembotrione decreased 17β-estradiol significantly in newborn females. The obtained results suggest that tembotrione might be considered a pro-estrogenic or estrogen agonistic compound under the exposure conditions applied in this investigation.

The aim of this paper was to evaluate whether symbiotic cooperation between green hydra (Hydra viridissima) and photoautotrophic alga gives higher resistance of the preservation of DNA integrity compared to brown hydra (Hydra oligactis). Norflurazon concentrations were 0.061 or 0.61 mg/L and UV-B light 254 nm, 0.023mWcm - 2 applied separately or simultaneously. By alkaline comet assay primary DNA damage was assessed and cytotoxicity by fluorescent staining. Norflurazon at 0.61 mg L - 1 significantly increased DNA damage in brown hydras compared to the control (6.17 ± 0.6 μm, 5.2 ± 1.7% vs. 2.9 ± 0.2 μm, 1.2 ± 0.2%). Cytotoxicity was significantly elevated, being higher in brown hydras (25.7 ± 3.5% vs. 8.2 ± 0.2%). UV-B irradiation induced significant DNA damage in brown hydras (13.5 ± 1.0 μm, 4.1 ± 1.0%). Simultaneous exposure to UV-B and norflurazon led to a synergistic DNA damaging. The frequency of cytotoxicity and hedgehog nucleoids was more pronounced in brown (78.3 ± 9.4%; 56.4 ± 6.0%) than in green hydras (34.7 ± 2.5%; 24.2 ± 0.6%). Evolutionary established symbiotic cooperation proved to provide resistance against cyto/genotoxicity., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

To contribute new information to the pyrethroid pesticide α-cypermethrin toxicity profile, we evaluated its effects after oral administration to Wistar rats at daily doses of 2.186, 0.015, 0.157, and 0.786 mg/kg bw for 28 days. Evaluations were performed using markers of oxidative stress, cholinesterase (ChE) activities, and levels of primary DNA damage in plasma/whole blood and liver, kidney, and brain tissue. Consecutive exposure to α-cypermethrin affected the kidney, liver, and brain weight of rats. A significant increase in concentration of the thiobarbituric acid reactive species was observed in the brain, accompanied by a significant increase in glutathione peroxidase (GPx) activity. An increase in GPx activity was also observed in the liver of all α-cypermethrin-treated groups, while GPx activity in the blood was significantly lower than in controls. A decrease in ChE activities was observed in the kidney and liver. Treatment with α-cypermethrin induced DNA damage in the studied cell types at almost all of the applied doses, indicating the highest susceptibility in the brain. The present study showed that, even at very low doses, exposure to α-cypermethrin exerts genotoxic effects and sets in motion the antioxidative mechanisms of cell defense, indicating the potential hazards posed by this insecticide.

The aim of this study was to investigate the genotoxic potential of low doses of chlorpyrifos (CPF) on blood and bone marrow cells in adult male Wistar rats. CPF was administered by oral gavage at daily doses of 0.010, 0.015, and 0.160 mg/kg of body weight (bw) for 28 consecutive days. Positive control (PC) was administered 300 mg/kg bw/day of ethyl methane sulphonate (EMS) for the final three days of the experiment. Toxic outcomes of exposure were determined with the in vivo micronucleus (MN) assay and alkaline comet assay. The 28-day exposure to the 0.015 mg/kg CPF dose, which was three times higher than the current value of acute reference dose (ARfD), reduced body weight gain in rats the most. The in vivo MN assay showed significant differences in number of reticulocytes per 1000 erythrocytes between PC and negative control (NC) and between all control groups and the groups exposed to 0.015 and 0.160 mg/kg bw/day of CPF. The number of micronucleated polychromatic erythrocytes per 2000 erythrocytes was significantly higher in the PC than the NC group or group exposed to 0.015 mg/kg bw/day of CPF. CPF treatment did not significantly increase primary DNA damage in bone marrow cells compared to the NC group. However, the damage in bone marrow cells of CPF-exposed rats was much higher than the one recorded in leukocytes, established in the previous research. Both assays proved to be successful for the assessment of CPFinduced genome instability in Wistar rats. However, the exact mechanisms of damage have to be further investigated and confirmed by other, more sensitive methods., (© 2022 Vilena Kašuba, Vedran Micek, Mirta Milić, Davor Želježić, and Anja Katić, published by Sciendo.)

Ochratoxin A (OTA) and citrinin (CTN) are nephrotoxic mycotoxins often found together in grain. The aim of this study was to measure their accumulation in the kidney and liver of adult male Wistar rats, see how it would be affected by combined treatment, and to determine if resveratrol (RSV) would decrease their levels in these organs. The rats received 125 or 250 mg/kg bw of OTA by gavage every day for 21 days and/or 20 mg/kg bw of CTN a day for two days. Two groups of rats treated with OTA+CTN were also receiving 20 mg/kg bw of RSV a day for 21 days. In animals receiving OTA alone, its accumulation in both organs was dose-dependent. OTA+CTN treatment resulted in lower OTA but higher CTN accumulation in both organs at both OTA doses. RSV treatment increased OTA levels in the kidney and liver and decreased CTN levels in the kidney. Our findings point to the competition between CTN and OTA for organic anion transporters 1 and 3., (© 2022 Dubravka Rašić, Srđan Stefanović, Dragan Milićević, Marin Mladinić, Davor Želježić, Alica Pizent, Paško Konjevoda, Maja Peraica, published by Sciendo.)

This paper assessed the potential of trans-placental and -lactational genotoxicity and oxidative stress induction of tembotrione, a naturally derived allelopathic herbicide. Several treatment protocols were applied to measure primary DNA damage by alkaline comet assay in leucocytes and liver. To address the oxidative stress induction, TBARS, ROS, SOD, CA, GSH-Px activity were recorded. The dams were treated from the first gestation day and pups sacrificed after birth. The second treatment protocol comprised treating the dams during gestation and lactation and sacrificing the pups at weaning. The third group of pups comprised offspring of dams that were treated in gestation and lactation and sacrificed in puberty. To address translactational genotoxicity, dams were treated in lactation only. Dams treated in gestation and lactation were sacrificed after reentering the estrous cycle and analyzed for DNA damage and oxidative stress. Tembotrione doses encountered in everyday human exposure, as estimated by the EFSA, were applied in dam treatment in consecutive days (ADI: 0.0004 mg/kg b.w./day, AOEL: 0.0007 mg/kg b.w./day, 1/500 LD 50 4.0 mg/kg b.w./day). Although we observed mitigated DNA integrity at the dose of 4.0 mg/kg/b.w./day in female pubertal rats, we can conclude that at the conditions employed in the study low doses of tembotrione do not pose a risk for DNA damage of the offspring of treated dams. Contrary to this, the highest dose significantly affected all the oxidative stress parameters in the liver and plasma of pubertal females, CAT and GSH-Px in the liver of males and ROS and CAT of dams., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The aim of this cross-sectional study was to investigate the occurrence of chromosomal abnormalities through the frequency of micronuclei and other genomic damage markers in patients with chronic generalized periodontitis and without periodontal disease. Micronucleus assay was performed in exfoliated gingival epithelial cells of 35 patients with generalized chronic periodontitis and 30 control subjects with healthy periodontium. Full mouth clinical examination was performed to define periodontal condition. The mean number of cells with micronuclei observed in chronic periodontitis and control groups was 1.8 (±1.49) and 2.0 (±1.34), respectively. Differences between the groups were not significant (p=0.574). Compared to control subjects, patients with chronic periodontitis showed a significant increase in the number of binucleated cells (p≤0.001) and number of cells with nucleoplasmic bridges (p=0.042). Study results indicated that chronic periodontitis was not associated with higher occurrence of chromosomal damage in gingival cells compared to individuals with healthy periodontium.

Imidacloprid is a neonicotinoid insecticide that acts selectively as an agonist on insect nicotinic acetylcholine receptors. It is used for crop protection worldwide, as well as for non-agricultural uses. Imidacloprid systemic accumulation in food is an important source of imidacloprid exposure. Due to the undisputable need for investigations of imidacloprid toxicity in non-target species, we evaluated the effects of a 28-day oral exposure to low doses of imidacloprid (0.06 mg/kg b. w./day, 0.8 mg/kg b. w./day and 2.25 mg/kg b. w./day) on cholinesterase activity, oxidative stress responses and primary DNA damage in the blood and brain tissue of male Wistar rats. Exposure to imidacloprid did not cause significant changes in total cholinesterase, acetylcholinesterase and butyrylcholinesterase activities in plasma and brain tissue. Reactive oxygen species levels and lipid peroxidation increased significantly in the plasma of rats treated with the lowest dose of imidacloprid. Activities of glutathione-peroxidase in plasma and brain and superoxide dismutase in erythrocytes increased significantly at the highest applied dose. High performance liquid chromatography with UV diode array detector revealed the presence of imidacloprid in the plasma of all the treated animals and in the brain of the animals treated with the two higher doses. The alkaline comet assay results showed significant peripheral blood leukocyte damage at the lowest dose of imidacloprid and dose-dependent brain cell DNA damage. Oral 28-day exposure to low doses of imidacloprid in rats resulted in detectable levels of imidacloprid in plasma and brain tissue that directly induced DNA damage, particularly in brain tissue, with slight changes in plasma oxidative stress parameters., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aim: The main purpose of this study was to assess the genotoxic and cytotoxic effect of fissure sealants on buccal epithelial cells., Material and Methods: The study was conducted on 45 patients (27 girls and 18 boys), seven to 16 years of age (age mean 12.09 ± 2.20). Buccal swabs were collected before (T0), seven (T1), 30 (T2) and 90 days (T3) consequently after fissure sealant placement (Helioseal F®, Equia Fil®, Constic®). Patients or legal guardians filled in the questionnaire regarding the demographic data (age, gender), dietary habits, health status, medication usage, and recent X-ray exposure. DNA damage was analyzed using the micronucleus test., Results: Statistically significant difference in the number of buccal cells with condensed chromatin was found between T0 (time before fissure sealant placement) and T3 (90 days after fissure sealant placement) period for Helioseal F® (P = 0.025). For the other two analyzed materials, no difference was observed during the tested period. There was no difference between materials in the same sampling time., Conclusion: Apart from an increase in cells with condensed chromatin 90 days after the placement of Helioseal F®, no other nuclear abnormalities were observed for tested fissure sealants. Although these sealants have now largely been used, it is of high importance that their biocompatibility is checked continuously, especially in in vivo clinical studies., Competing Interests: Conflict of interest: The authors declare no conflict of interest.

Manufacture of lead-containing products has long been associated with various health risks. To get an insight into the related genotoxic risks, we conducted a biomonitoring study in 50 exposed workers and 48 matched controls using a battery of endpoints that sensitively detect the extent of genome instability in peripheral blood lymphocytes. The levels of primary DNA damage were estimated with the alkaline comet assay, while cytogenetic abnormalities were determined with the cytokinesis-block micronucleus (CBMN) cytome assay. Additionally, CBMN slides of 20 exposed and 16 control participants were subjected to fluorescence in situ hybridisation (FISH), coupled with pancentromeric probes to establish the incidence of centromere-positive micronuclei, nuclear buds, and nucleoplasmic bridges. Blood lead levels (B-Pb) were measured with atomic absorption spectrometry. To further characterise cumulative effects of occupational exposure, we measured erythrocyte protoporphyrin (EP) concentrations and delta-aminolevulinic acid dehydratase (ALAD) activity in blood. We also assessed the influence of serum folate (S-folate) and vitamin B12 (S-B12) on genome stability. Compared to controls, occupationally exposed workers demonstrated significantly higher B-Pb (298.36±162.07 vs 41.58±23.02), MN frequency (18.71±11.06 vs 8.98±7.50), centromere positive MN (C+ MN) (8.15±1.8 vs 3.69±0.47), and centromere negative MN (C- MN) (14.55±1.80 vs 4.56±0.89). Exposed women had significantly higher comet tail intensity (TI) and length (TL) than control women. Furthermore, workers showed a positive correlation between age and nuclear buds and MN, between MN and years of exposure, and between S-B12 levels and TI and ALAD activity, while a negative correlation was found between TI and B-Pb. These findings suggest that occupational settings in the manufacture of lead-containing products pose significant genotoxic risks, which calls for developing more effective work safety programmes, including periodical monitoring of B-Pb and genetic endpoints.

Tembotrione is a rather novel pesticide, usually used for post-emergence weed control. Even though its use is rapidly growing, it is not followed by an adequate flow of scientific evidence regarding its toxicity towards non-target organisms. We evaluated the potential of low doses of tembotrione to induce oxidative stress and cytogenetic damage in blood and brain cells of adult male Wistar rats. Parameters of lipid peroxidation, glutathione levels, activities of antioxidant enzymes and primary DNA damage were assessed following 28-day repeated oral exposure to doses comparable with the currently proposed health-based reference values. The results of the alkaline comet assay showed that such low doses of tembotrione have the potency to inflict primary DNA damage in both peripheral blood leukocytes and brain of treated rats, even with only slight changes in the oxidative biomarker levels. The DNA damage in blood and brain cells of Wistar rats significantly increased at all applied doses, suggesting that tembotrione genotoxicity is mainly a result of direct interaction with DNA while the induction of oxidative stress responses contributes to DNA instability in a lesser extent. The findings of the present study call for further research using other sensitive biomarkers of effect and different exposure scenarios., Competing Interests: Declaration of competing interest None to declare., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The food enzyme is a d-xylose aldose-ketose-isomerase (EC 5.3.1.5) produced with the genetically modified Streptomyces rubiginosus strain DP-Pzn37 by Danisco US Inc. Although the production strain contains antibiotic resistance genes, the food enzyme was shown to be free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in an immobilised form for the isomerisation of glucose for the production of high fructose syrups. Residual amounts of total organic solids (TOS) are eliminated by the use of an immobilised food enzyme and further removed by the purification steps applied during the production of high fructose syrups using the immobilised enzyme; consequently, dietary exposure was not calculated. Genotoxicity tests did not raise safety concerns. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 85.2 mg TOS/kg body weight (bw) per day, the highest dose tested. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be low. Based on the data provided, the immobilisation process and the removal of total organic solids during the production of high fructose syrups, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use., (© 2020 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

DNA damage in the liver and kidney cells of adult male Wistar rats was studied using the comet assay after a 28-day oral administration of tembotrione at doses of 0.0007, 0.0013 and 0.7 mg/kg b.w./day [AOEL (acceptable operator exposure level), REL (residual exposure level) and 1000× AOEL]. As a descriptor of DNA damage, tail intensity was used. Antioxidant status was assessed by activity of glutathione peroxidase (GPx). Significant DNA damage was recorded in the kidney cells at all three doses as compared to negative control. In parenchymal liver cells, significant DNA damage was observed in AOEL and 1000× AOEL doses, while in non-parenchymal liver cells, only AOEL-treated group was significantly different compared to negative control. In both types of liver cells, REL and 1000× AOEL doses were significantly different from the AOEL dose. No significant changes in GPx activity compared to control were observed at any exposure level. The results of the present study suggest that repeated in vivo exposure to tembotrione led to low-level DNA instability in kidney and liver cells. Exposure to the highest tembotrione dose showed a relatively weak response with the alkaline comet assay. Further research should focus on the effects of this herbicide in other models along with different exposure scenarios.

The food enzyme glucan 1,4-α-maltotetraohydrolase (EC 3.2.1.8) is produced with the genetically modified Bacillus licheniformis strain DP-Dzf24 by Danisco US Inc. The production strain contains multiple copies of a known antimicrobial resistance gene. However, based on the absence of viable cells and DNA in the food enzyme, this is not considered to be a risk. The food enzyme is intended to be used in baking processes and starch processing for the production of glucose syrups. The residual amounts of the Total Organic Solids (TOS) in glucose syrups are removed by filtration and purification during starch processing. Consequently, dietary exposure was not calculated for this use. Based on the maximum use levels recommended for the baking processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-TOS was estimated to be up to 0.271 mg TOS/kg body weight per day in European populations. Toxicological tests with the food enzyme indicated that there was no concern with respect to genotoxicity or systemic toxicity. A no-observed-adverse-effect level (NOAEL) was identified in rats, which, compared with the dietary exposure, results in a margin of exposure of at least 347. The allergenicity was evaluated by searching for similarity of the amino acid sequence to those of known allergens; no match was found. The Panel considers that, under the intended conditions of use, the risk for allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered low. Based on the microbial source, genetic modifications, the manufacturing process, the compositional and biochemical data, the dietary exposure assessment and the findings in the toxicological studies, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use., (© 2019 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The food enzyme glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase; EC 1.1.3.4) is produced with a genetically modified Aspergillus niger strain ZGL by DSM Food Specialties B.V.. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and recombinant DNA. The glucose oxidase is intended to be used in baking processes. Based on the maximum use levels, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.004 mg TOS/kg body weight (bw) per day. The toxicity studies were carried out with an asparaginase from A. niger (strain ASP). The Panel considered this enzyme as a suitable substitute to be used in the toxicological studies, because they derive from the same recipient strain, the location of the inserts are comparable, no partial inserts were present and the production methods are essentially the same. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) at the highest dose of 1,038 and 1,194 mg TOS/kg bw per day (for males and females, respectively) that, compared with the estimated dietary exposure, results in a sufficiently high margin of exposure (MoE) (of at least 260,000). Similarity of the amino acid sequence to those of known allergens was searched and one match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use., (© 2019 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

Chlorpyrifos, imidacloprid, and α-cypermethrin are some of the most widely used insecticides in contemporary agriculture. However, their low-dose, nontarget genotoxic effects have not been extensively assayed. As one of the most relevant cancer biomarkers, we aimed to assess the aneuploidy due to chromosome missegregation during mitosis. To aim it we treated human lymphocytes in vitro with three concentrations of insecticides equivalents relevant for real scenario exposure assessed by regulatory agencies. We focused on chlorpyrifos as conventional and imidacloprid and α-cypermethrin as sustainable use insecticides. Cytokinesis-blocked micronucleus assay was performed coupled with fluorescence in situ hybridization (FISH) with directly labeled pancentromeric probes for chromosomes 9, 18, X and Y. None of the insecticides induced significant secondary DNA damage in terms of micronuclei (MN), nuclear buds (NB), or nucleoplasmic bridges (NPB). However, significant disbalances in chromosomes 9, 18, X and Y, and in insecticide-treated cells has been observed. According to recent studies, these disbalances in chromosome numbers may be atributted to defect sister chromatid cohesion which contribute to the increase of chromosome missegregation but not to micronuclei incidence. We conclude that tested insecticidal active substances exert chromosome missegregation effects at low concentrations, possibly by mechanism of sister chromatid cohesion. These findings may contribute to future risk assesments and understanding of insecticide mode of action on human genome. Environ. Mol. Mutagen. 60:72-84, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Objective: The aim of this study was to evaluate cytotoxic potencies of two light cured composite materials after heating on different temperatures and cured directly and through CAD/CAM overlay., Materials and Methods: Composite materials (microfilled-hybrid Gradia Direct Posterior and Beautifil II) were heated in a Calset warming unit at three different temperatures (T1:37°C, T2:54°C, T3:68°C). A small amount of heated composite material was placed in a round mold (diameter 6mm; 0.65mm thick), covered with Mylar sheet, pressed and polymerized with Bluephase LED unit. One group of samples were polymerized directly, and the other group through 2mm thick CAD/CAM ceramic-reinforced polymer (CRP) and CAD/CAM lithium disilicate ceramic (LDC) overlay for 20 and 40 seconds. The polymerized samples were placed immediately after curing in a lymphocyte cell culture. The viability of peripheral blood lymphocytes was evaluated using a dye exclusion technique by simultaneous staining with ethidium bromide and acridine orange. Quantitative assessments were made by determination of the percentage of viable, apoptotic and necrotic cells. The Pearson chi-square test was used for statistical analysis., Results: In case of 20 seconds polymerization, the highest number of viable cells polymerization were recorded when materials were heated at 37°C (T1), while in case of 40 seconds polymerization, the highest number of viable cells were recorded when the materials were heated at 54°C (T2). The samples polymerized through CAD/CAM overlays showed less cytotoxicity than samples polymerized directly., Conclusion: Apart from composite material composition, the cell viability was also influenced by curing time, temperature of pre-heating and polymerization pattern., Competing Interests: Conflict of interest: None declared

In this study, the influence of anthropogenic pollution on the aquatic environment of Plitvice Lakes National Park (PLNP) was investigated during 2011-2012 using a combination of chemical and cytogenetic analyses. Four groups of major contaminants [(volatile organic compounds: benzene, toluene, ethylbenzene, and xylenes (BTEX); persistent organochlorine pollutants: organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs); major and trace elements; anthropogenic radionuclides ( 90 Sr, 134 Cs, and 137 Cs)] were determined in three aquatic compartments (water, sediment, fish). Mass fractions of inorganic constituents in different compartments reflected the geological background of the area, indicating their origin from predominantly natural sources. Levels of volatile and persistent organic compounds in water and fish, respectively, were very low, at levels typical for remote pristine areas. Analysis of anthropogenic radionuclides in water and sediment revealed elevated activity concentrations of 137 Cs in water, and measurable 134 Cs in the upper sediment layers from April 2011, possibly as a consequence of the Fukushima nuclear accident in March 2011. The potential genotoxicity of river and lake water and lake sediment was assessed under laboratory conditions using the alkaline comet assay on human peripheral blood lymphocytes, and measured levels of primary DNA damage were within acceptable boundaries. The results showed that despite the protected status of the park, anthropogenic impact exists in both its terrestrial and aquatic components. Although contaminant levels were low, further monitoring is recommended to make sure that they will not rise and cause potentially hazardous anthropogenic impacts.

The food enzyme endo-1,4-β-xylanase (EC 3.2.1.8) is produced with the genetically modified Bacillus subtilis strain LMG S-24584 by Puratos N. V. The genetic modifications do not give rise to safety concerns. The Panel noted that, although the production strain was not detected in the food enzyme, recombinant DNA was present in all batches of the food enzyme tested. The food enzyme is intended to be used in baking processes. Based on the maximum use levels recommended for the baking processes and individual consumption data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-Total Organic Solids (TOS) was estimated to be up to 0.017 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rodents. A comparison of the no observed adverse effect level of 37 mg TOS/kg bw per day from this study with the dietary exposure results in a sufficiently high margin of exposure. The amino acid sequence of the food enzyme did not match those of known allergens. The Panel considered that, under the intended condition of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood of such reactions occurring is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use., (© 2018 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The food enzyme is an α-amylase (4-α-d-glucan glucanohydrolase; EC 3.2.1.1), produced with the genetically modified Aspergillus niger strain NZYM-SB by Novozymes A/S. The food enzyme does not contain the production organism or its DNA; therefore, there is no safety concern for the environment. The α-amylase is intended for use in starch processing, beverage alcohol (distilling) processes and baking processes. Residual amounts of total organic solids (TOS) are removed by distillation and by the purification steps applied during the production of glucose syrups (by > 99%). Consequently, dietary exposure was not calculated for these two uses. Based on the maximum use levels recommended for the baking processes and individual consumption data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-TOS was estimated to be up to 3.075 mg TOS/kg body weight per day in European populations. The food enzyme did not induce gene mutations in bacteria or micronuclei in human lymphocytes. Subchronic toxicity was assessed by means of a repeated-dose 90-day oral toxicity study in rodents. A no observed adverse effect level (NOAEL) was derived that, compared with the dietary exposure, resulted in a sufficiently high margin of exposure (MOE). Similarity of the amino acid sequence to those of known allergens was searched and two matches were found. The Panel considered that the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered low. Based on the genetic modifications, the manufacturing process, the compositional and biochemical data, the findings in the toxicological and genotoxicity studies, as well as the estimated dietary exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use., (© 2018 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

Terbuthylazine belongs to the chloro-s-triazine group of herbicides and acts primarily as a photosynthesis inhibitor. The mechanisms of action related to its exposure, relevant both in animals and humans, are still insufficiently investigated. This comprehensive study focused on the outcomes of terbuthylazine exposure at cell level in vitro, and a mice model in vivo. Experiments in vitro were conducted on whole human peripheral blood, isolated lymphocytes, and HepG2 cells exposed for 4 h to terbuthylazine at 8.00, 0.80, and 0.58 ng/mL, which is comparable with current reference values set by the European Commission in 2011. Terbuthylazine cytotoxicity was evaluated using dual fluorescent staining with ethidium bromide and acridine orange on lymphocytes, and CCK-8 colorimetric assay on HepG2 cells. The levels of DNA damage were measured using alkaline and hOGG1-modified comet assays. The potency of terbuthlyazine regarding induction of oxidative stress in vitro was studied using a battery of standard oxidative stress biomarkers. The in vivo experiment was conducted on Swiss albino mice exposed to terbuthlyazine in the form of an active substance and its formulated commercial product Radazin TZ-50 at a daily dose of 0.0035 mg/kg bw for 14 days. Following exposure, the DNA damage levels in leukocytes, bone marrow, liver, and kidney cells of the treated mice were measured using an alkaline comet assay. In vitro results suggested low terbuthylazine cytotoxicity in non-target cells. The highest tested concentration (8.00 ng/mL) reduced lymphocyte viability by 15%, mostly due to apoptosis, while cytotoxic effects in HepG2 cells at the same concentration were negligible. Acute in vitro exposure of human lymphocytes and HepG2 cells to terbuthylazine resulted in low-level DNA instability, as detected by the alkaline comet assay. Further characterization of the mechanisms behind the DNA damage obtained using the hOGG1-modified comet assay indicated that oxidative DNA damage did not prevail in the overall damage. This was further confirmed by the measured levels of oxidative stress markers, which were mostly comparable to control. Results obtained in mice indicate that both the active substance and formulated commercial product of terbuthylazine produced DNA instability in all of the studied cell types. We found that DNA in liver and kidney cells was more prone to direct toxic effects of the parent compound and its metabolites than DNA in leukocytes and bone marrow cells. The overall findings suggest the formation of reactive terbuthylazine metabolites capable of inducing DNA cross-links, which hinder DNA migration. These effects were most pronounced in liver cells in vivo and HepG2 cells in vitro. To provide a more accurate explanation of the observed effects, additional research is needed. Nevertheless, the present study provides evidence that terbuthylazine at concentrations comparable with current reference values possesses toxicological risk because it caused low-level DNA instability, both at cellular and animal organism level, which should be further established in forthcoming studies.

Pesticides are a highly diverse group of compounds and the most important chemical stressors in the environment. Mechanisms that could explain pesticide toxicity are constantly being studied and their interactions at the cellular level are often observed in well-controlled in vitro studies. Several pesticide groups have been found to impair the redox balance in the cell, but the mechanisms leading to oxidative stress for certain pesticides are only partly understood. As our scientific project "Organic pollutants in environment - markers and biomarkers of toxicity (OPENTOX)" is dedicated to studying toxic effects of selected insecticides and herbicides, this review is focused on reporting the knowledge regarding oxidative stress-related phenomena at the cellular level. We wanted to single out the most important facts relevant to the evaluation of our own findings from studies conducted on in vitro cell models.

The aim of this in vitro study was to evaluate cytotoxicity and genotoxicity of six different dental nanocomposite materials, three conventional ones and three flowable composite resin materials, in human lymphocytes. The following materials were tested: Tetric EvoCeram, Tetric EvoFlow, Filtek Ultimate, Filtek Ultimate Flow, G-aenial and G-aenial Flo. Cytotoxicity was evaluated for two mass concentrations (0.007 g/mL and 0.013 g/mL) of each material, non-cured and cured, after 4 hours and 24 hours. Genotoxicity was evaluated using micronucleus assay under the same conditions as applied during the investigation of cytotoxicity. Uncured forms of Tetric EvoCeram, Tetric EvoFlow and Filtek Ultimate Flow in higher mass concentration caused genotoxic effect. Uncured G-aenial Flo in higher mass concentration induced apoptosis and necrosis. Uncured Tetric EvoFlow and uncured Filtek Ultimate Flow in higher mass concentration induced early apoptosis after both test periods. None of the conventional composite resin materials tested showed cytotoxicity except for uncured G-aenial, which induced apoptosis in higher mass concentration in both test periods. In conclusion, under the conditions of this in vitro study, cured conventional composites did not show cytotoxic or genotoxic effect, which is important for clinical application of these materials, whereas uncured forms exhibited certain level of cytotoxicity and genotoxicity, mainly because of monomers in their composition.

Humans have used insecticides since ancient times. The spectrum and potency of available insecticidal substances has greatly expanded since the industrial revolution, resulting in widespread use and unforeseen levels of synthetic chemicals in the environment. Concerns about the toxic effects of these new chemicals on non-target species became public soon after their appearance, which eventually led to the restrictions of use. At the same time, new, more environmentally-friendly insecticides have been developed, based on naturally occurring chemicals, such as pyrethroids (derivatives of pyrethrin), neonicotinoids (derivatives of nicotine), and insecticides based on the neem tree vegetable oil (Azadirachta indica), predominantly azadirachtin. Although these new substances are more selective toward pest insects, they can still target other organisms. Neonicotinoids, for example, have been implicated in the decline of the bee population worldwide. This review summarises recent literature published on non-target toxicity of neonicotinoids, pyrethroids, and neem-based insecticidal substances, with a special emphasis on neonicotinoid toxicity in honeybees. We also touch upon the effects of pesticide combinations and documented human exposure to these substances.

In this 28 day-study, we evaluated the effects of herbicide glyphosate administered by gavage to Wistar rats at daily doses equivalent to 0.1 of the acceptable operator exposure level (AOEL), 0.5 of the consumer acceptable daily intake (ADI), 1.75 (corresponding to the chronic population-adjusted dose, cPAD), and 10 mg kg-1 body weight (bw) (corresponding to 100 times the AOEL). At the end of each treatment, the body and liver weights were measured and compared with their baseline values. DNA damage in leukocytes and liver tissue was estimated with the alkaline comet assay. Oxidative stress was evaluated using a battery of endpoints to establish lipid peroxidation via thiobarbituric reactive substances (TBARS) level, level of reactive oxygen species (ROS), glutathione (GSH) level, and the activity of glutathione peroxidase (GSH-Px). Total cholinesterase activity and the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were also measured. The exposed animals gained less weight than control. Treatment resulted in significantly higher primary DNA damage in the liver cells and leukocytes. Glyphosate exposure significantly lowered TBARS in the liver of the AOEL, ADI, and cPAD groups, and in plasma in the AOEL and cPAD group. AChE was inhibited with all treatments, but the AOEL and ADI groups significantly differed from control. Total ChE and plasma/liver ROS/GSH levels did not significantly differ from control, except for the 35 % decrease in ChE in the AOEL and ADI groups and a significant drop in liver GSH in the cPAD and 100xAOEL groups. AOEL and ADI blood GSH-Px activity dropped significantly, but in the liver it significantly increased in the ADI, cPAD, and 100xAOEL groups vs. control. All these findings show that even exposure to low glyphosate levels can have serious adverse effects and points to a need to change the approach to risk assessment of low-level chronic/sub-chronic glyphosate exposure, where oxidative stress is not necessarily related to the genetic damage and AChE inhibition.

The food enzyme considered in this opinion is aqualysin 1 (EC 3.4.21.111), produced from the genetically modified strain Bacillus subtilis LMGS 25520 by Puratos NV. The production strain was not detected in the food enzyme. Aqualysin 1 is intended to be used in baking processes. Based on the maximum use level recommended and individual consumption data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 2.13 mg TOS/kg body weight per day in European populations. Genotoxicity tests indicated no genotoxic concerns. The allergenicity was evaluated by searching for similarity of the amino acid sequence to those of known allergens and 23 matches were found (20 respiratory and 3 dermal allergens). However, the Panel considered that there are no indications for food allergic reactions to the food enzyme. The genetic modifications performed, the manufacturing process, the compositional and biochemical data, the allergenicity and the genotoxicity assessment did not raise safety concerns. The Panel considered the margin of exposure (MOE) calculated from the no observed adverse effect level (NOAEL) determined from the repeated dose 90-day oral toxicity study and the estimated dietary exposure as insufficient to conclude that there is no safety concern for this food enzyme under the intended conditions of use. The Panel noted that recombinant DNA was present in all batches of the food enzyme tested., (© 2018 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

A multimycotoxin analysis approach in grains results in frequent simultaneous findings of nephrotoxic mycotoxins ochratoxin A (OTA) and citrinin (CTN). The mechanism of CTN and OTA toxicities in biological systems is not fully understood but it is known that oxidative stress is involved. In this study, oxidative damage of DNA, lipids, and the concentration of glutathione (GSH), as well as possible antioxidative effects of resveratrol (RSV) were studied in vivo. Male adult Wistar rats were treated orally with OTA (0.125 and 0.250 mg kg -1  b.w.), RSV (20 mg kg -1  b.w.) for 21 days, CTN (20 mg kg -1  b.w.) for two days or with their combinations. The hOGG1 modified comet assay revealed kidneys and liver oxidative DNA damage in OTA + CTN treated animals, which was not reversed by RSV. CTN did not reduce glutathione (GSH) or increase malondialdehyde (MDA) concentration in any tissue, while OTA reduced kidneys GSH and increased kidneys and liver MDA. RSV increased GSH concentrations in all tissues and decreased MDA concentration in the liver only. Oxidative stress is involved in the toxicity of OTA and CTN but it seems that it differs significantly in organs. Most of the effects on GSH and MDA in combined toxicity may be attributed to the toxic effects of OTA. RSV was effective in restoring the depleted GSH in all tissues but had no effect on the MDA concentration and DNA damage., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The food enzyme is an endo-1,4-β-xylanase (EC 3.2.1.8) produced with a genetically modified strain of Aspergillus niger (strain XEA), by DSM Food Specialities B.V. The food enzyme is intended to be used in baking and brewing processes. Based on maximum use levels recommended for the food processes and individual consumption data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.310 mg TOS/kg body weight per day in European populations. Genotoxicity tests with the food enzyme did not indicate a genotoxic concern. A repeated dose 90-day oral toxicity study in rodents, carried out with this endo-1,4-β-xylanase, showed no concern with respect to systemic toxicity. The allergenicity was evaluated by searching for similarity of the amino acid sequence to those of known allergens; no match was found. The Panel considers that there are no indications for allergic sensitisation and elicitation reactions by dietary exposure to the food enzyme endo-1,4-β-xylanase. Based on the microbial source, the genetic modifications performed, the manufacturing process, the compositional and biochemical data provided, the dietary exposure assessment, the findings in the toxicological studies and the allergenicity assessment, the Panel concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use., (© 2018 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

This study investigated the levels and distribution of 17 polychlorinated biphenyls (PCBs) and organochlorine pesticides (HCB, α-HCH, β-HCH, γ-HCH, p,p'-DDE, p,p'-DDD, and p,p'-DDT) in placenta samples from women living in the coastal area of Croatia. During November 2012 to February 2013, 51 placenta samples were collected from healthy mothers. This study presents the first report about Croatian placenta samples. Each of the analysed compounds were found in all of the samples; all of the maximum values were < 1 ng g -1 w.w., and the highest median value found for PCB-28 was 11.2 pg g -1 w.w. PCBs and organochlorine pesticide (OCPs) present in placenta samples were tested for their genotoxic potential using the alkaline comet assay. The alkaline comet assay is one of the most reliable methods in assessing the DNA lesions that occurs in direct interaction of a chemical and the genome. The detected levels of PCBs and OCPs in the placenta did not pose a significant risk to the children's DNA during embryonic and foetal growth following short-term exposure. PCB and OCP concentrations in the placenta samples did not induce any significant primary damage to DNA in terms of DNA strand breaks and changes in the primary chemical structure, which could be detected by the alkaline comet assay.

The food enzyme considered in this opinion is an endo-1,4-β-xylanase (EC 3.2.1.8) produced with a genetically modified Bacillus subtilis strain from Puratos N.V. (Belgium). The genetic modifications do not raise safety concerns. The food enzyme contains neither the production organism nor recombinant DNA. The endo-1,4-β-xylanase is intended to be used in baking processes. Based on the maximum use levels recommended for the baking processes, dietary exposure to the food enzyme-total organic solids (TOS) was estimated on the basis of individual data from the EFSA Comprehensive European Food Consumption Database. This exposure estimate is up to 0.008 mg TOS/kg body weight per day in European populations. The food enzyme did not induce gene mutations in bacteria nor clastogenic activity in human lymphocytes. Therefore, there is no concern with respect to genotoxicity. The subchronic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rodents. A no observed adverse effect level was derived, which, compared with the dietary exposure, results in a sufficiently high margin of exposure. The allergenicity was evaluated by searching for similarity of the amino acid sequence to those of known allergens; no matches were found. The Panel considered that there are no indications for food allergic reactions to this xylanase. Based on the microbial source, genetic modifications performed, the manufacturing process, the compositional and biochemical data provided, the findings in the toxicological studies and allergenicity assessment, this food enzyme does not give rise to safety concerns under the intended conditions of use., (© 2018 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

In this 28 day-study, we evaluated the effects of the insecticide chlorpyrifos orally administered to Wistar rats at doses 0.160, 0.015, and 0.010 mg/kg b. w./day. Following treatment, total cholinesterase activity and activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were measured. Oxidative stress responses were evaluated using a battery of endpoints to establish lipid peroxidation, changes in total antioxidant capacity, level of reactive oxygen species (ROS), glutathione (GSH) level and activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase. Using HPLC-UV DAD analysis, levels of the parent compound and its main metabolite 3,5,6-trichloro-2-pyridinol in plasma and brain tissue were measured. The genotoxic effect was estimated using alkaline comet assay in leukocytes and brain tissue. The exposure did not result in significant effects on total cholinesterase, AChE and BChE activity in plasma and brain tissue. Lipid peroxidation slightly increased both in plasma and brain tissue. Total antioxidant capacity, ROS and GSH levels were marginally influenced by the exposure. Treatment led to significant increases of GSH-Px activity in blood, SOD activity in erythrocytes and a slight increase of catalase activity in plasma. HPLC-UV DAD analysis revealed the presence of both the parent compound and its main metabolite in the plasma of all of the experimental animals and brain tissue of the animals treated at the two higher doses. All of the tested doses of chlorpyrifos were slightly genotoxic, both to leukocytes and brain tissue. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The food enzyme considered in this opinion is a peroxidase (hydrogen-peroxide oxidoreductase; EC 1.11.1.7) obtained from hulls of soybeans ( Glycine max ) by the company Kerry Ingredients & Flavours. The compositional data provided were considered sufficient. The manufacturing process did not raise safety concerns. The enzyme is intended to be used in baking processes. Based on the maximum recommended use level, dietary exposure to the food enzyme total organic solids (TOS) was estimated on the basis of individual data from the EFSA Comprehensive European Food Consumption Database. This exposure estimate is lower than the exposure to the fraction of soybean comparable to the food enzyme TOS resulting from the consumption of whole soybean-derived foods by roughly an order of magnitude. As the food enzyme is derived from edible parts of soybean, in line with the requirements of the guidance document on food enzyme assessment, the Panel concluded that the provision of toxicological data was unnecessary. The potential allergenicity was evaluated by searching for similarity between the amino acid sequence of soybean peroxidase retrieved from the database Uniprot and the sequences of known food allergens; no match was found. Peroxidase from soybean hulls is not listed as an allergen in allergen databases. However, several soybean- and soybean hull proteins are known to be respiratory or food allergens. Based on the origin of the food enzyme from edible parts of soybean, the enzyme manufacturing process, the compositional and biochemical data provided, and the dietary exposure assessment, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. However, the Panel noted that this food enzyme may contain allergenic soybean proteins, thus, adverse reactions in susceptible soybean-allergic individuals cannot be ruled out., (© 2017 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

Background Diazepam, as one of the most frequent prescribed drug from 1,4-benzodiazepine group, has certain limitations in pharmaceutical technology due to its poor solubility in water. By forming inclusion complexes with 2-hydroxypropyl-β-cyclodextrin, diazepam's biopharmaceutical properties can be greatly improved. Aim Aim of this research was to in vitro evaluate genotoxicity of prepared novel complexes of diazepam and their influence on proliferation of human peripheral blood lymphocytes. Methods For identification of possible genotoxicity of diazepam inclusion complexes, cytokinesis-block micronucleus assay has been chosen. Evaluated concentrations of two diazepam inclusion complexes were 0.2 µg/mL, 0.5 µg/mL and 1.0 µg/mL in cell culture. For a reference, in vitro cytogenotoxicity evaluation of diazepam alone has been conducted as well. Results Neither one of the diazepam, complexed nor non-complexed, in given concentrations showed genotoxicity, induced genetic damage or loss of genetic material. Conclusions Nuclear division index values, as indicators of cytostaticity and cytotoxicity suggested that investigated inclusion diazepam complexes induced accelerated proliferation of human peripheral blood lymphocytes in vitro, therefore possibly shortening the duration and dynamics of the cell cycle., Competing Interests: Conflict of Interest: The authors declare that there is no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Terbuthylazine is a selective pre- and post-emergency chloro-triazine herbicide used for a broad spectrum of weed control. We evaluated the potential of low doses of terbuthylazine to induce oxidative stress and cytogenetic damage in peripheral blood samples of adult male Wistar rats. Following 28-day repeated oral exposure at 0.004 mg/kg b.w./day, 0.4 mg/kg b.w./day and 2.29 mg/kg b.w./day, parameters of lipid peroxidation, total antioxidant capacity, and activities of antioxidant enzymes were measured in blood samples. Alkaline comet assay on leukocytes and erythrocyte micronucleus assay were used to measure DNA damage. In addition, the concentration of terbuthylazine and its metabolite in urine and plasma were determined using high performance liquid chromatography with UV diode-array detector (HPLC-UV-DAD). The fraction of terbuthylazine excreted in urine was negligible and was not found in plasma. Deethylterbuthylazine was only compound detected in plasma samples. Exposure to terbuthylazine did not induce significant lipid peroxidation products. The significant changes in antioxidant enzyme activities and the elevated total antioxidant capacity indicated that terbuthylazine at experimental conditions applied has potential to disturb oxidative/antioxidant balance. Results regarding the alkaline comet assay as well as micronucleated reticulocyte frequency indicated that treatment led to low-level DNA instability. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios., (Copyright © 2017. Published by Elsevier Ltd.)

We studied the toxic effects of glyphosate in vitro on HepG2 cells exposed for 4 and 24 h to low glyphosate concentrations likely to be encountered in occupational and residential exposures [the acceptable daily intake (ADI; 0.5 μg/mL), residential exposure level (REL; 2.91 μg/mL) and occupational exposure level (OEL; 3.5 μg/mL)]. The assessments were performed using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell proliferation, alkaline comet assay and cytokinesis-block micronucleus (CBMN) cytome assay. The results obtained indicated effects on cell proliferation, both at 4 and 24 h. The levels of primary DNA damage after 4-h exposure were lower in treated vs. control samples, but were not significantly changed after 24 h. Using the CBMN assay, we found a significantly higher number of MN and nuclear buds at ADI and REL after 4 h and a lower number of MN after 24 h. The obtained results revealed significant oxidative damage. Four-hour exposure resulted in significant decrease at ADI [lipid peroxidation and glutathione peroxidase (GSH-Px)] and OEL [lipid peroxidation and level of total antioxidant capacity (TAC)], and 24-h exposure in significant decrease at OEL (TAC and GSH-Px). No significant effects were observed for the level of reactive oxygen species (ROS) and glutathione (GSH) for both treatment, and for 24 h for lipid peroxidation. Taken together, the elevated levels of cytogenetic damage found by the CBMN assay and the mechanisms of primary DNA damage should be further clarified, considering that the comet assay results indicate possible cross-linking or DNA adduct formation.

The food enzyme considered in this opinion is a β-amylase (EC 3.2.1.2) from soybean submitted by Nagase (Europa) GmbH. This β-amylase is intended to be used in the starch processing for maltose syrup production and the manufacture of a Japanese rice cake type. Based on the maximum use levels recommended for the respective food processes, dietary exposure to the food enzyme-total organic solids (TOS) was estimated on the basis of Japanese consumption data. Conservative average infant formula consumption, as reported in the EFSA Draft Guidance on risk assessment of substances present in food intended for infants below 16 weeks of age, was used to estimate the exposure to a fraction of soybean comparable to the food enzyme-TOS, resulting from the consumption of soybean-derived foods. The exposure estimate to the food enzyme-TOS was found to be lower than the comparable fraction from the source material. Potential allergenicity of the β-amylase was evaluated by searching for similarity of the amino acid sequence to those of known allergens, and no match was found. The β-amylase is produced from soybean, which is a known allergenic food. Japanese rice cake, consequently, may contain traces of soybean allergens, which may give rise to safety concerns in soybean-allergic consumers. Based on the origin of the food enzyme from edible parts of soybean, the manufacturing process, the compositional and biochemical data provided and the dietary intake estimates, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use, except that Japanese rice cake produced with this food enzyme may contain traces of soybean allergens., (© 2017 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

The food enzyme considered in this opinion is a 4-α-d-glucan maltohydrolase (EC 3.2.1.2) obtained from grain of barley ( Hordeum vulgare ), by the companies Genencor International B.V. and Senson Oy. This β-amylase is intended to be used in several food-manufacturing processes: baking and brewing processes, distilled alcohol production, and starch processing for the production of glucose syrups. The compositional data provided for the food enzyme were considered sufficient. The manufacturing process did not raise safety concerns. Based on the maximum use levels recommended for the respective food processes, dietary exposure to the food enzyme-total organic solids (TOS) was estimated on the basis of individual data from the EFSA Comprehensive European Food Consumption Database. This exposure estimate is similar to or lower than the exposure to a fraction of barley comparable to the food enzyme-TOS, resulting from the consumption of barley-derived foods. As the food enzyme is derived from edible parts of barley, in line with the requirements of the guidance document on food enzyme assessment, the Panel accepted that there was no need for the provision of toxicological data for this food enzyme and the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. Considering the potential for allergenicity, the gluten content of the food enzyme was below the detection limit of the analytical method, which is well below the level of 20 mg/kg for 'gluten-free' products. The amino acid sequence of the β-amylase was compared to those of known allergens and no match was found. The food enzyme β-amylase from barley is an occupational respiratory allergen and may contain low levels of other allergenic barley proteins that may trigger adverse reactions upon oral challenges in individuals with an oral sensitisation to cereals. The Panel considers that dietary exposure to the food enzyme β-amylase from barley may result in incidental cases of food allergic reactions., (© 2017 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.)

Nanoparticle use has increased radically raising concern about possible adverse effects in humans. Zinc oxide nanoparticles (ZnO NPs) are among the most common nanomaterials in consumer and medical products. Several studies indicate problems with their safe use. The aim of our study was to see at which levels ZnO NPs start to produce adverse cytogenetic effects in human lymphocytes as an early attempt toward establishing safety limits for ZnO NP exposure in humans. We assessed the genotoxic effects of low ZnO NP concentrations (1.0, 2.5, 5, and 7.5 μg mL-1) in lymphocyte cultures over 14 days of exposure. We also tested whether low and high-density lymphocytes differed in their ability to accumulate ZnO NPs in these experimental conditions. Primary DNA damage (measured with the alkaline comet assay) increased with nanoparticle concentration in unseparated and high density lymphocytes. The same happened with the fragmentation of TP53 (measured with the comet-FISH). Nanoparticle accumulation was significant only with the two highest concentrations, regardless of lymphocyte density. High-density lymphocytes had significantly more intracellular Zn2+ than light-density ones. Our results suggest that exposure to ZnO NPs in concentrations above 5 μg mL-1 increases cytogenetic damage and intracellular Zn2+ levels in lymphocytes.

This study evaluated the cyto- and genotoxic effects of three pesticides: α-cypermethrin, chlorpyrifos and imidacloprid applied in vitro to human lymphocytes and HepG2 cells for exposure times of 4 and 24 h at concentrations corresponding to OEL, ADI and REL. Assessments were made using oxidative stress biomarkers and the alkaline comet, cytokinesis-block micronucleus cytome and cell viability assays. Low doses of all three pesticides displayed DNA damaging potential, both in lymphocytes and HepG2 cells. At the tested concentrations, all three compounds induced lymphocyte apoptosis, though α-cypermethrin and chlorpyrifos were generally more cyto- and genotoxic than imidacloprid. At the tested concentrations, oxidative stress biomarkers were not significantly altered, and the effects mediated indirectly through free radicals may not have a key role in the formation of DNA damage. It is likely that the DNA damaging effects were caused by direct interactions between the tested compounds and/or their metabolites that destabilized the DNA structure. The tested pesticides had the potential for MN, NB and NPB formation and to disturb cell cycle kinetics in both cell types. There were also indications that exposure to α-cypermethrin led to the formation of crosslinks in DNA, though this would require more detailed study in the future., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tembotrione is a triketone herbicide, usually used for post-emergence weed control in corn. Currently, there is little or no published data on its genotoxicity to human cells either in vitro or in vivo. This study evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione [corresponding to the acceptable daily intake (0.17 μg/mL), residential exposure level (0.002 μg/mL) and acceptable operator exposure level (0.0012 μg/mL)] on human hepatocellular carcinoma cell line HepG2, using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell viability, alkaline comet assay, and cytokinesis-block micronucleus "cytome" assay. Tembotrione applied at concentrations likely to be encountered in occupational and residential exposures induced cytogenetic outcomes in non-target cells despite non-significant changes in the values of oxidative stress biomarkers. We assume that the observed effects were mainly the consequence of impaired metabolic pathways in HepG2 cells due to the inhibition of the enzyme 4-hydroxyphenyl-pyruvate-dioxygenase by tembotrione, which possibly caused a depletion of folate levels leading to excess formation of nuclear buds in the affected cells. Regardless of the fact that tembotrione was previously reported negative for mutations and chromosome aberrations in vitro, our findings call for more precaution in its use., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The aim of our study was to investigate how exposure to heavy metal-rich waters from gypsum mining affects the morphology and levels of primary DNA damage in Gammarus balcanicus. Chemical analysis revealed increased concentrations of metals in water and sediment collected at a site impacted by gypsum mine wastewaters. The specimens also showed elevated total tissue metal levels when compared with the organisms collected at the reference site. The most prominent increase was observed for strontium, followed by iron, nickel, vanadium, aluminium, and manganese. The major pathway of entry for these toxic substances was through the degraded exoskeleton as a consequence of excessive strontium input (unbalanced calcium/strontium ratio) and altered permeability. Disturbed exoskeleton integrity was observed only in individuals collected downstream of the gypsum mine, which was confirmed by electron microscopy. Levels of primary DNA damage were evaluated using the alkaline comet assay in the haemolymph of the specimens., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

This study investigates the effects of oxime K048 (730, 200, and 7.3nM) on the viability and chromosome stability of human peripheral blood lymphocytes (PBLs) after a 30min exposure in vitro. Cytotoxicity was tested by a viability assay with ethidium bromide and acridine orange. For the evaluation of the genotoxic potential, we used comet assays, cytokinesis-blocked micronucleus (CBMN) assay, and chromosome aberration (CA) analysis. We found acceptable cytotoxicity for K048 (9.7±2.1% non-viable PBL at highest concentration vs. 7.3±2.5% in control; apoptosis dominated over necrosis). Overall primary DNA damage was low and not significantly different from controls. The hOGG1-comet assay showed a slight increase in the level of oxidative DNA damage. In oxime treated PBLs, we found 13-19 MN compared to 15 MN in control cultures. The frequencies and types of CA in oxime-treated PBLs did not significantly differ from controls. K048 showed acceptable biocompatibility at the level of cell viability and chromatin/chromosome integrity. Since no increase in secondary genome damage was detected, the primary DNA lesions may have resulted from treatment-induced cell stress, subsequently becoming repaired and not fixed as chromosome aberrations. The toxicity profile of K048 should be further studied and compared with other clinically relevant oximes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A conjugate of pyridine-4-aldoxime and atropine (ATR-4-OX) was synthesized and its antidotal efficiency was tested in vitro on tabun- or paraoxon-inhibited acetylcholinesterase (AChE) of human erythrocytes as well as in vivo using soman-, tabun- or paraoxon-poisoned mice. Its genotoxic profile was assessed on human lymphocytes in vitro and was found acceptable for further research. ATR-4-OX showed very weak antidotal activity, inadequate for soman or tabun poisoning. Conversely, it was effective against paraoxon poisoning both in vitro and in vivo. All animals treated with 5 % or 25 % LD(50) doses of the new oxime survived after administration of 10.0 or 16.0 LD(50) doses of paraoxon, respectively. Based on the persistence of toxicity symptoms in mice, the atropine moiety had questionable effects in attenuating such symptoms. It appears that ATR-4-OX has a therapeutic effect related to the reactivation of phosphylated AChE, but not to receptor antagonization.