According to Organisation for Economic Co-operation and Development (OECD) test guideline no. (TG) 487 for the in vitro mammalian cell micronucleus test (MNT), short (pulse) treatment of primary human lymphocytes should be followed immediately by a sampling period of 1.5 - 2.0 cell cycle lengths in the presence of cytochalasin B. However, TG 487 allows extending the sampling period "if it is known or suspected that the test chemical affects the cell cycling time". It is often unknown in advance whether a test substance induces cell cycle delays, and therefore whether an extension of the sampling period is warranted. Here, we investigate whether extending the sampling time after pulse treatment - by including a recovery period prior to cytochalasin B treatment - affects the sensitivity of the in vitro MNT. Primary human lymphocytes were exposed to eleven mutagens and assessed using two protocols ("recovery" and "no recovery") in parallel from the same treatment culture. Including a recovery period after pulse treatment generally allowed testing of higher concentrations without excessive cytotoxicity. In addition, cultures subjected to a recovery period showed generally higher micronucleus rates at concentrations with acceptable levels of cytotoxicity, compared to cultures not having undergone a recovery period. To compare the sensitivity of both protocols at non-cytotoxic concentrations, benchmark concentration analysis was performed. Here, the "recovery method" proved to be at least as sensitive as the "no recovery method". The reproducibility of these findings was demonstrated in an interlaboratory ring trial using a subset of up to five mutagens. In conclusion, inclusion of a recovery period after pulse treatment per se covers potential substance-induced cell cycle delays without any loss in sensitivity towards the induced mutagenic effect. The "recovery" protocol can be used for assessing the mutagenic potential of test substances affecting and not affecting the cell cycle.

Metabolism-based category formation for the prioritisation of genotoxicity hazard assessment for plant protection product residues (Part 5): Acetyl CoA carboxylase inhibitors.

The organotellurane RF07 (RF07) is an organic compound containing tellurium, a rare semi-metal, and its leishmanicidal and antimalarial activity has already been reported in previous studies. This study evaluated the toxic effects of RF07 at 0.42, 21.37, and 42.75mg/kg using comet assay, micronucleus test, and hematological/biochemical tests in Mus musculus. Female Swiss mice received intraperitoneal RF07. Blood was collected at 24, 48, and 72h, followed by euthanasia for bone marrow analysis. Results showed RF07 increased DNA damage, clastogenic/aneugenic effects via micronuclei formation, and cytotoxicity through apoptosis in bone marrow erythroid precursors and altering the ratio of polychromatic to normochromatic erythrocytes. RF07 also caused myelosuppression and hepatotoxicity. To our knowledge, this is the first study explore these toxic effects. However, the mechanism remains unclear, requiring further investigation.

The edge effect alters microclimatic conditions and increases heavy metal pollution in monoculture landscapes, thereby exposing wildlife to toxic substances. This highlights the need for genotoxicity assessments in fragmented environments. The micronucleus test is a valuable biomarker for detecting DNA damage, though its use in wild mammals is recent. In this pioneering study, we applied the text to exfoliated buccal mucosa cells of Gracilinanus agilis, a South American marsupial, using the micronucleus test. Individuals were captured in a semideciduous forest in southern Goiás, central Brazil. We collected buccal cells from 41 individuals (13 females and 28 males) across 13 sampling sites. We identified six types of nuclear abnormalities: micronuclei, karyolysis, pyknosis, binucleated cells, karyorrhexis, and nuclear buds. Karyolysis, pyknosis, and binucleated cells were most frequent at the forest edge compared to the interior, indicating greater genotoxic stress in edge habitats. Our findings support the use of the micronucleus test as a non-invasive tool for monitoring DNA damage in wild populations and demonstrate that marsupials inhabiting forest edges are more susceptible to genotoxic effects.

We describe the impact of lifestyle related factors on the formation of micronuclei (MN) in buccal cells. It is known that consumption of alcohol and of other drugs including tobacco and other plants cause damage of the genetic material and leads to adverse health effects. Overall, 235 articles were evaluated in total. Tobacco smoking, i.e. consumption of cigarettes (75 publications), water pipes (OECD (2023) In Vitro Mammalian Cell Micronucleus Test. OECD, Paris) and crack smoking (Ravanat, J.L., Cadet, J., and Douki, T. (2012) Oxidatively generated DNA lesions as potential biomarkers of in vivo oxidative stress. Curr Mol Med, 12, 655-671) caused mainly positive effects, while negative results were obtained with pure marijuana (WHO (2025) Preventing cancer. WHO, Geneva). Chewing habits (smokeless tobacco (Wultsch, G., Nersesyan, A., Misik, M., Ferk, F., Schelch, K., Scharnagl, M., Grusch, M., and Knasmuller, S. (2025) The Micronucleus Assay in Exfoliated Buccal Cells for Occupational Exposure Studies: An Overview. Mutagenesis) and betel with and without tobacco (Fenech, M., Holland, N., Zeiger, E., Chang, P.W., Kirsch-Volders, M., Bolognesi, C., Stopper, H., Knudsen, L.E., Knasmueller, S., Nersesyan, A., Thomas, P., Dhillon, V., Deo, P., Franzke, B., Andreassi, M.G., Laffon, B., Wagner, K.H., Norppa, H., da Silva, J., Volpi, E.V., Wilkins, R., and Bonassi, S. (2024) Objectives and achievements of the HUMN project on its 26th anniversary. Mutat Res Rev Mutat Res, 794, 108511) caused induction of MN in cells from the oral cavity (numbers indicate the number of publications). A study concerning khat chewing yielded a positive finding while reduction of MN was seen in consumers of coca leaves. Investigations concerning the effects of alcohol (OECD (2023) In Vitro Mammalian Cell Micronucleus Test. OECD, Paris), meat consumption (Huang, R., and Zhou, P.K. (2021) DNA damage repair: historical perspectives, mechanistic pathways and clinical translation for targeted cancer therapy. Signal Transduct Target Ther, 6, 254) and studies concerning the impact of the body mass index and body fat (Mateuca, R.A., Decordier, I., and Kirsch-Volders, M. (2012) Cytogenetic methods in human biomonitoring: principles and uses. Methods Mol Biol, 817, 305-334) led to inconsistent results. No evidence of MN induction was observed in studies on continuous physical exercise (WHO (2025) Preventing cancer. WHO, Geneva). Furthermore, exhaustive sporting activities also showed no indications of chromosomal damage (WHO (2025) Preventing cancer. WHO, Geneva). A number of studies (in total 22) was realized to investigate the effects of mobile phone specific radiation. The exposure was not adequately assessed in these investigations with questionnaires, furthermore, frequently inadequate stains (DNA-unspecific) were used (OECD (2023) In Vitro Mammalian Cell Micronucleus Test. OECD, Paris). The findings are controversial, and a clear negative result was obtained in a recent controlled intervention study. Overall the results indicate that the MN assay with buccal cells can be used to monitor chromosomal damage caused by smoking and chewing habits and to predict adverse health effects in the respiratory system and on the upper gastrointestinal tract. To draw firm conclusions about the effects of nutrition, mobile phone use, physical exercise, and body weight, further well-controlled trials are needed.

This study aimed to comprehensively assess the edible safety and in vivo bioactivities of recombinant humanized type III collagen (rhCol III). A systematic toxicological evaluation was conducted, including subchronic toxicity tests, bacterial reverse mutation assays, mammalian erythrocyte micronucleus tests, and chromosome aberration tests. Concurrently, efficacy was evaluated using a zebrafish model to investigate multiple biological endpoints. The safety assessment revealed no toxicologically significant abnormalities, confirming a favorable safety profile for long-term oral consumption. In the zebrafish model, rhCol III significantly inhibited the expression of inflammatory factors, reduced the accumulation of advanced glycation end products (AGEs), alleviated oxidative stress damage, and improved molecular indicators associated with skin barrier function. The results demonstrate that rhCol III possesses multifaceted bioactivities, including anti-inflammatory, anti-glycation, antioxidant, and anti-aging properties. This research provides a robust scientific foundation for the development and application of rhCol III in high-value-added oral nutricosmetic products.

Selective serotonin reuptake inhibitors (SSRIs) are first-line therapies for depressive and anxiety disorders and are often used for years, including in reproductive-age patients and during pregnancy. This has intensified concern about possible genotoxicity and DNA damage. Citalopram is emphasized because it has the most extensive and consistently positive invivo genotoxicity evidence among SSRIs, particularly in rodent comet and micronucleus endpoints. This review summarizes genotoxicity data for SSRIs, focusing on citalopram, across invitro assays, animal studies, human biomonitoring, and environmental research. We briefly define mutagenicity and clastogenic and aneugenic effects, describe major DNA lesions, and review commonly applied methods: comet, micronucleus, and chromosomal aberration tests, plus biomarker approaches such as γH2AX and measures of oxidative DNA damage. Potential off-target mechanisms are considered in light of SSRI pharmacology and pharmacokinetics, including direct DNA interactions, mitochondrial dysfunction, reactive oxygen species generation, and disruption of chromosome segregation. Comparative findings indicate that citalopram and fluoxetine show the most consistent experimental genotoxic signals, whereas paroxetine and escitalopram are less frequently implicated. For citalopram, rodent studies report DNA strand breaks, combined clastogenic/aneugenic effects in bone marrow, germ cell DNA damage, and reproductive toxicity, with additional positive findings in non-mammalian models. Human studies are few and provide mixed, largely indirect evidence. Work suggests antioxidants can reduce SSRI-related oxidative stress and DNA damage in some models. Environmental studies raise concern for aquatic organisms. Overall, citalopram shows genuine experimental genotoxic potential, but clinical relevance in humans remains uncertain and warrants robust longitudinal investigation and careful risk-benefit assessment in practice.

Overweight and obesity have been increasing drastically in recent years due to the growing consumption of fast food. Obese individuals exhibit reduced antioxidant defenses, which can lead to DNA damage. Thus, studies have been conducted to mitigate obesity-related complications. Animal research has used the cafeteria diet (CAF) as an obesity induction model, as it mimics human consumption of ultra-processed foods. Currently, complementary dietary strategies are being explored to prevent and/or alleviate obesity-related complications, particularly through natural compounds with anti-obesity effects. Among these, melatonin (MEL) has gained attention due to its antioxidant and anti-inflammatory properties. This study aimed to evaluate the effects of melatonin supplementation on biochemical, genotoxic, and inflammatory parameters in mice fed a CAF diet. A total of 60 male Swiss mice were divided into six experimental groups (n=10): (1) Standard Diet (SD) - fed standard chow for 21 weeks; (2) SD + MEL - fed standard chow and supplemented with melatonin for 24 weeks; (3) CAF - fed CAF for 21 weeks; (4) CAF + MEL - fed CAF and supplemented with melatonin; (5) CAF / CAF + MEL - fed CAF for the first 17 weeks, then continued CAF while starting melatonin supplementation for the last four weeks, totaling 17 weeks of CAF; (6) CAF + MEL / CAF - fed CAF and supplemented with melatonin for the first 17 weeks, then stopped melatonin supplementation for the last four weeks, totaling 21 weeks. Blood samples were collected at 17 and 21 weeks to assess DNA damage, lipid profile (triglycerides, total cholesterol, and HDL), liver function (ALT and AST), inflammatory markers (TNF-α and IL-10), fasting glucose, and insulin tolerance in all six groups. At the end of the experiment, animals were euthanized, and liver, kidney, adipose tissue, and bone marrow were collected for further analyses, including the Comet Assay, Micronucleus Test, oxidative stress evaluation, and Western blot. Results showed that CAF induced an inflammatory state, characterized by increased TNF-α and decreased IL-10, along with alterations in lipid and liver profiles. Additionally, CAF led to DNA damage in multiple tissues and insulin resistance. MEL supplementation for 17 weeks reversed these changes. In the last four weeks of the experiment, CAF was associated with oxidative stress and damage in the liver, kidney, and bone marrow. MEL effectively attenuated these obesity-related alterations, primarily by modulating proteins involved in homologous and non-homologous DNA repair pathways. In conclusion, the findings demonstrate that MEL is a potent antioxidant and may be a promising candidate for reducing biochemical and genetic alterations associated with obesity.

The effluents generated during the process of hair dyeing exhibit a complex composition, comprising chemical compounds with varying toxicity levels. While the adverse impact of hair dyes on human health is acknowledged, there is a notable absence of studies addressing the toxicity associated with effluents produced during these activities. The primary objective of this study was to assess two effluents emanating from beauty salons after brown hair dyeing: one resulting from hair washing with water, shampoo, and conditioner, referred to as the complete effluent (CE), and the other from washing the dyed hair solely with water, excluding surfactants, referred to as the dye effluent (DE). Invitro bioassays were conducted with the human hepatoma cell line (HepG2/C3A). Cytotoxicity was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan Blue tests, while genotoxicity and mutagenicity were assessed by comet assay and cytokinesis-block micronucleus test, respectively. The cells were exposed for 4 h to various dilutions of the two sampled effluents [100.0% (1); 50.0% (2); 25.0% (3); 12.5% (4); 6.25% (5); 3.125% (6)]. Cytotoxicity was induced by CE-1, DE-1, DE-2, and DE-3 dilutions as indicated by both assays, whereas CE-2 dilution exhibited cytotoxicity solely through the MTT assay. These findings suggest impaired cell membrane integrity, permeability, and mitochondrial activity. Nontoxic dilutions (4, 5, and 6) were viable for the comet assay and micronucleus test, revealing genotoxicity without mutagenic potential. Consequently, residual concentrations of effluents were found to induce nonlethal and reparable primary DNA damage. Moreover, the effluents decreased the cytokinesis-block proliferation index and the cell replication index, indicating interference and arrestment in the cell cycle. These outcomes highlight the potential threat posed by residual concentrations of hair DEs to environmental quality and human health, emphasizing the imperative need for pre-disposal treatments in salon settings.

Both everyday electrical devices and high voltage transmission lines produce electric and magnetic fields with a frequency of 50 Hz in Europe and most other countries. Although several studies have already been investigating the effects of extremely low frequency magnetic field (ELF-MF) exposure on biological material, this topic is still debated. High-quality research is still needed to keep the available data up to date and to decrease the low-quality study design bias. We investigated the effects of 50 Hz magnetic fields with an intensity of 200 µT (rms) on HaCaT cells, an immortal keratinocyte cell line derived from adult human skin cells. The exposure system allowed standard in vitro cultivation and blinded experimental design with simultaneous exposure and sham-exposure for 2 or 24 h. The biological endpoints were measured using the WST-1 assay (cell viability), the alkaline comet assay (DNA integrity), and the micronucleus test (chromosomal distribution). The results show no significant effects of the parameters mentioned. Our data support the assessment that 50 Hz ELF-MF up to 200 µT do not cause health effects. This study contributes valuable knowledge to the existing pool of evidence for the effects of ELF-MF on human cells.

Combined assessment of transgenic rodent gene mutation and micronuclei formation by benzo[a]pyrene, N-ethyl-N-nitrosourea, 2,4-diaminotoluene and ethyl carbamate.

Environmental contamination by hazardous metals poses significant risks to aquatic ecosystems and human health. Heavy metals such as cadmium (Cd), mercury (Hg), and lead (Pb) induce oxidative stress (OS) through the excessive generation of reactive oxygen species (ROS), resulting in behavioural, biochemical, genetic, and histological damage. To evaluate the protective efficacy of ascorbic acid (AA) against acute Cd, Hg, and Pb toxicity in Heteropneustes fossilis using an integrated behavioural, biochemical, oxidative stress, and genotoxicity assessment framework, focusing on its antioxidant properties and ability to scavenge free radicals, we used an integrated behavioural, biochemical, oxidative stress, and genotoxicity assessment framework. Adult H. fossilis were randomly allocated into treatment groups (n = 10/group) and exposed for 96 h to Cd (50.4 mg/L), Hg (0.70 mg/L), Pb (280.04 mg/L), AA (100 mg/L), or metal + AA combinations. Blinded observers recorded behavioural and histological outcomes. Biochemical and oxidative stress markers (SOD, CAT, GSH, MDA, AChE) were quantified using validated assays. Data were tested for normality (Shapiro-Wilk), outliers, and homogeneity of variance, followed by a one-way ANOVA with post hoc multiple comparisons (Tukey). Results are reported as mean ± SEM. AChE activity showed a significant increase in Cd- and Hg-exposed groups (p < 0.05), confirming neurotoxicity, whereas AA co-treatment restored values toward control levels, likely through its free radical scavenging activity. Antioxidant enzymes (SOD, CAT, and GSH) were significantly reduced across the Cd, Hg, and Pb groups (p < 0.01), while MDA levels were elevated, indicating lipid peroxidation. No significant differences were observed between the control, AA alone, and HMs + AA groups for most biochemical markers, indicating that AA plays a protective role by mitigating oxidative damage. Micronucleus test results showed a marked increase in micronuclei frequency in all HMs groups (p < 0.001), with a significant reduction after AA co-exposure, suggesting genoprotective effects. Histopathological alterations in gills, liver, kidney, and brain were pronounced in metal-exposed fish but minimal when co-treated with AA. Ascorbic acid significantly mitigates heavy metal-induced neurotoxicity, oxidative stress, genotoxicity, and tissue damage in H. fossilis. These findings support AA as a potent bioactive antioxidant capable of reducing acute heavy metal toxicity in fish, with potential relevance for other aquatic species.

Subchronic oral toxicity and genotoxicity of Aurantii Fructus Immaturus water extract.

Safety evaluation of honey truffle sweet protein produced from Komagataellaphaffii.

Withania somnifera, commonly known as Ashwagandha, is a widely recognized medicinal plant in India, belonging to the family Solanaceae, used in Ayurveda due to its diverse therapeutic properties. The roots of Ashwagandha are considered the most active part of the plant, for its biological and pharmacological effects. However, very little scientific evidence regarding its safety assessment has been published. Thus, the objective of the present study was to assess the safety of the standardized extract of Ashwagandha, known as Shagandha, which is prepared from the roots of Ashwagandha containing 2.5% Withanolides, analysed using a USP method (HPLC). The GLP studies for acute, subacute, subchronic, reproductive, bacterial reverse mutation assay, and mammalian erythrocyte micronucleus test were conducted following the test guidelines established by the Organization for Economic Cooperation and Development (OECD). Treatment with Shagandha (Ashwagandha Root Extract-ARE) did not result in any toxicologically significant changes regarding abnormal clinical signs or behavioral changes, body weight, reproductive and developmental parameters, or gross and histopathological changes. Additionally, the results of genotoxicity as evaluated by the invitro reverse mutation assay and invivo micronucleus test in mice demonstrated that ARE did not induce any genotoxic effects. These findings indicate that the oral administration of ARE is safe in rodents, non-mutagenic, with no adverse effects under experimental conditions.

The potential carcinogenic and genotoxic effects of radiofrequency electromagnetic fields, particularly those emitted by mobile communication systems, have raised public health concerns. A previous study by the U.S. National Toxicology Program suggested increased incidences of gliomas and cardiac schwannomas in rats exposed to high levels of RF radiation. To evaluate these findings, an international collaborative study was initiated between Japan and Korea. Male Hsd:Sprague Dawley SD rats were exposed to 900 MHz CDMA-modulated RF-EMFs at a whole-body specific absorption rate of 4 W/kg for 18 h and 20 min daily over 2 yr. The study included a 28-d preliminary toxicity study, genotoxicity assays (alkaline comet and micronucleus tests), and a 2-yr carcinogenicity assessment. All procedures followed OECD guidelines and Good Laboratory Practice. No statistically significant increases in the incidences of neoplastic or non-neoplastic lesions were found in any major organ, including the brain, heart, and adrenal glands. Genotoxicity assays revealed no evidence of DNA damage or chromosomal aberrations in RF-exposed rats. A higher survival rate in the RF-exposed group, likely due to lower body weight and food consumption, was observed. This study, performed in Japan, jointly planned and executed by Japan and Korea, provides strong evidence that long-term exposure to 900 MHz RF-EMFs did not produce reproducible carcinogenic or genotoxic effects in male rats. Combined with data from the Korean counterpart study, these results are expected to contribute to future international assessments of the carcinogenic potential of electromagnetic radiation.

Image-based morphological characterization of the erythrocytic shape of green turtle (Chelonia mydas) for its potential use as an environmental biomarker.

Suitability of the use of the intraperitoneal route in the in vivo micronucleus test to evaluate the genotoxicity of hair dyes.

How many mutagens are missed under REACH due to limited low tonnage information requirements?

Dynamic subcellular localization of HMGB1 in colorectal cancer cells following exposure to environmental mutagens.