Assessment of acute toxicity, genotoxicity, and anti-inflammatory activity of SteLL, a lectin from Schinus terebinthifolia Raddi. Leaves, in mice.

ETHNOPHARMACOLOGY RELEVANCE: Schinus terebinthifolia Raddi (Anacardiaceae), known as Brazilian pepper tree, stands out as a medicinal plant widely used in traditional medicine. The leaves are popularly used as anti-inflammatory agent and to relieve inflammatory conditions such as bronchitis, ulcers, and wounds, for example. AIM OF THE STUDY: The present study evaluated the acute toxicity, genotoxicity, and anti-inflammatory activity of S. terebinthifolia leaf lectin (SteLL) in mice (Mus musculus). MATERIALS AND METHODS: In the acute toxicity assay, the animals were treated intraperitoneally (i.p.) or orally (per os) with a single dose of 100 mg/kg. Genotoxicity was assessed by the comet and micronucleus assays. Carrageenan-induced peritonitis and paw edema models were used to evaluate the anti-inflammatory effects of SteLL (1, 5 and 10 mg/kg, i.p.). RESULTS: No animal died and no signs of intoxication or histopathological damage were observed in the acute toxicity assay. Genotoxic effect was not detected. In peritonitis assay, SteLL reduced in 56-69% leukocyte migration to the peritoneal cavity; neutrophil count decreased by 25-32%, while mononuclear cell count increased by 67-74%. SteLL promoted a notable reduction of paw edema after 4 h (61.1-63.4%). Morphometric analysis showed that SteLL also decreased the thickness of epidermal edema (30.2-40.7%). Furthermore, SteLL decreased MPO activity, plasma leakage, NO release, and modulated cytokines in both peritoneal fluid and paw homogenate. CONCLUSION: SteLL did not induce acute toxicity or genotoxicity in mice and stands out as a promising candidate in the development of new phytopharmaceuticals with anti-inflammatory action.

The use of effect biomarkers in chemical mixtures risk assessment - Are they still important?

Human epidemiological studies with biomarkers of effect play an invaluable role in identifying health effects with chemical exposures and in disease prevention. Effect biomarkers that measure genetic damage are potent tools to address the carcinogenic and/or mutagenic potential of chemical exposures, increasing confidence in regulatory risk assessment decision-making processes. The micronucleus (MN) test is recognized as one of the most successful and reliable assays to assess genotoxic events, which are associated with exposures that may cause cancer. To move towards the next generation risk assessment is crucial to establish bridges between standard approaches, new approach methodologies (NAMs) and tools for increase the mechanistically-based biological plausibility in human studies, such as the adverse outcome pathways (AOPs) framework. This paper aims to highlight the still active role of MN as biomarker of effect in the evolution and applicability of new methods and approaches in human risk assessment, with the positive consequence, that the new methods provide a deeper knowledge of the mechanistically-based biology of these endpoints.

Assessment of genotoxicity of air pollution in urban areas using an integrated model of passive biomonitoring.

Atmospheric pollution is a major public health issue and has become increasingly critical for human health. Urban atmospheric pollution is typically assessed through physicochemical indicators aligned with environmental legislation parameters, providing data on air quality levels. While the effects of pollution on sensitive organisms serve as a warning for public health decision-makers, there remains a need to explore the interpretation of environmental data on pollutants. The use of species adapted to urban environments as sentinels enables continuous and integrated monitoring of environmental pollution implications on biological systems. In this study, we investigated the use of the plant species Tradescantia pallida as a biomonitor to evaluate the genotoxic effects of atmospheric pollution under diverse vehicular traffic conditions. T. pallida was strategically planted at the leading urban intersections in Uberlândia, Brazil. During COVID-19 pandemic lockdowns, we compared indicators such as physical, biological, and traffic data at different intersections in residential and commercial zones. The reduction in vehicular traffic highlighted the sensitivity of plant species to changes in air and soil pollutants. T. pallida showed bioaccumulation of heavy metals Cd and Cr in monitored areas with higher traffic levels. Additionally, we established a multiple linear regression model to estimate genotoxicity using the micronucleus test, with chromium concentration in the soil (X1) and particulate matter (PM) in the atmosphere (X2) identified as the primary independent variables. Our findings provide a comprehensive portrait of the impact of vehicular traffic changes on PM and offer valuable insights for refining parameters and models of Environmental Health Surveillance.

Assessment of genotoxicity biomarkers in gasoline station attendants due to occupational exposure.

Gasoline station attendants are exposed to numerous chemicals that might have genotoxic and carcinogenic potential, such as benzene in fuel vapor and particulate matter and polycyclic aromatic hydrocarbons in vehicle exhaust emission. According to IARC, benzene and diesel particulates are Group 1 human carcinogens, and gasoline has been classified as Group 2A "possibly carcinogenic to humans." At gas stations, self-service is not implemented in Turkey; fuel-filling service is provided entirely by employees, and therefore they are exposed to those chemicals in the workplace during all working hours. Genetic monitoring of workers with occupational exposure to possible genotoxic agents allows early detection of cancer. We aimed to investigate the genotoxic damage due to exposures in gasoline station attendants in Turkey. Genotoxicity was evaluated by the Comet, chromosomal aberration, and cytokinesis-block micronucleus assays in peripheral blood lymphocytes. Gasoline station attendants (n = 53) had higher tail length, tail intensity, and tail moment values than controls (n = 61). In gasoline station attendants (n = 46), the frequencies of chromatid gaps, chromosome gaps, and total aberrations were higher compared with controls (n = 59). Increased frequencies of micronuclei and nucleoplasmic bridges were determined in gasoline station attendants (n = 47) compared with controls (n = 40). Factors such as age, duration of working, and smoking did not have any significant impact on genotoxic endpoints. Only exposure increased genotoxic damage in gasoline station attendants independently from demographic and clinical characteristics. Occupational exposure-related genotoxicity risk may increase in gasoline station attendants who are chronically exposed to gasoline and various chemicals in vehicle exhaust emissions.

Subjective biosafety assessment of bisdemethoxycurcumin from the rhizomes of Curcuma longa.

Introduction/Background: Curcuma longa, a plant native to the Indian subcontinent has a variety of biological activities. Curcumin is the most abundant and biologically active compound with many therapeutic properties. Demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) - the two other bioactive components present in Curcuma longa, besides curcumin, are collectively termed curcuminoids. Apart from the well-known curcumin, BDMC also has been reported to possess promising biological and pharmacological effects, but very little scientific evidence on its safety assessment has been published.Objective: The present study was undertaken to determine the safety of pure BDMC from Curcuma longa extract in rodents which comprises of general toxicity (both four weeks and three months duration), reproductive/developmental toxicity and genotoxicity studies.Methods: The Good Laboratory Practice studies were carried out in accordance with the test guidelines established by the Organization for Economic Cooperation and Development.Results: No treatment-related adverse findings were seen in general toxicity testing and a no observed adverse effect level (NOAEL) of 1000 mg/kg/day was established after four weeks (sub-acute) and three-months (sub-chronic) dosing. Evaluation of fertility, embryo-fetal, and post-natal reproductive and developmental parameters also showed no adverse findings with a NOAEL of 1000 mg/kg/day established. The results of genotoxicity as evaluated by in vitro reverse mutation assay, and in vivo micronucleus test in mice indicate that BDMC did not induce any genotoxic effects.Conclusion: Oral administration of BDMC is safe in rodents and non-mutagenic, with no adverse effects under experimental conditions.

Adapting the in vitro micronucleus assay (OECD Test Guideline No. 487) for testing of manufactured nanomaterials: recommendations for best practices.

The current Organisation for Economic Co-Operation and Development test guideline number 487 (OECD TG No. 487) provides instruction on how to conduct the in vitro micronucleus assay. This assay is one of the gold standard approaches for measuring the mutagenicity of test items; however, it is directed at testing low molecular weight molecules and may not be appropriate for particulate materials (e.g. engineered nanoparticles [ENPs]). This study aimed to adapt the in vitro micronucleus assay for ENP testing and underpins the development of an OECD guidance document. A harmonized, nano-specific protocol was generated and evaluated by two independent laboratories. Cell lines utilized were human lymphoblastoid (TK6) cells, human liver hepatocytes (HepG2) cells, Chinese hamster lung fibroblast (V79) cells, whole blood, and buffy coat cells from healthy human volunteers. These cells were exposed to reference ENPs from the Joint Research Council (JRC): SiO2 (RLS-0102), Au5nm and Au30nm (RLS-03, RLS-010), CeO2 (NM212), and BaSO4 (NM220). Tungsten carbide-cobalt (WC/Co) was used as a trial particulate positive control. The chemical controls were positive in all cell cultures, but WC/Co was only positive in TK6 and buffy coat cells. In TK6 cells, mutagenicity was observed for SiO2- and both Au types. In HepG2 cells, Au5nm and SiO2 showed sub-two-fold increases in micronuclei. In V79 cells, whole blood, and buffy coat cells, no genotoxicity was detected with the test materials. The data confirmed that ENPs could be tested with the harmonized protocol, additionally, concordant data were observed across the two laboratories with V79 cells. WC/Co may be a suitable particulate positive control in the in vitro micronucleus assay when using TK6 and buffy coat cells. Detailed recommendations are therefore provided to adapt OECD TG No. 487 for testing ENP.

Safety assessment of high fructose corn syrup and fructose used as sweeteners in foods.

High Fructose Corn Syrup (HFCS) and Fructose (FR) are widely used sweeteners in many foods and beverages. This study aimed at investigating the cytotoxic effects of HFCS (5%-30%) and FR (62.5-2000 µg/mL) using MTT assay in Human Hepatocellular Carcinoma (HepG2) cells, and genotoxic effects of using Chromosome Aberrations (CAs), Sister Chromatid Exchanges (SCEs), Micronuclei (MN) and comet assays in human lymphocytes. HFCS significantly reduced the cell viability in HepG2 cells at between 7.5% and 30% for 24 and 48 h. 30% HFCS caused a very significant toxic effect. FR had a cytotoxic effect in HepG2 cells at all treatments. However, as fructose concentration decreased, the cell viability decreased. HFCS (10%-20%) and FR (250-2000 µg/mL) decreased the mitotic index at higher concentrations. IC50 value was found to be a 15% for 48 h. IC50 value of FR was detected as 62.5 µg/mL for 24 h and 48 h. HFCS significantly increased CAs frequency at 15% and 20%. FR significantly increased the frequency of CAs at 250, 1000, and 2000 µg/mL for 48 h. Both sweeteners increased the frequency of SCEs at all concentrations. HFCS (15% and 20%) and FR (250, 1000, and 2000 µg/mL) induced MN frequency at higher concentrations. HFCS caused DNA damage in comet assay at 10% -30%. FR increased tail intensity and moment at 125-2000 µg/mL and tail length at 62.5, 250 and 500 µg/mL. Therefore, HFCS and FR are clearly seen to be cytotoxic and genotoxic, especially at higher concentrations.

HFCS and FR exhibited cytotoxic effect at HepG2 and human lymphocytes at higher concentrations.Both sweeteners increased the frequencies of CAs and SCEs at higher concentrations.HFCS caused DNA damage at 10% -30% concentrations.HFCS (15% and 20%) and FR (250, 1000, and 2000 µg/mL) induced MN frequency.

Assessment of the three-test genetic toxicology battery for groundwater metabolites.

The two-test in vitro battery for genotoxicity testing (Ames and micronucleus) has in the majority of cases replaced the three-test battery (as two-test plus mammalian cell gene mutation assay) for the routine testing of chemicals, pharmaceuticals, cosmetics, and agrochemical metabolites originating from food and feed as well as from water treatment. The guidance for testing agrochemical groundwater metabolites, however, still relies on the three-test battery. Data collated in this study from 18 plant protection and related materials highlights the disparity between the often negative Ames and in vitro chromosome aberration data and frequently positive in vitro mammalian cell gene mutation assays. Sixteen of the 18 collated materials with complete datasets were Ames negative, and overall had negative outcomes in in vitro chromosome damage tests (weight of evidence from multiple tests). Mammalian cell gene mutation assays (HPRT and/or mouse lymphoma assay (MLA)) were positive in at least one test for every material with this data. Where both MLA and HPRT tests were performed on the same material, the HPRT seemed to give fewer positive responses. In vivo follow-up tests included combinations of comet assays, unscheduled DNA synthesis, and transgenic rodent gene mutation assays, all gave negative outcomes. The inclusion of mammalian cell gene mutation assays in a three-test battery for groundwater metabolites is therefore not justified and leads to unnecessary in vivo follow-up testing.

Assessment of buccal mucosa genotoxicity in insecticide-exposed urban fumigators in Cali, Colombia.

OBJECTIVES: This study aimed to evaluate cytogenetic damage in the buccal mucosa of non-exposed subjects (N = 33) and insecticide-exposed fumigators (N = 31) in the urban area of Cali, Colombia. MATERIAL AND METHODS: Through a questionnaire sociodemographic data, anthropometric measurements, state of health, and lifestyle were collected. Buccal micronucleus cytome (BMCyt) assay was using for evaluate cytogenetic damage. RESULTS: The study showed that all fumigators used adequate personal protective equipment (PPE) and had low alcohol consumption. The authors did not find significant differences in BMCyt biomarkers between the groups (p > 0.05). Multivariate analysis showed a 13% increase in micronucleus (MN) frequency for every year of increasing age (OR = 1.13, p = 0.029), and higher MN with the decrease in daily fruit consumption (OR = 4.71, p = 0.084), without statistical significance. CONCLUSIONS: The results between groups could be related to healthy habits and PPE use among the subjects. Int J Occup Med Environ Health. 2024;37(1):128-37.

A toxicological assessment of spermidine trihydrochloride produced using an engineered strain of Saccharomyces cerevisiae.

Spermidine is a polyamine consumed in the diet, endogenously biosynthesized in most cells, and produced by the intestinal microbiome. A variety of foods contribute to intake of spermidine along with other polyamines. Spermidine trihydrochloride (spermidine-3HCl) of high purity can be produced using an engineered strain of Saccharomyces cerevisiae. Spermidine has a demonstrated history of safe use in the diet; however, limited information is available in the public literature to assess the potential toxicity of spermidine-3HCl. To support a safety assessment for this spermidine-3HCl as a dietary source of spermidine, authoritative guideline and good laboratory practice (GLP) compliant in vitro genotoxicity assays (bacterial reverse mutation and mammalian micronucleus assays) and a 90-day oral (dietary) toxicity study in rats were conducted with spermidine-3HCl. Spermidine-3HCl was non-genotoxic in the in vitro assays, and no adverse effects were reported in the 90-day oral toxicity study up to the highest dose tested, 12500 ppm, equivalent to 728 mg/kg bw/day for males and 829 mg/kg bw/day for females. The subchronic no observed adverse effect level (NOAEL) is 728 mg/kg bw/day.

Assessment of imidacloprid induced genotoxicity in Pethia conchonius (Rosy barb), a common freshwater fish of India.

Imidacloprid is one of the highly efficient, globally used neonicotinoid groups of insecticides. The indiscriminate use of imidacloprid is contaminating large water bodies affecting not only the target organisms but also non-target organisms including fish. The present study aimed to assess the extent of nuclear DNA damage by imidacloprid in Pethia conchonius a freshwater fish in India using comet and micronucleus assays. The LC50 value of imidacloprid was estimated to be 227.33 mg L-1. Based on the LC50-96 h value, three sub-lethal concentrations of imidacloprid, SLC I -18.94 mg L-1, SLC II -28.41 mg L-1 and SLC III -56.83 mg L-1 were used to detect its genotoxic effect at DNA and cellular level. The imidacloprid exposed fishes exhibited higher DNA damage and nuclear abnormalities (p < 0.05) than the control. The %head DNA, %tail DNA, tail length and the frequency of micronuclei with other nuclear abnormalities like blebbed and notched nuclei were significantly higher than the control in a time and concentration-dependent manner. The DNA damage parameters such as %head DNA (29.107 ± 1.843), %tail DNA (70.893 ± 1.843), tail length (361.431 ± 8.455) micronucleus (1.300 ± 0.019), notched (0.844 ± 0.011) and blebbed (0.811 ± 0.011) nuclei were found to be highest for SLC III (56.83 mg L-1) at 96 h. The findings indicate that IMI is highly genotoxic in fish and other vertebrates leading to mutagenic/clastogenic effects. The study will be helpful in optimization of the imidacloprid use.

A comparative study on chromium-induced micronuclei assessment in the peripheral blood of Hsd:ICR mice.

This study investigated the genotoxic effects of chromium (Cr) in Hsd:ICR mice, considering factors such as oxidative state, apoptosis, exposure pathway, duration, pregnancy, and transplacental exposure. Genotoxicity was assessed using the erythrocytes' micronucleus (MN) assay, while apoptosis was evaluated in nucleated blood cells. The results showed that Cr(III) (CrK(SO4 )2 and CrCl3 ) did not induce any marked genotoxic damage. However, Cr(VI) (CrO3 , K2 Cr2 O7 , Na2 Cr2 O7 , and K2 CrO4 ) produced varying degrees of genotoxicity, with CrO3 being the most potent. MN frequencies increased following 24-h exposure, with a greater effect in male mice. Administering 20 mg/kg of CrO3 via gavage did not lead to significant effects compared to intraperitoneal administration. Short-term oral treatment with a daily dose of 8.5 mg/kg for 49 days elevated MN levels only on day 14 after treatment. Pregnant female mice exposed to CrO3 on day 15 of pregnancy exhibited reduced genotoxic effects compared to nonpregnant animals. However, significant increases in MN levels were found in their fetuses starting 48 h after exposure. In summary, data indicate the potential genotoxic effects of Cr, with Cr(VI) forms inducing higher genotoxicity than Cr(III). These findings indicate that gender, exposure route, and pregnancy status might influence genotoxic responses to Cr.

Assessment of the genotoxic and mutagenic effects induced by T-2 mycotoxin in HepG2 cells.

The T-2 toxin is a mycotoxin produced by molds belonging to Fusarium. Among the Fusarium mycotoxins, trichothecenes are frequently reported in food and feed, being the T-2 toxin (T-2) the mycotoxin which possesses the highest toxicity. According to EFSA, T-2 is found in various cereal grains used in food and feed products, mainly in oats, and it has a high environmental impact due to its mechanisms of toxicity. However, recent information on its genotoxic and mutagenic effects is lacking. This work aimed to evaluate the genotoxic and mutagenic potential of T-2 in vitro. For this purpose, HepG2 cells were exposed to 15, 30, and 60 nM T-2 for 24 h, then the DNA damage was evaluated by the micronucleus and the comet assays. In addition, point mutation analysis was performed by the bacterial reverse mutation test using 0.15-60 nM of T-2 concentrations. The results showed chromosomal damage at 60 nM T-2 since significantly more MN appeared at this concentration than in the control samples. Regarding the comet assay, DNA double helix breaks appeared at all concentrations tested and, in a concentration-dependent manner. However, no mutagenic effects were observed at any of the concentrations tested for the Salmonella typhimurium (S. Typhimurium) strains TA98, TA100, TA1535, TA1537, or the Escherichia coli (E. Coli) WP2 strain in the absence or presence of a metabolic activation system. Therefore, these results showed that T-2 mycotoxin produced genotoxic effects by MN and comet assay, while no mutagenicity was observed. However, further research simulating different metabolic activation pathways and the combined exposure of this mycotoxin with other mutagenic chemicals that could be present in the diet is necessary to discard the mutagenic potential of T-2 fully. These results highlight the carcinogenic potential and danger associated with T-2 exposure and should be considered to prevent associated food risks for the human population.

Genotoxicity assessment of 1,4-anhydro-4-seleno-D-talitol (SeTal) in human liver HepG2 and HepaRG cells.

1,4-Anhydro-4-seleno-D-talitol (SeTal) is a highly water-soluble selenosugar with interesting antioxidant and skin-tissue-repair properties; it is highly stable in simulated gastric and gastrointestinal fluids and is a potential pharmaceutical ingredient that may be administered orally. Hepatic toxicity is often a major problem with novel drugs and can result in drug withdrawal from the market. Predicting hepatotoxicity is therefore essential to minimize late failure in the drug-discovery process. Herein, we report in vitro studies to evaluate the cytotoxic and genotoxic potential of SeTal in HepG2 and hepatocyte-like differentiated HepaRG cells. Except for extremely high concentrations (10 mM, 68 h-treatment in HepG2), SeTal did not affect the viability of each cell type. While the highest examined concentrations (0.75 and 1 mM in HepG2; 1 mM in HepaRG) were observed to induce primary DNA damage, SeTal did not exhibit clastogenic or aneugenic activity toward either HepG2 or HepaRG cells. Moreover, no significant cytostasis variations were observed in any experiment. The clearly negative results observed in the CBMN test suggest that SeTal might be used as a potential active pharmaceutical ingredient. The present study will be useful for the selection of non-toxic concentrations of SeTal in future investigations.

Toxicological assessment of minoxidil: A drug with therapeutic potential besides alopecia.

Minoxidil is regularly prescribed for alopecia, and its therapeutic potential has expanded in recent times. However, few studies have been conducted to evaluate its toxicity, and controversial findings regarding its mutagenic activities remain unsolved. This study aimed to access cytotoxic, genotoxic, and mutagenic properties of minoxidil using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, comet assay, and micronucleus test in mouse fibroblast (L929) cells and its point mutation induction potential in the Salmonella/microsome assay. Furthermore, an in vivo toxicity assessment was conducted in Caenorhabditis elegans. Minoxidil showed cytotoxicity at 2.0 mg/mL in MTT assay. Genotoxicity was observed after 3 h treatment in L929 cells using comet assay. No mutagenic effect was observed in both the micronucleus test and the Salmonella/microsome assay. The lethal dose 50 in C. elegans was determined to be 1.75 mg/mL, and a delay in body development was detected at all concentrations. In conclusion, minoxidil induces DNA damage only in early treatment, implying that this DNA damage may be repairable. This observation corroborates the absence of mutagenic activities observed in L929 cells and Salmonella typhimurium strains. However, the toxicity of minoxidil was evident in both C. elegans and L929 cells, underscoring the need for caution in its use.

Assessment of Micronuclei Frequency in the Peripheral Blood of Adult and Pediatric Patients Receiving Fractionated Total Body Irradiation.

The cytokinesis-block micronucleus (CBMN) assay is an established method for assessing chromosome damage in human peripheral blood lymphocytes resulting from exposure to genotoxic agents such as ionizing radiation. The objective of this study was to measure cytogenetic DNA damage and hematology parameters in vivo based on MN frequency in peripheral blood lymphocytes (PBLs) from adult and pediatric leukemia patients undergoing hematopoietic stem cell transplantation preceded by total body irradiation (TBI) as part of the conditioning regimen. CBMN assay cultures were prepared from fresh blood samples collected before and at 4 and 24 h after the start of TBI, corresponding to doses of 1.25 Gy and 3.75 Gy, respectively. For both age groups, there was a significant increase in MN yields with increasing dose (p < 0.05) and dose-dependent decrease in the nuclear division index (NDI; p < 0.0001). In the pre-radiotherapy samples, there was a significantly higher NDI measured in the pediatric cohort compared to the adult due to an increase in the percentage of tri- and quadri-nucleated cells scored. Complete blood counts with differential recorded before and after TBI at the 24-h time point showed a rapid increase in neutrophil (p = 0.0001) and decrease in lymphocyte (p = 0.0006) counts, resulting in a highly elevated neutrophil-to-lymphocyte ratio (NLR) of 14.45 ± 1.85 after 3.75 Gy TBI (pre-exposure = 4.62 ± 0.49), indicating a strong systemic inflammatory response. Correlation of the hematological cell subset counts with cytogenetic damage, indicated that only the lymphocyte subset survival fraction (after TBI compared with before TBI) showed a negative correlation with increasing MN frequency from 0 to 1.25 Gy (r = -0.931; p = 0.007). Further, the data presented here indicate that the combination of CBMN assay endpoints (MN frequency and NDI values) and hematology parameters could be used to assess cytogenetic damage and early hematopoietic injury in the peripheral blood of leukemia patients, 24 h after TBI exposure.

Genotoxicity assessment of titanium dioxide nanoparticles using a standard battery of in vivo assays.

As one representative of nanometal oxides, titanium dioxide nanoparticles (TiO2-NPs) have been widely used, particularly in the food industry. The genotoxicity of TiO2-NPs has attracted great attention over the years. This study was undertaken to investigate the chromosome and DNA damage effects of TiO2-NPs (0, 50, 150, and 500 mg/kg BW) using rodent models. After a comprehensive characterization, we conducted a standard battery of in vivo genotoxicity tests, including the chromosomal aberration test (CA), micronucleus (MN) test, and the comet test. The results of all these tests were negative. There were no structural or numerical chromosomal abnormalities in mice bone marrow cells, no increase in the frequency of micronucleated polychromatic erythrocytes in mice bone marrow cells, and no elevation in % tail DNA in rat hepatocytes. This indicated that TiO2-NPs did not cause chromosomal damage or have a direct impact on DNA. These findings suggested that TiO2-NPs did not exhibit genotoxicity and provided valuable data for risk assessment purposes.

Genotoxicity assessment in HepaRG™ cells as a new approach methodology follow up to a positive response in the human TK6 cell micronucleus assay: Naphthalene case study.

We are evaluating the use of metabolically competent HepaRG™ cells combined with CometChip® for DNA damage and the micronucleus (MN) assay as a New Approach Methodology (NAM) alternative to animals for follow up genotoxicity assessment to in vitro positive genotoxic response. Naphthalene is genotoxic in human TK6 cells inducing a nonlinear dose-response for the induction of micronuclei in the presence of rat liver S9. of naphthalene. In HepaRG™ cells, naphthalene genotoxicity was assessed using either 6 (CometChip™) or 12 concentrations of naphthalene (MN assay) with the top dose used for assessment of genotoxicity for the Comet and MN assay was 1.25 and 1.74 mM respectively, corresponding to approximately 45% cell survival. In contrast to human TK6 cell with S9, naphthalene was not genotoxic in either the HepaRG™ MN assay or the Comet assay using CometChip®. The lack of genotoxicity in both the MN and comet assays in HepaRG™ cells is likely due to Phase II enzymes removing phenols preventing further bioactivation to quinones and efficient detoxication of naphthalene quinones or epoxides by glutathione conjugation. In contrast to CYP450 mediated metabolism, these Phase II enzymes are inactive in rat liver S9 due to lack of appropriate cofactors causing a positive genotoxic response. Rat liver S9-derived BMD10 over-predicts naphthalene genotoxicity when compared to the negative genotoxic response observed in HepaRG™ cells. Metabolically competent hepatocyte models like HepaRG™ cells should be considered as human-relevant NAMs for use genotoxicity assessments to reduce reliance on rodents.

Replacing animal-derived components in in vitro test guidelines OECD 455 and 487.

The evaluation of single substances or environmental samples for their genotoxic or estrogenic potential is highly relevant for human- and environment-related risk assessment. To examine the effects on a mechanism-specific level, standardized cell-based in vitro methods are widely applied. However, these methods include animal-derived components like fetal bovine serum (FBS) or rat-derived liver homogenate fractions (S9-mixes), which are a source of variability, reduced assay reproducibility and ethical concerns. In our study, we evaluated the adaptation of the cell-based in vitro OECD test guidelines TG 487 (assessment of genotoxicity) and TG 455 (detection of estrogenic activity) to an animal-component-free methodology. Firstly, the human cell lines A549 (for OECD TG 487), ERα-CALUX® and GeneBLAzer™ ERα-UAS-bla GripTite™ (for OECD TG 455) were investigated for growth in a chemically defined medium without the addition of FBS. Secondly, the biotechnological S9-mix ewoS9R was implemented in comparison to the induced rat liver S9 to simulate in vivo metabolism capacities in both OECD test guidelines. As a model compound, Benzo[a]pyrene was used due to its increased genotoxicity and endocrine activity after metabolization. The metabolization of Benzo[a]Pyrene by S9-mixes was examined via chemical analysis. All cell lines (A549, ERα-CALUX® and GeneBLAzer™ Erα-UAS-bla GripTite™) were successfully cultivated in chemically defined media without FBS. The micronucleus assay could not be conducted in chemically defined medium due to formation of cell clusters. The methods for endocrine activity assessment could be conducted in chemically defined media or reduced FBS content, but with decreased assay sensitivity. The biotechnological ewoS9R showed potential to replace rat liver S9 in the micronucleus in FBS-medium with A549 cells and in the ERα-CALUX® assay in FBS- and chemically defined medium. Our study showed promising steps towards an animal-component free toxicity testing. After further improvements, the new methodology could lead to more reproducible and reliable results for risk assessment.

Safety assessment of a solid lipid curcumin particle preparation: In vitro and in vivo genotoxicity studies.

Curcumin, one of the three principal curcuminoids found within turmeric rhizomes, has long been associated with numerous physiologically beneficial effects; however, its efficacy is limited by its inherently low bioavailability. Several novel formulations of curcumin extracts have been prepared in recent years to increase the systemic availability of curcumin; Longvida®, a solid lipid curcumin particle preparation, is one such formulation that has shown enhanced bioavailability compared with standard curcuminoid extracts. As part of a safety assessment of Longvida® for use as a food ingredient, a bacterial reverse mutation test (OECD TG 471) and mammalian cell erythrocyte micronucleus test (OECD TG 474) were conducted to assess its genotoxic potential. In the bacterial reverse mutation test, Longvida® did not induce base-pair or frame-shift mutations at the histidine locus in the genome of Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537, in the presence or absence of exogenous metabolic activation. Additionally, two gavage doses (24 h apart) of Longvida® to Swiss albino mice at 500, 1000, or 2000-mg/kg body weight/day did not cause structural or numerical chromosomal damage in somatic cells in the mammalian erythrocyte micronucleus test. It was therefore concluded that Longvida® is non-genotoxic.