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Regulatory Toxicology and Pharmacology... Mar 2024The alkaline comet assay is frequently used as in vivo follow-up test within different regulatory environments to characterize the DNA-damaging potential of different...
The alkaline comet assay is frequently used as in vivo follow-up test within different regulatory environments to characterize the DNA-damaging potential of different test items. The corresponding OECD Test guideline 489 highlights the importance of statistical analyses and historical control data (HCD) but does not provide detailed procedures. Therefore, the working group "Statistics" of the German-speaking Society for Environmental Mutation Research (GUM) collected HCD from five laboratories and >200 comet assay studies and performed several statistical analyses. Key results included that (I) observed large inter-laboratory effects argue against the use of absolute quality thresholds, (II) > 50% zero values on a slide are considered problematic, due to their influence on slide or animal summary statistics, (III) the type of summarizing measure for single-cell data (e.g., median, arithmetic and geometric mean) may lead to extreme differences in resulting animal tail intensities and study outcome in the HCD. These summarizing values increase the reliability of analysis results by better meeting statistical model assumptions, but at the cost of information loss. Furthermore, the relation between negative and positive control groups in the data set was always satisfactorily (or sufficiently) based on ratio, difference and quantile analyses.
Topics: Animals; Comet Assay; DNA Damage; Reproducibility of Results; Mutation; Research Design
PubMed: 38401761
DOI: 10.1016/j.yrtph.2024.105583 -
Ecotoxicology and Environmental Safety Mar 20242-Methylfuran (2-MF) is an important member of the furan family generated during food thermal processing. An in-vivo multiple endpoint genotoxicity assessment system was...
2-Methylfuran (2-MF) is an important member of the furan family generated during food thermal processing. An in-vivo multiple endpoint genotoxicity assessment system was applied to explore the genotoxic mode of action and threshold of 2-MF. Male Sprague-Dawley rats received 2-MF by oral gavage at doses of 0.16, 0.625, 2.5, and 10 mg/kg.bw/day for 120 days. An additional 15 days were granted for recovery. The Pig-a gene mutation frequency of RET and RBC showed significant increases among the 2-MF groups on day 120. After a 15-day recovery period, the Pig-a gene mutation frequency returned to levels similar to those in the vehicle control. The tail intensity (TI) values of peripheral blood cells at a dose of 10 mg/kg.bw/day significantly increased from day 4 and remained at a high level after the recovery period. No statistical difference was found in the micronucleus frequency of peripheral blood between any 2-MF dose group and the corn oil group at any timepoint. 2-MF may not induce the production of micronuclei, but it could cause DNA breakage. It could not be ruled out that 2-MF may accumulate in vivo and cause gene mutations. Hence, DNA, other than the spindle, may be directly targeted. The mode of action of 2-MF may be that it was metabolized by EPHX1 to more DNA-active metabolites, thus leading to oxidative and direct DNA damage. The point of departure (PoD) of 2-MF-induced genotoxicity was derived as 0.506 mg/kg bw/day.
Topics: Rats; Animals; Male; Rats, Sprague-Dawley; Micronucleus Tests; Reticulocytes; DNA Damage; Furans; DNA; Mutagenicity Tests
PubMed: 38394755
DOI: 10.1016/j.ecoenv.2024.116125 -
Toxins Feb 2024Water kefir grains (WKGs), the starter used to develop a traditional beverage named water kefir, consist of a symbiotic mixture of probiotics with diverse bioactivities,...
Water kefir grains (WKGs), the starter used to develop a traditional beverage named water kefir, consist of a symbiotic mixture of probiotics with diverse bioactivities, but little is known about their abilities to remove mycotoxins that have serious adverse effects on humans and animals. This study investigated the ability of WKGs to remove aflatoxin B1 (AFB1), one of the most toxic mycotoxins, under different settings, and determined the mechanism of absorption mediated by WKGs and the effect of WKGs on the toxicity induced by AFB1 and the reduction in AFB1 in cow milk and tea soups. The results showed the WKGs used herein were dominated by , , , , , , and . HPLC analysis demonstrated that the WKGs effectively removed AFB1 at concentrations ranging from 1 to 5 µg/mL, pH values ranging from 3 to 9, and temperatures ranging from 4 to 45 °C. Additionally, the removal of AFB1 mainly depended on absorption, which was consistent with the Freundlich and pseudo-second-order kinetic models. Moreover, only 49.63% of AFB1 was released from the AFB1-WKG complex after four washes when the release of AFB1 was non-detectable. Furthermore, WKG treatment caused a dramatic reduction in the mutagenicity induced by AFB1 according to an Ames test and reduced more than 54% of AFB1 in cow milk and three tea soups. These results suggested that WKGs can act as a potential bio-absorbent with a high binding ability to detoxify AFB1 in food and feed via a chemical action step and multi-binding sites of AFB1 absorption in a wide range of scenarios.
Topics: Animals; Female; Cattle; Humans; Aflatoxin B1; Kefir; Lactobacillus; Probiotics; Tea
PubMed: 38393185
DOI: 10.3390/toxins16020107 -
Genes and Environment : the Official... Feb 2024Carbendazim (methyl 2-benzimidazolecarbamate, CASRN: 10605-21-7) exhibits spindle poisoning effects and is widely used as a fungicide. With respect to genotoxicity,...
BACKGROUND
Carbendazim (methyl 2-benzimidazolecarbamate, CASRN: 10605-21-7) exhibits spindle poisoning effects and is widely used as a fungicide. With respect to genotoxicity, carbendazim is deemed to be non-mutagenic in vitro, but it causes indicative DNA damage in vivo and chromosome aberrations in vitro and in vivo. In this study, we examined the mutagenicity of carbendazim in vivo.
RESULTS
MutaMice were treated with carbendazim orally at doses of 0 (corn oil), 250, 500, and 1,000 mg/kg/day once a day for 28 days. A lacZ assay was used to determine the mutant frequency (MF) in the liver and glandular stomach of mice. MutaMice were administered up to the maximum dose recommended by the Organization for Economic Co-operation and Development Test Guidelines for Chemicals No. 488 (OECD TG488). The lacZ MFs in the liver and glandular stomach of carbendazim-treated animals were not significantly different from those in the negative control animals. In contrast, positive control animals exhibited a significant increase in MFs in both the liver and glandular stomach.
CONCLUSIONS
Carbendazim is non-mutagenic in the liver and glandular stomach of MutaMice following oral treatment.
PubMed: 38378650
DOI: 10.1186/s41021-024-00299-4 -
International Journal of Molecular... Jan 2024The Ames/quantitative structure-activity relationship (QSAR) International Challenge Projects, held during 2014-2017 and 2020-2022, evaluated the performance of various...
The Ames/quantitative structure-activity relationship (QSAR) International Challenge Projects, held during 2014-2017 and 2020-2022, evaluated the performance of various predictive models. Despite the significant insights gained, the rules allowing participants to select prediction targets introduced ambiguity in model performance evaluation. This reanalysis identified the highest-performing prediction model, assuming a 100% coverage rate (COV) for all prediction target compounds and an estimated performance variation due to changes in COV. All models from both projects were evaluated using balance accuracy (BA), the Matthews correlation coefficient (MCC), the F1 score (F1), and the first principal component (PC1). After normalizing the COV, a correlation analysis with these indicators was conducted, and the evaluation index for all prediction models in terms of the COV was estimated. In total, using 109 models, the model with the highest estimated BA (76.9) at 100% COV was MMI-VOTE1, as reported by Meiji Pharmaceutical University (MPU). The best models for MCC, F1, and PC1 were all MMI-STK1, also reported by MPU. All the models reported by MPU ranked in the top four. MMI-STK1 was estimated to have F1 scores of 59.2, 61.5, and 63.1 at COV levels of 90%, 60%, and 30%, respectively. These findings highlight the current state and potential of the Ames prediction technology.
Topics: Humans; Quantitative Structure-Activity Relationship; Mutagenicity Tests; Correlation of Data
PubMed: 38338650
DOI: 10.3390/ijms25031373 -
Toxicology Reports Jun 2024N-nitrosamines, a very heterogeneous class of chemicals, may enter humans in small amounts through various sources and are produced endogenously, too. Some are known to...
N-nitrosamines, a very heterogeneous class of chemicals, may enter humans in small amounts through various sources and are produced endogenously, too. Some are known to be mutagenic carcinogens and have recently been detected as impurities in several marketed pharmaceuticals. Despite their known mutagenic properties, the suitability of the bacterial reverse mutation (Ames) assay and in particular the use of induced rat liver S9 to detect their mutagenic potential, is often discussed. Recently, it could be demonstrated that induced rat liver S9 is capable of metabolizing small alkyl nitrosamines to exert their mutagenic potential (Bringezu & Simon, 2022). In this project, the mutagenic potential of nitrosamines in vitro under different S9 conditions applying the preincubation protocol and OECD 471-compliant standard Ames test recommendations was investigated. These conditions included various amounts of S9 fraction from hamster and rat, uninduced or induced with Aroclor 1254 or Phenobarbital/beta-Naphthoflavone (PB/NF). The findings indicated that in addition to induced S9, uninduced hamster S9 also demonstrated effectiveness. Moreover, both rat and hamster S9 fractions exhibited suitable responses in terms of mutation frequencies. Increasing the S9 content did not increase the sensitivity of the Ames test. However, above 20% S9, reduced mutation frequency was observed in the higher concentration range suggesting cytotoxicity to the bacteria. Thus, limiting the S9 content to 10% provides reliable results and relates to a lower number of animals required for S9 production which is in concordance with the 3R principles (reduce, refine, replace) for animal testing. In addition, results obtained show that uninduced and induced hamster S9 are similarly effective, doubting the requirement of pretreating animals with enzyme inducers. Further investigations to compare mutagenicity data and rat and hamster S9 proteome analyses are ongoing.
PubMed: 38322170
DOI: 10.1016/j.toxrep.2024.01.012 -
Mutagenesis Mar 2024Chemical safety testing plays a crucial role in product and pharmacological development, as well as chemoprevention; however, in vitro genotoxicity safety tests do not... (Review)
Review
Chemical safety testing plays a crucial role in product and pharmacological development, as well as chemoprevention; however, in vitro genotoxicity safety tests do not always accurately predict the chemicals that will be in vivo carcinogens. If chemicals test positive in vitro for genotoxicity but negative in vivo, this can contribute to unnecessary testing in animals used to confirm erroneous in vitro positive results. Current in vitro tests typically evaluate only genotoxicity endpoints, which limits their potential to detect non-genotoxic carcinogens. The frequency of misleading in vitro positive results can be high, leading to a requirement for more informative in vitro tests. It is now recognized that multiple-endpoint genotoxicity testing may aid more accurate detection of carcinogens and non-carcinogens. The objective of this review was to evaluate the utility of our novel, multiple-endpoint in vitro test, which uses multiple cancer-relevant endpoints to predict carcinogenic potential. The tool assessed micronucleus frequency, p53 expression, p21 expression, mitochondrial respiration, cell cycle abnormalities and, uniquely, cell morphology changes in human lymphoblastoid cell lines, TK6 and MCL-5. The endpoints were used to observe cellular responses to 18 chemicals within the following categories: genotoxic carcinogens, non-genotoxic carcinogens, toxic non-carcinogens, and misleading in vitro positive and negative agents. The number of endpoints significantly altered for each chemical was considered, alongside the holistic Integrated Signature of Carcinogenicity score, derived from the sum of fold changes for all endpoints. Following the calculation of an overall score from these measures, carcinogens exhibited greater potency than non-carcinogens. Genotoxic carcinogens were generally more potent than non-genotoxic carcinogens. This novel approach therefore demonstrated potential for correctly predicting whether chemicals with unknown mechanism may be considered carcinogens. Overall, while further validation is recommended, the test demonstrates potential for the identification of carcinogenic compounds. Adoption of the approach could enable reduced animal use in carcinogenicity testing.
Topics: Animals; Humans; Carcinogens; Carcinogenicity Tests; Mutagenicity Tests; Carcinogenesis; DNA Damage; In Vitro Techniques
PubMed: 38301659
DOI: 10.1093/mutage/geae004 -
Microorganisms Dec 2023Urban air pollution is recognized as a critical problem for public health and is classified as a carcinogen for humans. A great number of studies have focused on the...
BACKGROUND
Urban air pollution is recognized as a critical problem for public health and is classified as a carcinogen for humans. A great number of studies have focused on the monitoring of urban air mutagenicity. One of the best-known and applied methods for assessing mutagenicity is the Ames test, a bacterial reverse mutation test. The classic protocol for assessing air mutagenicity involves the concentration of particulate matter (PM) on filters and subsequent extraction using organic solvents. This work aimed to develop a method for the evaluation of air mutagenicity directly impacted by air on microbial plates already containing an Ames' microbial sensor.
METHODS
A specific six-month sampling campaign was carried out in Turin in a period with high air pollution. Samples were tested for mutagenicity on strains TA98, TA100, and YG1024 with the traditional method and with the new direct method.
RESULTS
The new protocol is able to evaluate the mutagenicity of the sampled air and obtain repeatable results. The final sensitivity is similar to the traditional method (≈10 net revertants/m); however, the mutagenic response is due to the complete air pollution mixture, including volatile and semivolatile pollutants avoiding the concentration of filters and the following laborious extraction procedures.
CONCLUSIONS
Despite some critical issues in contamination control, the method is easier, faster, and less expensive than traditional methods.
PubMed: 38276172
DOI: 10.3390/microorganisms12010003 -
Toxicology Research Feb 2024The safety of a rhamnogalacturonan-I-enriched pectin extract (G3P-01) from pumpkin ( var. Dickinson) was evaluated for use as an ingredient in food and dietary...
The safety of a rhamnogalacturonan-I-enriched pectin extract (G3P-01) from pumpkin ( var. Dickinson) was evaluated for use as an ingredient in food and dietary supplements. G3P-01 was tested in a battery of genetic toxicity studies including reverse mutagenicity and micronucleus assay. In addition, Sprague-Dawley rats were randomized and orally dosed with G3P-01 incorporated in animal diet at concentrations of 0, 9000, 18,000, and 36,000 ppm daily for 13-weeks (n=10/sex/group) in line with OECD guidelines (TG 408). The results of the bacterial reverse mutation assay and micronucleus assay in TK6 cells demonstrated a lack of genotoxicity. The 13-week oral toxicity study in Sprague-Dawley rats demonstrated that the test article, G3P-01 was well tolerated; there were no mortalities and no adverse effects on clinical, gross pathology, hematology, blood chemistry, and histological evaluation of the essential organs of the animals. The present study demonstrates that G3P-01 is non-genotoxic and is safe when ingested in diet at concentrations up to 36, 000 ppm. The subchronic no-observed-adverse-effect level (NOAEL) for G3P-01 was concluded to be 36,000 ppm, equivalent to 1,899 and 2,361 mg/kg/day for male and female rats respectively.
PubMed: 38274036
DOI: 10.1093/toxres/tfae004 -
Mutation Research. Genetic Toxicology... Jan 20245-Aminoisophthalic acid and 5-nitroisophthalic acid (5-NIPA) are potential impurities in preparations of 5-amino-2,4,6-triiodoisophthalic acid, which is a key...
Mutagenicity assessment of two potential impurities in preparations of 5-amino-2,4,6 triiodoisophthalic acid, a key intermediate in the synthesis of the iodinated contrast agent iopamidol.
5-Aminoisophthalic acid and 5-nitroisophthalic acid (5-NIPA) are potential impurities in preparations of 5-amino-2,4,6-triiodoisophthalic acid, which is a key intermediate in the synthesis of the iodinated contrast agent iopamidol. We have studied their mutagenicity in silico (quantitative structure-activity relationships, QSAR) and by the bacterial reverse mutation assay (Ames test). First, the compounds were screened with the tools Derek Nexus™ and Leadscope®. Both compounds were flagged as potentially mutagenic (class 3 under ICH M7). However, contrary to the in silico prediction, neither chemical was mutagenic in the Ames test (plate incorporation method) with or without S9 metabolic activation.
Topics: Mutagens; Contrast Media; Iopamidol; Computer Simulation; Mutagenicity Tests
PubMed: 38272634
DOI: 10.1016/j.mrgentox.2023.503720