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.

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.

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.

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 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.

Assessment of Genetic Damage in Coal Miners of Punjab, Pakistan.

Coal miners are continuously exposed to coal mine dust and airborne particulate that act as a potential risk to their health. The present study evaluates the DNA damage in coal miners using the Buccal Micronucleus Cytome (BMCyt) assay. The samples of the blood and buccal epithelial cells of 40 coal miners and 20 control subjects were taken from coal mines of Pail and Padhrar, Pakistan, to establish buccal anomaly frequencies of metal levels in the blood. Besides this, work history and duration hours were also analyzed. Results revealed that micronucleus frequencies positively correlated with the metal concentrations in the miner's blood. The change in the extent of nuclear damage per unit change in the year was 0.170 for micronuclei; however, with addition in each year of working experience, nuclear buds and broken egged nuclei increased by 0.316 and 0.194 units, where each year increases karyolysis by 0.349 units and karyorrhexis by 0.308 units, respectively. An increase in work hours and working years was positively correlated with cytogenetic damage. Nuclear damage in coal miners due to occupational exposure is obvious and increases with increasing work experience. Hence, the Buccal Micronucleus Cytome assay has proved to be an effective cytogenetic biomonitoring tool for assessing genetic and nuclear damage in coal miners.

A pilot biomonitoring study of air pollution in the urban area of Sarajevo, Bosnia and Herzegovina: genotoxicity assessment in buccal cells.

Air pollution, recognized as a human carcinogen, is a significant cause of death in industrial and developing countries, and Bosnia and Herzegovina (B&H) is one of the leading countries for air pollution-caused death rate and has the poorest urban air quality in Europe. Despite a population decrease, urban air pollution in B&H has increased due to traffic pollution and still intensive use of solid fuel for heating and cooking. Human biomonitoring studies, regarding the described air pollution, have not been conducted before, and particularly have not been conducted in the region of Sarajevo. Good health, well-being, and environmental protection are part of the 17 defined Sustainable Development Global Goals. Accordingly, this study aimed to determine baseline levels of DNA damage in a group of Sarajevo citizens and to compare seasonal variations in DNA damage in relation to the reported levels of air pollution. From 33 individuals included in the study, samples were collected in the summer and winter seasons. The buccal micronucleus cytome (BMCyt) assay and comet assay in leucocytes isolated from saliva were performed. Mean values and standard deviations of log-transformed tail intensity (%), tail length (µm), and tail moment results in winter were 1.14 ± 0.23, 2.20 ± 0.14, and 1.03 ± 0.29, respectively, while in the summer season those values were 1.19 ± 0.19, 2.25 ± 0.17, and 1.07 ± 0.25, respectively. No significant differences were found for the comet assay parameters. Nevertheless, BMCyt results showed significant increases in micronuclei (P = .008), binuclear cells (P = .04), karyolysis (P = .0003), condensed chromatin (P = .03), and pyknosis (P = .002) in winter. Although the results of comet and BMCyt assays are not in accordance, this study contributes to the human air pollution biomonitoring in Sarajevo, B&H, and based on the genotoxic effects of air pollution evidenced by the BMCyt biomarker further studies of this kind are necessary.

Determination of potential thresholds for N-ethyl-N-nitrosourea and ethyl methanesulfonate based on a multi-endpoint genotoxicity assessment platform in rats.

The main goal of the study was to investigate the genotoxic response of N-ethyl-N-nitrosourea (ENU) and ethyl methanesulfonate (EMS) at low doses in a multi-endpoint genotoxicity assessment platform in rats and to derive potential thresholds and related metrics. Male Sprague-Dawley rats were treated by daily oral gavage for 28 consecutive days with ENU (0.25 ~ 8 mg/kg bw) and EMS (5 ~ 160 mg/kg bw), both with six closely spaced dose levels. Pig-a gene mutation assay, micronucleus test, and comet assay were performed in several timepoints. Then, the dose-response relationships were analyzed for possible points of departure (PoD) using the no observed genotoxic effect level and benchmark dose (BMD) protocols with different critical effect sizes (CES, 0.05, 0.1, 0.5, and 1SD). Overall, dose-dependent increases in all investigated endpoints were found for ENU and EMS. PoDs varied across genetic endpoints, timepoints, and statistical methods, and selecting an appropriate lower 95% confidence limit of BMD needs a comprehensive consideration of the mode of action of chemicals, the characteristics of tests, and the model fitting methods. Under the experimental conditions, the PoDs of ENU and EMS were 0.0036 mg/kg bw and 1.7 mg/kg bw, respectively.

Characterization of benzo[a]pyrene and colchicine based on an in vivo repeat-dosing multi-endpoint genotoxicity quantitative assessment platform.

Two prototypical genotoxicants, benzo[a]pyrene (B[a]P) and colchicine (COL), were selected as model compounds to deduce their quantitative genotoxic dose-response relationship at low doses in a multi-endpoint genotoxicity assessment platform. Male Sprague-Dawley rats were treated with B[a]P (2.5-80 mg/kg bw/day) and COL (0.125-2 mg/kg bw/day) daily for 28 days. The parameters included were as follows: comet assay in the peripheral blood and liver, Pig-a gene mutation assay in the peripheral blood, and micronucleus test in the peripheral blood and bone marrow. A significant increase was observed in Pig-a mutant frequency in peripheral blood for B[a]P (started at 40 mg/kg bw/day on Day 14, started at 20 mg/kg bw/day on Day 28), whereas no statistical difference for COL was observed. Micronucleus frequency in reticulocytes of the peripheral blood and bone marrow increased significantly for B[a]P (80 mg/kg bw/day on Day 4, started at 20 mg/kg bw/day on Days 14 and 28 in the blood; started at 20 mg/kg bw/day on Day 28 in the bone marrow) and COL (started at 2 mg/kg bw/day on Day 14, 1 mg/kg bw/day on Day 28 in the blood; started at 1 mg/kg bw/day on Day 28 in the bone marrow). No statistical variation was found in indexes of comet assay at all time points for B[a]P and COL in the peripheral blood and liver. The dose-response relationships of Pig-a and micronucleus test data were analyzed for possible point of departures using three quantitative approaches, i.e., the benchmark dose, breakpoint dose, and no observed genotoxic effect level. The practical thresholds of the genotoxicity of B[a]P and COL estimated in this study were 0.122 and 0.0431 mg/kg bw/day, respectively, and our results also provided distinct genotoxic mode of action of the two chemicals.

Lymphocyte-based challenge DNA-repair assays for personalized health risk assessment.

Combinations of genetic and environmental factors are responsible for the development of many human diseases, such as cancer, as demonstrated using various biomarkers. Within this scenario, DNA repair holds a gate-keeper position which determines outcomes after appearance of DNA damage and, therefore, adverse cellular consequences, e.g., initiation of carcinogenesis. DNA repair deficiency and some of the subsequent events can be validated from studies using live cells from cancer patients. However, these deficiencies/events are difficult to demonstrate in live cells from normal individuals because individual variations in DNA repair capacities (DRC) are too low to be measured easily. Such lack of information has been hindering progress in developing personalized disease prevention and intervention protocols, especially among exposed populations. However, using a variety of challenge assays as biomarkers, variations in individual's DRC can be amplified in live cells and be determined. Furthermore, evidence indicates that DRC are not only inherited but can also be modified by environmental factors (e.g., nutritional status and exposure to genotoxic substances). Using these challenge assays, e.g., in live lymphocytes, individual's DRC can be holistically and functionally determined as well as quantitated. With the more precise information, assessment of health risk can be better determined on an individual rather than on a population basis. This review provides a succinct summary on the development and application of recent challenge assays in lymphocytes which can provide measurements of individuals' DRC, and on the latest data for more precise disease prevention and intervention.

A new imaging platform (iScreen) allows for the concurrent assessment of micronucleus induction and genotoxic mode of action in human A375 cells.

Genotoxicity testing guidelines require the assessment of the clastogenic and aneugenic potential of compounds. While in vitro micronucleus assays detect both types of endpoints, it requires labor-intensive microscopic scoring and does not discriminate between the two modes of actions. Here, we present a novel high-content imaging platform in A375 human cells that addresses the need for rapid scoring while providing additional mechanistic information. We evaluated the new platform with 12 compounds, three compounds from each mechanistic class (clastogen, aneugen tubulin binder, aneugen aurora inhibitor, and nongenotoxicant) following 4- and 24-h compound treatments. The approach we developed is first discriminating between genotoxicant and nongenotoxicant using an image analysis algorithm to quantify micronucleus induction below a 60% cytotoxicity cutoff. Then it uses centromere protein A (CENPA) staining for the genotoxic compounds to discriminate between aneugens and clastogens. Lastly, we use phosphorylated histone H2AX Ser139 (γH2AX) staining to confirm clastogenicity and changes in phosphorylated histone 3 Ser10 (pH 3) and increases in polyploidy in mitotic cells to discriminate between aneugens that bind tubulin from those that affect aurora kinases. All compounds were correctly classified, and we showed by using benchmark dose-response analysis that the imaging platform in A375 cells is at least as sensitive as the MicroFlow® assay in TK6 cells for genotoxicant but appears to be more specific for the nongenotoxicants. A detailed comparison of the cell lines and a more comprehensive validation with a much larger compound set, predictive and dose-response modeling will be presented in the future.

Comprehensive interpretation of in vitro micronucleus test results for 292 chemicals: from hazard identification to risk assessment application.

Risk assessments are increasingly reliant on information from in vitro assays. The in vitro micronucleus test (MNvit) is a genotoxicity test that detects chromosomal abnormalities, including chromosome breakage (clastogenicity) and/or whole chromosome loss (aneugenicity). In this study, MNvit datasets for 292 chemicals, generated by the US EPA's ToxCast program, were evaluated using a decision tree-based pipeline for hazard identification. Chemicals were tested with 19 concentrations (n = 1) up to 200 µM, in the presence and absence of Aroclor 1254-induced rat liver S9. To identify clastogenic chemicals, %MN values at each concentration were compared to a distribution of batch-specific solvent controls; this was followed by cytotoxicity assessment and benchmark concentration (BMC) analyses. The approach classified 157 substances as positives, 25 as negatives, and 110 as inconclusive. Using the approach described in Bryce et al. (Environ Mol Mutagen 52:280-286, 2011), we identified 15 (5%) aneugens. IVIVE (in vitro to in vivo extrapolation) was employed to convert BMCs into administered equivalent doses (AEDs). Where possible, AEDs were compared to points of departure (PODs) for traditional genotoxicity endpoints; AEDs were generally lower than PODs based on in vivo endpoints. To facilitate interpretation of in vitro MN assay concentration-response data for risk assessment, exposure estimates were utilized to calculate bioactivity exposure ratio (BER) values. BERs for 50 clastogens and two aneugens had AEDs that approached exposure estimates (i.e., BER < 100); these chemicals might be considered priorities for additional testing. This work provides a framework for the use of high-throughput in vitro genotoxicity testing for priority setting and chemical risk assessment.

Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances.

The micronucleus (MN) assay is widely used as part of a battery of tests applied to evaluate the genotoxic potential of chemicals, including new food additives and novel food ingredients. Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, biochemistry and genomic events in the gut, the site of first contact for ingested materials. Here we have adapted and validated the MN endpoint assay protocol for use with complex 3D reconstructed intestinal microtissues; we have named this new protocol the reconstructed intestine micronucleus cytome (RICyt) assay. Our data suggest the commercial 3D microtissues replicate the physiological, biochemical and genomic responses of native human small intestine to exogenous compounds. Tissues were shown to maintain log-phase proliferation throughout the period of exposure and expressed low background MN. Analysis using the RICyt assay protocol revealed the presence of diverse cell types and nuclear anomalies (cytome) in addition to MN, indicating evidence for comprehensive DNA damage and mode(s) of cell death reported by the assay. The assay correctly identified and discriminated direct-acting clastogen, aneugen and clastogen requiring exogenous metabolic activation, and a non-genotoxic chemical. We are confident that the genotoxic response in the 3D microtissues more closely resembles the native tissues due to the inherent tissue architecture, surface area, barrier effects and tissue matrix interactions. This proof-of-concept study highlights the RICyt MN cytome assay in 3D reconstructed intestinal microtissues is a promising tool for applications in predictive toxicology.

Assessment of genetic damage induced by gadolinium-based radiocontrast agents.

BACKGROUND: Today, although gadolinium based contrast agents have been frequently used in the field of medicine, there is limited data available whether gadolinium based agents affect the genome. AIM: The present study aimed to investigate the genotoxic and cytotoxic potentials of gadoteric acid and gadoversetamide used as gadolinium-based contrast agents for magnetic resonance (MR) imaging. MATERIAL AND METHODS: The cytokinesis-block micronucleus assay was applied to human peripheral blood lymphocytes to assess the genotoxicity measured as micronucleus (MN), nucleoplasmic bridge (NPBs) and nuclear bud (NBUDs) frequencies. Furthermore, cytokinesis-block proliferation index (CBPI) was calculated to determine cytostasis. Lymphocytes were treated with gadoteric acid at concentrations of 1.0, 2.5, 5.0, and 25 mM and with gadoversetamide at concentrations of 0.25, 1.0, 2.5, and 5.0 mM for 48 h. RESULTS: Gadoteric acid did not cause significant increase in MN, NBPs and NBUDs frequencies and CBPI values at any concentration. Gadoversetamide induced significantly increase MN formation at concentration of 2.5 mM, NBP formation at concentrations of 1.0 and 2.5 mM, and NBUD formation at concentrations of 0.25, 1.0 and 2.5 mM. Additionally, gadoversetamide exposure resulted in statistically significant decrease in CBPI values compared to the control at concentrations of 2.5 and 5.0 mM. In addition, CBPI levels in response to concentrations of gadoversetamide was negatively and significantly associated with concentration. CONCLUSION: These findings show that gadoteric acid does not have genotoxic or cytotoxic potential, while gadoversetamide might have both genotoxic and cytotoxic potential on human peripheral blood lymphocytes. As a comparison, gadoversetamide was found more genotoxic and cytotoxic.

Genotoxicity test battery - An assessment of its utility in early drug development.

Formal requirements for genotoxicity testing of drug candidates to support clinical entry have been in place since the issue of initial regulatory guidance over 25 years ago and subsequent update a decade ago. An evaluation of such testing, supporting first clinical entry of 108 small molecule drug candidates over the last decade, showed that the most common approach (75 % of tested compounds) was for a Good Laboratory Practice test battery in the form of 2 in vitro (a bacterial reverse mutation and a mammalian cell) assays and one in vivo assay. The majority of other tested compounds involved in vitro testing only in bacterial reverse mutation and mammalian cell assays. Testing using a bacterial reverse mutation assay and an in vivo assessment of genotoxicity with 2 different tissues was limited to 2 occasions. For in vitro mammalian cell testing, the chromosome aberration test was most commonly used (70 % occasions), followed by a micronucleus test (16 % occasions) or a mouse lymphoma assay (14 % occasions). For in vivo evaluation, the most common test was a rodent bone marrow micronucleus test (87 % occasions). A positive in vitro mammalian cell assay result was seen on 13 % occasions but was not confirmed with further in vivo testing and the drug candidates were taken into the clinic. In conclusion, the present evaluation showed that the current test battery paradigm for genotoxicity testing has an integral part in supporting clinical entry to confirm candidate drugs taken into the clinic are unlikely to have genotoxic activity.

Assessment of Peripheral Blood Lymphocytes in Parents of Autistic Children by Cytokinesis Block Micronucleus Assay.

Autism spectrum disorders are neurodevelopmental complex diseases with causative de-novo and inherited genetic factors. They include a range of cognitive and behavioral conditions such as pervasive developmental disorder, Asperger's syndrome, and autism. Cytokinesis-block micronucleus assay, as a cytogenetic study has been considered as one of the indicators of chromosomal damage in peripheral blood. This study aimed to investigate the frequency of micronucleus (MN) in peripheral blood lymphocytes of parents with autistic children. The study was case-control and the cases were parents of autistic children referring to the psychiatric department of the Ali-Asghar Hospital of Tehran. The total number of samples was 60 cases and 30 controls. The results showed that autistic children's parents had a significant increase in MN frequency in binucleated lymphocytes. Further researches are suggested to analyze the environmental and genetic reasons for MN increase in autistic children parents.

Indigenous Fish-Based Assessment of Genotoxic Potentials of the Helong Reservoir in Guangzhou, China.

The present study was conducted to assess the genotoxic potential of water from the Helong Reservoir, which was designated as a strategic drinking water source by the Guangdong Provincial Government of China in October 2016. Four kinds of common indigenous fish samples (Labeo rohita, Cirrhinus molitorella, red tilapia, and Oreochromis niloticus) were collected at 6 sampling sites during the period from July to November 2020. Fish from the clean drinking water source of the upper reaches of the Liuxihe Reservoir in Guangzhou were collected as the control. Both the alkaline single cell gel electrophoresis assay and the micronucleus test were used to detect DNA damage and the micronucleus rate in erythrocytes of fish samples, respectively. The results indicated that there was a significant increase in comet tail length, Olive tail moment, and micronucleus rates of all fish samples compared with those of the control (p < 0.05). The order of sensitivity to DNA damage and micronucleus formation was Labeo rohita > Cirrhinus molitorella > red tilapia > Oreochromis niloticus. The results of the 2 kinds of experiments were in perfect agreement with each other. We conclude that there are obvious genotoxic effects from the water in the Helong Reservoir. As a strategic drinking water source, the safety of the Reservoir water quality should be considered. The local government should put the restoration of the Helong Reservoir water quality on the agenda as soon as possible. Environ Toxicol Chem 2021;40:1919-1927. © 2021 SETAC.

Transforming early pharmaceutical assessment of genotoxicity: applying statistical learning to a high throughput, multi end point in vitro micronucleus assay.

To provide a comprehensive analysis of small molecule genotoxic potential we have developed and validated an automated, high-content, high throughput, image-based in vitro Micronucleus (IVM) assay. This assay simultaneously assesses micronuclei and multiple additional cellular markers associated with genotoxicity. Acoustic dosing (≤ 2 mg) of compound is followed by a 24-h treatment and a 24-h recovery period. Confocal images are captured [Cell Voyager CV7000 (Yokogawa, Japan)] and analysed using Columbus software (PerkinElmer). As standard the assay detects micronuclei (MN), cytotoxicity and cell-cycle profiles from Hoechst phenotypes. Mode of action information is primarily determined by kinetochore labelling in MN (aneugencity) and γH2AX foci analysis (a marker of DNA damage). Applying computational approaches and implementing machine learning models alongside Bayesian classifiers allows the identification of, with 95% accuracy, the aneugenic, clastogenic and negative compounds within the data set (Matthews correlation coefficient: 0.9), reducing analysis time by 80% whilst concurrently minimising human bias. Combining high throughput screening, multiparametric image analysis and machine learning approaches has provided the opportunity to revolutionise early Genetic Toxicology assessment within AstraZeneca. By multiplexing assay endpoints and minimising data generation and analysis time this assay enables complex genotoxicity safety assessments to be made sooner aiding the development of safer drug candidates.

Assessment of the Genotoxic Potential of Cizolirtine a Substance-P and Calcitonin Gene-Related Peptide Release Modulator.

The analysis of the genotoxic potential of cizolirtine, a compound being developed as a drug for analgesia and for urinary incontinence, was carried out using a battery of in vitro and in vivo assays as recommended in the guidelines for medicinal products. Negative results were obtained in an Ames test (up to 5000 µg/plate), in a Mouse Lymphoma assay (up to 2000 µg/ml) and in a single dose mouse bone marrow micronucleus assay (up to 300 mg/kg). In a human lymphocyte chromosome aberration assay, a slight statistical increase in the frequency of cells with chromosome aberrations including gaps was reported for the concentrations of 200 and 1600 µg/ml at the 24-h sampling time. This minor increase in chromosome aberrations was considered of questionable biological relevance since it was moderate, was within the laboratory historical control values, did no show a dose-dependent effect and was not observed at similar concentrations in a repeat assay. Taking into considerations the results obtained in the different in vitro and in vivo assays and a weight-of-evidence analysis, it suggests that cizolirtine would not pose a genotoxic risk when administered to humans.