Assessment of mutations induced by cold atmospheric plasma jet treatment relative to known mutagens in Escherichia coli.

The main bactericidal components of cold atmospheric plasma (CAP) are thought to be reactive oxygen and nitrogen species (RONS) and UV-radiation, both of which have the capacity to cause DNA damage and mutations. Here, the mutagenic effects of CAP on Escherichia coli were assessed in comparison to X- and UV-irradiation. DNA damage and mutagenesis were screened for using a diffusion-based DNA fragmentation assay and modified Ames test, respectively. Mutant colonies obtained from the latter were quantitated and sequenced. CAP was found to elicit a similar mutation spectrum to X-irradiation, which did not resemble that for UV implying that CAP-produced RONS are more likely the mutagenic component of CAP. CAP treatment was also shown to promote resistance to the antibiotic ciprofloxacin. Our data suggest that CAP treatment has mutagenic effects that may have important phenotypic consequences.

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.

Follow-up genotoxicity assessment of Ames-positive/equivocal chemicals using the improved thymidine kinase gene mutation assay in DNA repair-deficient human TK6 cells.

Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, the in vitro genotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species. For better understanding the mechanistic implications regarding conflict outcomes between different assay systems, it is necessary to develop in vitro genotoxicity testing approaches with higher specificity towards DNA-damaging reagents. We have recently established an improved thymidine kinase (TK) gene mutation assay (TK assay) i.e. deficient in DNA excision repair system using human lymphoblastoid TK6 cells lacking XRCC1 and XPA (XRCC1-/-/XPA-/-), the core factors of base excision repair (BER) and nucleotide excision repair (NER), respectively. This DNA repair-deficient TK6 cell line is expected to specifically evaluate the genotoxic potential of chemical substances based on the DNA damage. We focussed on four reagents, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA), p-phenylenediamine (PPD), auramine and malachite green (MG) as the Ames test-positive chemicals. In our assay, assessment using XRCC1-/-/XPA-/- cells revealed no statistically significant increase in the mutant frequencies after treatment with NEDA, PPD and MG, suggesting the chemicals to be non-genotoxic in humans. The observations were consistent with that of the follow-up in vivo studies. In contrast, the mutant frequency was markedly increased in XRCC1-/-/XPA-/- cells after treatment with auramine. The results suggest that auramine is the genotoxic reagent that preferentially induces DNA damages resolved by BER and/or NER in mammals. Taken together, BER/NER-deficient cell-based genotoxicity testing will contribute to elucidate the mechanism of genotoxicity and therefore play a pivotal role in the accurate safety assessment of chemical substances.

Design and Synthesis of Non-Covalent Imidazo[1,2-a]quinoxaline-Based Inhibitors of EGFR and Their Anti-Cancer Assessment.

A series of 30 non-covalent imidazo[1,2-a]quinoxaline-based inhibitors of epidermal growth factor receptor (EGFR) were designed and synthesized. EGFR inhibitory assessment (against wild type) data of compounds revealed 6b, 7h, 7j, 9a and 9c as potent EGFRWT inhibitors with IC50 values of 211.22, 222.21, 193.18, 223.32 and 221.53 nM, respectively, which were comparable to erlotinib (221.03 nM), a positive control. Furthermore, compounds exhibited excellent antiproliferative activity when tested against cancer cell lines harboring EGFRWT; A549, a non-small cell lung cancer (NSCLC), HCT-116 (colon), MDA-MB-231 (breast) and gefitinib-resistant NSCLC cell line H1975 harboring EGFRL858R/T790M. In particular, compound 6b demonstrated significant inhibitory potential against gefitinib-resistant H1975 cells (IC50 = 3.65 µM) as compared to gefitinib (IC50 > 20 µM). Moreover, molecular docking disclosed the binding mode of the 6b to the domain of EGFR (wild type and mutant type), indicating the basis of inhibition. Furthermore, its effects on redox modulation, mitochondrial membrane potential, cell cycle analysis and cell death mode in A549 lung cancer cells were also reported.

Filling the gap between risk assessment and molecular determinants of tumor onset.

In the past two decades, a ponderous epidemiological literature has causally linked tumor onset to environmental exposure to carcinogens. As consequence, risk assessment studies have been carried out with the aim to identify both predictive models of estimating cancer risks within exposed populations and establishing rules for minimizing hazard when handling carcinogenic compounds. The central assumption of these works is that neoplastic transformation is directly related to the mutational burden of the cell without providing further mechanistic clues to explain increased cancer onset after carcinogen exposure. Nevertheless, in the last few years, a growing number of studies have implemented the traditional models of cancer etiology, proposing that neoplastic transformation is a complex process in which several parameters and crosstalk between tumor and microenvironmental cells must be taken into account and integrated with mutagenesis. In this conceptual framework, the current strategies of risk assessment that are solely based on the 'mutator model' require an urgent update and revision to keep pace with advances in our understanding of cancer biology. We will approach this topic revising the most recent theories on the biological mechanisms involved in tumor formation in order to envision a roadmap leading to a future regulatory framework for a new, protective policy of risk assessment.

Al-Tolerant Barley Mutant hvatr.g Shows the ATR-Regulated DNA Damage Response to Maleic Acid Hydrazide.

ATR, a DNA damage signaling kinase, is required for cell cycle checkpoint regulation and detecting DNA damage caused by genotoxic factors including Al3+ ions. We analyzed the function of the HvATR gene in response to chemical clastogen-maleic acid hydrazide (MH). For this purpose, the Al-tolerant barley TILLING mutant hvatr.g was used. We described the effects of MH on the nuclear genome of hvatr.g mutant and its WT parent cv. "Sebastian", showing that the genotoxic effect measured by TUNEL test and frequency of cells with micronuclei was much stronger in hvatr.g than in WT. MH caused a significant decrease in the mitotic activity of root cells in both genotypes, however this effect was significantly stronger in "Sebastian". The impact of MH on the roots cell cycle, analyzed using flow cytometry, showed no differences between the mutant and WT.

Development of molecular assays for the rapid and cost-effective determination of fluoroquinolone, macrolide and lincosamide susceptibility of Mycoplasma synoviae isolates.

Mycoplasma synoviae infection occurs worldwide, leading to considerable economic losses in the chicken and turkey industry due to infectious synovitis, respiratory diseases and eggshell apex abnormalities. Control programs against M. synoviae infection are based on eradication, vaccination and medication with antimicrobial agents. Prudent use of antibiotics can be improved greatly by the determination of antibiotic susceptibility prior to the treatment. However, the conventional broth or agar microdilution is very labor-intensive and time-consuming method. Thus, there is an increasing need for rapid antimicrobial susceptibility tests in order to guide antibiotic therapy more effectively. The aim of this study was to develop mismatch amplification mutation assays (MAMAs) to detect resistance-associated mutations in M. synoviae. M. synoviae strains with previously determined minimal inhibitory concentrations (MICs) and whole genomes (n = 92) were used for target selection and assay specification. For the evaluation of the developed assays, 20 clinical samples and an additional 20 M. synoviae isolates derived from these specimens were also included in this study. MIC values of these 20 isolates were determined by broth microdilution method. Five MAMAs were designed to identify elevated MICs of fluoroquinolones, while three MAMAs were developed to detect decreased susceptibility to macrolides and lincomycin. The sensitivity of the MAMA tests varied between 102-104 template copy number/reaction depending on the assay. Clinical samples showed identical genotype calls with the M. synoviae isolates derived from the corresponding specimens in each case. Supporting the results of conventional in vitro sensitivity tests, our approach provides a feasible tool for diagnostics. Rapidity, robustness and cost-effectiveness are powerful advantages of the developed assays. Supporting prudent antibiotic usage instead of empirical treatment, the use of this method can reduce significantly the economic impact of M. synoviae in the poultry industry and decrease bacterial resistance-related public health concerns.

Genotoxicity as a toxicologically relevant endpoint to inform risk assessment: A case study with ethylene oxide.

The purpose of the present investigation is to analyze the in vivo genotoxicity dose-response data of ethylene oxide (EO) and the applicability of the derived point-of-departure (PoD) values when estimating permitted daily exposure (PDE) values. A total of 40 data sets were identified from the literature, and benchmark dose analyses were conducted using PROAST software to identify a PoD value. Studies employing the inhalation route of exposure and assessing gene or chromosomal mutations and chromosomal damage in various tissues were considered the most relevant for assessing risk from EO, since these effects are likely to contribute to adverse health consequences in exposed individuals. The PoD estimates were screened for precision and the values were divided by data-derived adjustment factors. For gene mutations, the lowest PDE was 285 parts per trillion (ppt) based on the induction of lacI mutations in the testes of mice following 48 weeks of exposure to EO. The corresponding lowest PDE value for chromosomal mutations was 1,175 ppt for heritable translocations in mice following 8.5 weeks of EO exposure. The lowest PDE for chromosomal aberrations was 238 ppt in the mouse peripheral blood lymphocytes following 48 weeks of inhalation exposure. The diverse dose-response data for EO-induced genotoxicity enabled the derivation of PoDs for various endpoints, tissues, and species and identified 238 ppt as the lowest PDE in this retrospective analysis.

Ultra-accurate Duplex Sequencing for the assessment of pretreatment ABL1 kinase domain mutations in Ph+ ALL.

Mutations of ABL1 are the dominant mechanism of relapse in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL). We performed highly accurate Duplex Sequencing of exons 4-10 of ABL1 on bone marrow or peripheral blood samples from 63 adult patients with previously untreated Ph + ALL who received induction with intensive chemotherapy plus a BCR-ABL1 TKI. We identified ABL1 mutations prior to BCR-ABL1 TKI exposure in 78% of patients. However, these mutations were generally present at extremely low levels (median variant allelic frequency 0.008% [range, 0.004%-3.71%] and did not clonally expand and lead to relapse in any patient, even when the pretreatment mutation was known to confer resistance to the TKI received. In relapse samples harboring a TKI-resistant ABL1 mutation, the corresponding mutation could not be detected pretreatment, despite validated sequencing sensitivity of Duplex Sequencing down to 0.005%. In samples under the selective pressure of ongoing TKI therapy, we detected low-level, emerging resistance mutations up to 5 months prior to relapse. These findings suggest that pretreatment ABL1 mutation assessment should not guide upfront TKI selection in Ph + ALL, although serial testing while on TKI therapy may allow for early detection of clinically actionable resistant clones.

Genotoxic Assessment of the Dry Decoction of Myracrodruon urundeuva Allemão (Anacardiaceae) Leaves in Somatic Cells of Drosophila melanogaster by the Comet and SMART Assays.

Medicinal plants are worldwide used as an efficient treatment of many diseases. Myracrodruon urundeuva Allemão (Anacardiaceae) is widely used Brazilian folk medicine to treat inflammations and infections of the female genital tract, conditions of the stomach and throat, and to heal wounds on the skin and mucous membranes. Several pharmacological properties of extracts and compounds isolated from M. urundeuva are found in the literature, corroborating its uses as antiulcer and gastroprotective, anti-inflammatory and analgesic, as well as antimicrobial. Despite these many uses in traditional herbal medicine, there are few reports of its toxic-genetic effect. This work aimed to investigate the genotoxic and mutagenic potential in vivo of the dry decoction of M. urundeuva leaves on somatic cells of Drosophila melanogaster, through the Comet assay and somatic mutation and recombination test (SMART). Six concentrations (0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 mg/mL) were studied after feeding individuals for 24 hr in culture medium hydrated with extracts of M. urundeuva. In the Comet assay, all concentrations showed a genotoxic effect significantly higher than the negative control group, treated with distilled water. The two highest concentrations were also superior to the positive control group, treated with cyclophosphamide (1 mg/mL). In the SMART, there was a mutagenic effect at all concentrations tested, with a clear dose-dependent relationship. Both recombination and mutation account for these mutagenic effects. The set of results indicate that the dry decoction of M. urundeuva leaves is genotoxic and mutagenic for D. melanogaster under the experimental conditions of this study. Environ. Mol. Mutagen. 61:329-337, 2020. © 2019 Wiley Periodicals, Inc.

Considerations for the Use of Mutation as a Regulatory Endpoint in Risk Assessment.

Assessment of a chemical's potential to cause permanent changes in the genetic code has been a common practice in the industry and regulatory settings for decades. Furthermore, the genetic toxicity battery of tests has typically been employed during the earliest stages of the research and development programs of new product development. A positive outcome from such battery has a major impact on the chemical's utility, industrial hygiene, product stewardship practices, and product life cycle analysis, among many other decisions that need to be taken by the industry, even before the registration of a chemical is undertaken. Under the prevailing regulatory paradigm, the dichotomous (yes/no) evaluation of the chemical's genotoxic potential leads to a conservative, linear no-threshold (LNT) risk assessment, unless compelling and undeniable data to the contrary can be provided to satisfy regulators, typically in a number of different global jurisdictions. With the current advent of predictive methods, new testing paradigms, mode-of-action/adverse outcome pathways, and quantitative risk assessment approaches, various stakeholders are starting to employ these state-of-the-science methodologies to further the conversation on decision making and advance the regulatory paradigm beyond the dominant LNT status quo. This commentary describes these novel methodologies, relevant biological responses, and how these can affect internal and regulatory risk assessment approaches. Environ. Mol. Mutagen. 61:84-93, 2020. © 2019 Wiley Periodicals, Inc.

Quantitative Interpretation of Genetic Toxicity Dose-Response Data for Risk Assessment and Regulatory Decision-Making: Current Status and Emerging Priorities.

The screen-and-bin approach for interpretation of genotoxicity data is predicated on three false assumptions: that genotoxicants are rare, that genotoxicity dose-response functions do not contain a low-dose region mechanistically characterized by zero-order kinetics, and that genotoxicity is not a bona fide toxicological endpoint. Consequently, there is a need to develop and implement quantitative methods to interpret genotoxicity dose-response data for risk assessment and regulatory decision-making. Standardized methods to analyze dose-response data, and determine point-of-departure (PoD) metrics, have been established; the most robust PoD is the benchmark dose (BMD). However, there are no standards for regulatory interpretation of mutagenicity BMDs. Although 5-10% is often used as a critical effect size (CES) for BMD determination, values for genotoxicity endpoints have not been established. The use of BMDs to determine health-based guidance values (HBGVs) requires assessment factors (AFs) to account for interspecies differences and variability in human sensitivity. Default AFs used for other endpoints may not be appropriate for interpretation of in vivo mutagenicity BMDs. Analyses of published dose-response data showing the effects of compensatory pathway deficiency indicate that AFs for sensitivity differences should be in the range of 2-20. Additional analyses indicate that the AF to compensate for short treatment durations should be in the range of 5-15. Future work should use available data to empirically determine endpoint-specific CES values; similarly, to determine AF values for BMD adjustment. Future work should also evaluate the ability to use in vitro dose-response data for risk assessment, and the utility of probabilistic methods for determination of mutagenicity HBGVs. Environ. Mol. Mutagen. 61:66-83, 2020. © 2019 Her Majesty the Queen in Right of Canada.

A comparative study on erlotinib & gefitinib therapy in non-small cell lung carcinoma patients.

Background & objectives: Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) have been evaluated in patients with advanced non-small cell lung cancer (NSCLC). Erlotinib and gefitinib are the first-generation EGFR-TKIs for patients with NSCLC. However, there is a paucity of studies comparing the effectiveness of these two drugs. Hence, this study was aimed to compare the effectiveness and safety of erlotinib and gefitinib in NSCLC patients. Methods: This study included 71 NSCLC patients who received EGFR-TKIs between 2013 and 2016. Adverse drug reaction of both erlotinib (n=37) and gefitinib (n=34) was determined and graded according to Common Terminology Criteria for Adverse Events grading system. Effectiveness was measured using response evaluation criteria in solid tumours and progression-free survival (PFS). Pharmacoeconomic analysis was performed by cost-effective analysis. Results: When comparing safety profile, both the drugs had similar adverse events except for dermal side effects such as acneiform eruption (51.4%), rash (54.05%) and mucositis (59.5%) for erlotinib and 20.6, 26.5 and 29.4 per cent for gefitinib, respectively. The PFS of the two drugs was compared to differentiate the effectiveness of erlotinib and gefitinib. There was no significant difference between the effectiveness of the two drugs. The pharmacoeconomic analysis showed that gefitinib was more cost-effective than erlotinib. Interpretation & conclusions: This study showed that erlotinib and gefitinib had similar effectiveness but gefitinib had a better safety profile compared to erlotinib. Therefore, gefitinib could be considered a better option for NSCLC patients compared to erlotinib. However, further studies need to be done with a large sample to confirm these findings.

Understanding the importance of low-molecular weight (ethylene oxide- and propylene oxide-induced) DNA adducts and mutations in risk assessment: Insights from 15 years of research and collaborative discussions.

The interpretation and significance of DNA adduct data, their causal relationship to mutations, and their role in risk assessment have been debated for many years. An extended effort to identify key questions and collect relevant data to address them was focused on the ubiquitous low MW N7-alkyl/hydroxyalkylguanine adducts. Several academic, governmental, and industrial laboratories collaborated to gather new data aimed at better understanding the role and potential impact of these adducts in quantifiable genotoxic events (gene mutations/micronucleus). This review summarizes and evaluates the status of dose-response data for DNA adducts and mutations from recent experimental work with standard mutagenic agents and ethylene oxide and propylene oxide, and the importance for risk assessment. This body of evidence demonstrates that small N7-alkyl/hydroxyalkylguanine adducts are not pro-mutagenic and, therefore, adduct formation alone is not adequate evidence to support a mutagenic mode of action. Quantitative methods for dose-response analysis and derivation of thresholds, benchmark dose (BMD), or other points-of-departure (POD) for genotoxic events are now available. Integration of such analyses of genetox data is necessary to properly assess any role for DNA adducts in risk assessment. Regulatory acceptance and application of these insights remain key challenges that only the regulatory community can address by applying the many learnings from recent research. The necessary tools, such as BMDs and PODs, and the example datasets, are now available and sufficiently mature for use by the regulatory community. Environ. Mol. Mutagen. 60: 100-121, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Integrated In Vivo Genotoxicity Assessment of Procarbazine Hydrochloride Demonstrates Induction of Pig-a and LacZ Mutations, and Micronuclei, in MutaMouse Hematopoietic Cells.

Procarbazine hydrochloride (PCH) is a DNA-reactive hematopoietic carcinogen with potent and well-characterized clastogenic activity. However, there is a paucity of in vivo mutagenesis data for PCH, and in vitro assays often fail to detect the genotoxic effects of PCH due to the complexity of its metabolic activation. We comprehensively evaluated the in vivo genotoxicity of PCH on hematopoietic cells of male MutaMouse transgenic rodents using a study design that facilitated assessments of micronuclei and Pig-a mutation in circulating erythrocytes, and lacZ mutant frequencies in bone marrow. Mice were orally exposed to PCH (0, 6.25, 12.5, and 25 mg/kg/day) for 28 consecutive days. Blood samples collected 2 days after cessation of treatment exhibited significant dose-related induction of micronuclei in both immature and mature erythrocytes. Bone marrow and blood collected 3 and 70 days after cessation of treatment also showed significantly elevated mutant frequencies in both the lacZ and Pig-a assays even at the lowest dose tested. PCH-induced lacZ and Pig-a (immature and mature erythrocytes) mutant frequencies were highly correlated, with R2 values ≥0.956, with the exception of lacZ vs. Pig-a mutants in mature erythrocytes at the 70-day time point (R2 = 0.902). These results show that PCH is genotoxic in vivo and demonstrate that the complex metabolism and resulting genotoxicity of PCH is best evaluated in intact animal models. Our results further support the concept that multiple biomarkers of genotoxicity, especially hematopoietic cell genotoxicity, can be readily combined into one study provided that adequate attention is given to manifestation times. Environ. Mol. Mutagen. 60:505-512, 2019. © 2018 Her Majesty the Queen in Right of Canada.

Assessment of the Antigenotoxic Activity of Poly(d,l-lactic- co-glycolic acid) Nanoparticles Loaded with Caffeic Acid Phenethyl Ester Using the Ames Salmonella/Microsome Assay.

In the present study, the antigenotoxic activity of poly(d,l-lactic- co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with caffeic acid phenethyl ester (CAPE) was investigated in comparison to free CAPE using the Ames Salmonella/microsome assay. Additionally, to elucidate the impacts of the type of solvent effect on antigenotoxic activity, the following systems were tested: CAPE in water (poor solvent), ethyl alcohol (good solvent), and PLGA NPs (unknown). The effect of the NP system on solubility was investigated for the first time by assessing the antigenotoxic potential. In this study, the CAPE/PLGA NPs were synthesized using an oil-in-water (o/w) single-emulsion solvent evaporation method with an average size of 206.2 ± 1.2 nm, ζ potential of -19.8 ± 2.5 mV, encapsulation efficiency of 87.2 ± 2.5%, and drug loading of 53.3 ± 1.8%. According to the results of the antigenotoxic activity, the highest antimutagenic activity in both applied strains was found for CAPE in ethanol, and the lowest activity was detected for CAPE in water. Our study has shown that NP systems exhibit high antigenotoxic activity, which is similar to the results of CAPE dissolved in ethanol. These results have shown that NP systems increase biological activity of hydrophobic substances by increasing their solubility and that the use of PLGA instead of organic solvents in drug production may provide an increase in their medical utility.

Assessment of mutagenic, recombinogenic and carcinogenic potential of titanium dioxide nanocristals in somatic cells of Drosophila melanogaster.

Nanoparticles have been widely used in several sectors and their long-term effect on the body and environment remains unknown. To evaluate the mutagenic, recombinogenic and carcinogenic potential of 11 nm titanium dioxide nanocrystals (TiO2 NCs), the Somatic Mutation and Recombination Test (SMART) and the Test for Detection of Epithelial Tumors Clones (Warts-Wts) were used, both in Drosophila melanogaster. Third-instar larvae (72 + 4 h), obtained in both tests, were treated with different concentrations of TiO2 NCs ranging from 6.25 to 100 mM. Ultrapure water and urethane were used as negative and positive controls, respectively. At ST cross, all concentrations of TiO2 NCs showed a significant increase in the frequencies of mutant spots, demonstrating higher recombination rates. At the HB cross, only the 50 mM concentration showed a negative result. In the Wts Test, all used concentrations were carcinogenic, except for the 100 mM one, which was toxic. No relationship was demonstrated between the used concentrations and the obtained responses. There was no interference of the cytochrome P450 enzyme complex in the induction of mutant spots.

Genotoxicity assessment of piperitenone oxide: An in vitro and in silico evaluation.

Piperitenone oxide, a natural flavouring agent also known as rotundifolone, has been studied for the genotoxicity assessment by an integrated in vitro and in silico experimental approach, including the bacterial reverse mutation assay, the micronucleus test, the comet assay and the computational prediction by Toxtree and VEGA tools. Under our experimental conditions, the monoterpene showed to induce both point mutations (i.e. frameshift, base-substitution and/or oxidative damage) and DNA damage (i.e. clastogenic or aneuploidic damage, or single-strand breaks). Computational prediction for piperitenone oxide agreed with the toxicological data, and highlighted the presence of the epoxide function and the α,ß-unsaturated carbonyl as possible structural alerts for DNA damage. However, improving the toxicological libraries for natural occurring compounds is required in order to favour the applicability of in silico models to the toxicological predictions. Further in vivo evaluations are strictly needed in order to evaluate the role of the bioavailability of the substance and the metabolic fate on its genotoxicity profile. To the best of our knowledge, these data represent the first evaluation of the genotoxicity for this flavour compound and suggest the need of further studies to assess the safety of piperitenone oxide as a flavouring agent.