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

https://doi.org/10.1016/j.fct.2017.06.021Get rights and content

Highlights

  • An integrated in vitro and in silico genotoxicity assessment for the natural flavouring agent piperitenone oxide.

  • Experiments carried out in vitro by Ames test, micronucleus and comet assays, and in silico by Toxtree and VEGA tools.

  • Test substance induced point mutations and DNA damage, including micronuclei and single-strand breaks.

  • Epoxide and α,β-unsaturated carbonyl found as structural alerts for mutagenicity at computational analysis.

Abstract

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,β-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.

Introduction

Piperitenone oxide (1-methyl-4-propan-2-ylidene-7-oxabicyclo[4.1.0]heptan-5-one; C10H14O2; MW 166), also named rotundifolone, is a naturally occurring oxygenated monoterpene (Fig. 1). It was firstly isolated from Mentha rotundifolia Ehrh. and then found to be a major component (over 50%) of the essential oils from many Mentha species, including M. suaveolens, M. spicata, Calamintha nepeta and C. incana (Garzoli et al., 2015). It is currently used as a flavouring agent in different commercial products (viz. creams, lotions, detergents, and various other personal and household products). Furthermore, interesting biological activities have been highlighted over the years (Božović et al., 2015). Both the essential oil from Mentha spp. and piperitenone oxide have been found to possess antiparasitic activity (Matos-Rocha et al., 2013, De Sousa et al., 2016) and insecticidal properties against mosquitoes and weevils (Tripathi et al., 2004, Lima et al., 2014, Zekri et al., 2013). Conversely, the monoterpene weakly contributed to the cytotoxicity of the Mentha villosa essential oil against human cancer cell lines (Amaral et al., 2015). Interestingly, it exhibited antibacterial, antiviral and antifungal activities (Arruda et al., 2006; Pietrella et al., 2011; Civitelli et al., 2014). In addition, hypothensive, bradicardic and myorelaxant effects, likely due to the block of calcium current by inhibiting L-type Cav channels, were highlighted (Sousa et al., 1997; Silva et al., 2011). Piperitenone oxide and its structural analogues also exhibited antinociceptive properties, in which the epoxide group and the substituents on the ring carbon seem to play a pivotal role (De Sousa et al., 2007).

Being piperitenone oxide (FL no. 16.004; Flavouring Group Evaluation, FGE.213) classified as a flavouring agent used in foodstuffs, the European Commission asked the European Food Safety Authority (EFSA) Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF Panel) to give a scientific opinion on its implications and concerns for human health, by applying the procedure of Commission Regulation EC No 1565/2000 (EFSA CEF Panel, 2012). Particularly, due to the lack of supporting information provided by Flavour Industry, a toxicity assessment for piperitenone oxide has been reported to be pending (EFSA CEF Panel, 2014, EFSA CEF Panel, 2015).

The safety evaluation of a fragrance material includes a broad range of toxicological information, both for the compound itself and for structurally related chemicals belonging to the same chemical group (Bickers et al., 2003). Among toxicological information, genotoxicity is a systemic consideration, as it can be related to carcinogenicity (Di Sotto et al., 2008). Normally, to evaluate a potential genotoxic risk due to a chemical exposition, in vitro assays for detecting point mutations (Ames test) and extended treatment (e.g., micronucleus assay, single cell gel electrophoresis assay or comet assay) are used in the first instance (EMEA, 2008, Di Sotto et al., 2013). If the results of these studies are positive, in vivo studies, for example a mammalian cytogenetic study, are performed. Recently, also a computational approach has been proposed by the regulatory Agencies to complete the toxicity profile of a compound by in silico predictions (EFSA, 2014).

In this context, in order to provide some toxicological data for the genotoxicity assessment of piperitenone oxide, present study was aimed at evaluating the ability of this flavouring compound to induce in vitro point mutations in bacteria by the Ames test, and in mammalian cells by both the micronucleus and comet assays, so allowing to detect different potential genotoxic endpoints. Particularly, the cytokinesis-block micronucleus technique, with an extended exposure treatment (24 h), was applied for detecting clastogenic and aneugenic effects (Kirsch-Volders et al., 2011, OECD Organisation for Economic Cooperation and Development, 2016); moreover, the alkaline protocol was used for the comet assay (Tice et al., 2000). For both tests, the extended treatment precludes the inclusion of the exogenous metabolic activator, due to the cytotoxicity of S9 mix and to the short half-life of the enzymatic system (Kirsch-Volders et al., 2011, OECD Organisation for Economic Cooperation and Development, 2016).

In addition, a computational evaluation of the piperitenone oxide genotoxicity potential has been performed by using the freely available in silico Toxtree (Estimation of Toxic Hazard - A Decision Tree Approach) and VEGA tools, based on toxicity and QSAR database respectively. In fact, Toxtree can estimate toxic hazards using a decision tree-based approach (Patlewicz et al., 2008). For evaluating a potential mutagenicity, the decision tree is based on the Benigni/Bossa rules and on the structural alerts for genotoxic carcinogens available in the literature (Benigni and Bossa, 2011). VEGA is a QSAR statistical model which relates the chemical structure to mutagenicity by mathematical relationships (Bakhtyari et al., 2013). For the mutagenic predictions, CAESAR (developed by Polytechnic of Milan, Milan, Italy), SarPy/IRFMN (developed by Polytechnic of Milan and “Mario Negri” Institute, Milan, Italy), ISS (developed by Superior Institute of Health, Rome, Italy) and KNN/Read-Across (developed by “Mario Negri” Institute, Milan, Italy) tools were applied.

Section snippets

Extraction and purification of piperitenone oxide

Piperitenone oxide has been isolated from the Mentha suaveolens L. (Fam. Lamiaceae) essential oil, obtained by 4-h hydrodistillation of the mintleaves in Clevenger-type apparatus, as previously described (Angiolella et al., 2010). The analysis of the essential oil was performed by gas chromatography and mass spectroscopy (DMePe BETA PS086, 0.25 mm film, 25 m column length, 0.25 mm diameter, operating temperature of 220 °C, elution with helium) and the constituents were identified by comparison

Bacterial reverse mutation assay

Piperitenone oxide did not give any precipitate up to the highest concentration of 60 μmol/plate (corresponding to 22.6 mM), while produced cytotoxic effects up to the concentration of 6 μmol/plate in all strains tested (Table S1). Mutagenicity was evaluated at nontoxic concentrations (from 0.6 to 5.1 μmol/plate, 1:1.4 dilution factor). When tested in S. typhimurium hisD3052 strains, despite a lack of mutagenicity in the precursor TA1538, the substance produced a statically significant and

Discussion

Piperitenone oxide is a natural fragrance, commonly used as food and cosmetic additive in commercial products. Due to its natural origin and the low amount used as flavouring agent, a relatively scanty and safe consumer exposure has been expected, then its toxicity has been perceived as of minor concern. However, a risk of overexposure due to its natural presence in several essential oils and its widespread use as flavouring additive should not be excluded.

Recently, in order to characterize the

Author contributions

Study design: Di Sotto A. and Mazzanti G.; extraction and purification of piperitenone oxide: Rino Ragno, Mijat Bozovic; chemical analysis of the test substance: Fabio Barile; in vitro experiments: Di Sotto A., Di Giacomo S., Abete L., Parisi O.A., Vitalone A.; in silico genotoxicity predictions: Di Sotto A.; writing the manuscript: Di Sotto A., Di Giacomo S., Izzo A.A., Mazzanti G.; Final approval: all authors.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by “Enrico and Enrica Sovena” Foundation. The Authors thank Prof. Luca Romanelli for him valuable suggestions and advice.

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