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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

In vitro genotoxicity testing of carvacrol and thymol using the micronucleus and mouse lymphoma assays

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Sara Maisanaba a,∗ , Ana I. Prieto a , Maria Puerto a , Daniel Gutiérrez-Praena a , Es¸ref Demir b , Ricard Marcos c,d , Ana M. Cameán a a

Area of Toxicology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain Department of Biology, Faculty of Arts and Sciences, Akdeniz University, Antalya, Turkey c Group of Mutagenesis, Department of Genetics and Microbiology, University Autonoma of Barcelona, Cerdanyola del Vallès, Barcelona, Spain d CIBER Epidemiology and Public Health, Instituto de Salud Carlos III, Spain b

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Article history: Received 13 February 2015 Received in revised form 7 May 2015 Accepted 10 May 2015 Available online xxx

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Keywords: Carvacrol Thymol Micronucleus test Mouse lymphoma assay Food packaging

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1. Introduction

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Currently, antimicrobial additives derived from essential oils (Eos) extracted from plants or spices, such as Origanum vulgare, are used in food packaging. Thymol and carvacrol, the major EO compounds of O. vulgare, have demonstrated their potential use as active additives. These new applications use high concentrations, thereby increasing the concern regarding their toxicological profile and especially their genotoxic risk. The aim of this work was to investigate the potential in vitro genotoxicity of thymol (0–250 ␮M) and carvacrol (0–2500 ␮M) at equivalent doses to those used in food packaging. The micronucleus (MN) test and the mouse lymphoma (MLA) assay on L5178Y/Tk± mouse lymphoma cells were used. The negative results for thymol with the MN with and without the S9 fraction and also with the MLA assay reinforce the view that this compound is not genotoxic in mammalian cells. However, carvacrol presented slight genotoxic effects, but only in the MN test at the highest concentration assayed (700 ␮M) and in the absence of metabolic activation. The lack of genotoxic response in the MLA assay after 4 and 24 h of exposure indicates a low genotoxic potential for carvacrol. Alternatively, the general negative findings observed in both assays suggest that the MN results of carvacrol are marginal data without biological relevance. These results can be useful to identify the appropriate concentrations of these substances to be used as additives in food packaging. © 2015 Published by Elsevier B.V.

Essential oils (Eos) and their main components are used as additives in the food industry due to their flavour, antimicrobial, 27 and antioxidant properties [1–4]. The use of Eos as food preserva28 tives has been limited because high concentrations are required to 29 reach sufficient activity [8]. Active packaging is a new preservation 30 system where volatile compounds from Eos create a preservative 31 atmosphere. This is considered a good alternative to the direct 32 incorporation of these oils into food, avoiding undesirable sec33 34Q2 ondary effects [6–7]. Eos derived from oregano species, including its major com35 ponents carvacrol and thymol, are currently among the most 36 frequently used in active food packaging. At present, the usefulness 37 26

∗ Corresponding author at: Area of Toxicology, Faculty of Pharmacy, University of Sevilla, C/Profesor García González n◦ 2, 41012 Sevilla, Spain. Tel.: +34 954 556762; fax: +34 954 556422. E-mail address: [email protected] (A.M. Cameán).

of these compounds as substitutes of the common synthetic antioxidants (butylated hydroxyanisole and butylated hydroxytoluene) is under discussion [8]. Because the use of carvacrol and thymol and Eos in general requires high doses, there is increasing concern regarding potential exposure to these compounds. Consequently, more research is needed to establish effective and safe concentrations of Eos and their components [9–10]. In accordance with the Guidelines of the Scientific Committee on Food for Safety Assessment of Substances Used in Food Contact Materials, the core set of tests used to construct a profile of the risks/benefits associated with the use of Eos and their components as food contact materials consists of the following 3 in vitro genotoxicity studies: (1) a test of the induction of gene mutations in bacteria; (2) a test for the induction of gene mutations in mammalian cells (preferably the mouse lymphoma Tk assay); and (3) a test for the induction of chromosomal mutations (chromosome aberrations, CA or micronucleus MN tests) in mammalian cells [11–13]. Among the different in vitro mammalian gene-mutation assays the mouse lymphoma assay (MLA) is the most widely used [14]. Similarly, the in vitro MN assay is increas-

http://dx.doi.org/10.1016/j.mrgentox.2015.05.005 1383-5718/© 2015 Published by Elsevier B.V.

Please cite this article in press as: S. Maisanaba, et al., In vitro genotoxicity testing of carvacrol and thymol using the micronucleus and mouse lymphoma assays, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2015), http://dx.doi.org/10.1016/j.mrgentox.2015.05.005

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ingly used in the evaluation of Eos [15–17] instead of the classical CA assay [18–20]. Regarding the genotoxic potential of thymol and carvacrol, several studies have been conducted with different results, depending on the assay system and the range of concentrations used [21–27]. In bacterial systems, negative results were obtained for thymol using the Ames test [27], whereas for carvacrol, the results were contradictory [28–29]. Recent studies from our laboratory found that carvacrol exhibited mutagenic potential, being more active in the presence of a metabolic source [27]. When the comet assay was used, conflicting results were reported. Although Aydin et al. [21] demonstrated that carvacrol and thymol induced DNA damage in human lymphocytes, Horváthová et al. [23] concluded that the compounds were not associated with a DNA-damaging effects on human hepatoma HepG2 and colonic Caco-2 cells. Taking these facts into account, the aim of the present work was to investigate the potential in vitro genotoxicity/mutagenicity of carvacrol and thymol at relevant concentrations, according to their possible use in food packaging. MN and MLA tests using the L5178Y/Tk± mouse lymphoma cell-line were performed. Because no previous data exist for the selected genotoxicity assays, the obtained data are relevant to assess or confirm their potential health hazards for humans.

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2. Material and methods

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2.1. Chemicals

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Thymol (2-isopropyl-5-methylphenol, 99.5%; CAS No. 89-83-8), carvacrol (5-isopropyl-2-methylphenol, 98%; CAS No. 499-75-2), mitomycin (MMC, CAS No. 50-07-7), cyclophosphamide (CP, CAS No. 6055-19-2), methyl methanesulfonate (MMS, 99% purity; CAS No. 66-27-3), thiazolyl blue tetrazolium bromide (MTT, 99.7% purity; CAS No. 298-93-1), trifluorothymidine (TFT, ≥99% purity; CAS No. 70-00-8), thymidine (CAS No. 4449-43-8), hypoxanthine (99% purity; CAS No. 68- 94-0), methotrexate (CAS No. 59-05-2), glycine (≥99% purity; CAS No. 56-40-6), cythochalasin B (Cyt-B, 98%, CAS No. 14,930-96-2), Giemsa stain (CAS No. 51,811-82-6), and trypan blue solution 0.4% (CAS No. 72-57-1) were purchased from Sigma–Aldrich (Madrid, Spain). RPMI 1640 medium, horse serum, l-glutamine solution (CAS No. 56-85-9), penicillin/streptomycin solution, sodium pyruvate solution (CAS No. 113-24-6), and amphotericin B solution (CAS No. 1397-89-3) were purchased from Gibco (Biomol, Sevilla, Spain).

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2.2. Cells and culture conditions

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L5178Y/Tk± mouse lymphoma cells were used for both assays and were provided by Dr. Olivier Gillardeux (Safoni-Synthélabo, Paris, France). Cells were confirmed as free of mycoplasma by PCR. L5178Y/Tk± cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, 2 mM l-glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, 1 mM sodium pyruvate, and 2.5 g/mL amphotericin B. Cells were routinely diluted at 2 × 105 cells/mL each day to prevent overgrowth. Cell density was determined with an automated cell counter (Invitrogen® ). Cultures were maintained in a humidified incubator with 5% CO2 at 37 ◦ C.

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2.3. Test solutions

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The concentration ranges of thymol and carvacrol were selected considering the content of these active compounds in packaging materials and their possible migration to food, which represents the worst case scenario of exposure [30]. Stock solutions of thymol (0.5 M) and carvacrol (2.5 M) were prepared in dimethyl sulfoxide (DMSO). RPMI 1640 medium was used

to prepare the different tested solutions, in which the final quantity of DMSO was always less than 0.1%. RPMI 1640 medium served as a negative control, and MMC (0.0625 ␮g/mL), CP (8 ␮g/mL), and MMS (10 ␮g/mL) were used as positive controls. Each solution was prepared immediately prior to its use. 2.4. Micronucleus test (MN) This assay was performed according to the procedure OECD 487 from the Guideline for the Testing of Chemicals [31]. Two duplicate cultures/concentrations were stablished in T25 flasks in the absence or presence of the S9 metabolic activation system. 2.4.1. EC50 determination for the MN test L5178Y/Tk± cells were exposed to different concentrations of thymol (0–250 ␮M) or carvacrol (0–2500 ␮M), and the viability was assessed using the trypan blue exclusion test [32] to establish the half maximal effective concentration (EC50 ). Cells were seeded at 2 × 105 cells/mL and were exposed for 24 h when the metabolic S9 fraction was not used or for 4 h when this fraction was used. 2.4.2. Micronucleus assay L5178Y/Tk± cells were seeded at a concentration of 2 × 105 cells/mL and were incubated for 24 h at 37 ◦ C in a 5% CO2 atmosphere. Then, culture media were removed from the flask by centrifugation (600 rpm, 6 min), and the cells were treated with five different concentrations of thymol (0–250 ␮M) or carvacrol (0–700 ␮M), selected according to the EC50 (without or with S9) previously obtained. RPMI medium was used as a negative control, and MMC 0.0625 ␮g/mL (in absence of S9) or CP 8 ␮g/mL(in presence of S9) were used as positive controls. The exposure times were 24 h in absence of S9 or 4 h in the presence of S9, according to the OECD guidelines [31]. After these periods, cells were exposed to Cyt-B (6 ␮g/mL) for 20 h. Then, cultures were centrifuged (600 rpm, 6 min), the supernatant was removed, and cells were subjected to a hypotonic treatment (KCl 0.051 M, 20 min, room temperature). After this time, cells were centrifuged and fixed with methanol/acetic acid (3:1). The resultant pellets were resuspended and dropped on microscope slides. After drying, cells were stained with Giemsa 10% solution for 3–5 min. All slides were encoded by a person unconnected with the study, and the scoring was conducted by a different person using an Olympus BX61 light microscope at 100X. Micronuclei frequencies were analysed in at least 2000 binucleated cells per concentration. In addition, cells were scored to evaluate the nuclear division index (NDI) [33]. 2.5. Mouse lymphoma thymidine-kinase assay (MLA) For a successful outcome of the MLA, L5178Y/Tk± cells were subjected to a cleansing to purge excess possible Tk−/− mutants by culturing cells for 24 h in THMG medium (thymidine 9 ␮g/mL, hypoxantine 15 ␮g/mL, methotrexate 0.3 ␮g/mL, glycine 22.5 ␮g/mL). Then, cells were transferred to THG medium (THMG without methotrexate) for 2 days. The purged cultures were checked for a low background of Tk−/− mutants and were stored in liquid nitrogen. The MLA was performed according to Soriano et al. [34]. Preliminary experiments were conducted to determine the cytotoxicity of thymol or carvacrol. Cytotoxicity was determined by the relative total growth (RTG) after a treatment of 4 h with thymol (0–250 ␮M) or 4 h and 24 h with carvacrol (0–2500 ␮M), without an external metabolic activation source. The recommended highest concentration was one with an RTG of 20% [35]. Each main experiment comprised a negative control (RPMI 1640 medium), a positive

Please cite this article in press as: S. Maisanaba, et al., In vitro genotoxicity testing of carvacrol and thymol using the micronucleus and mouse lymphoma assays, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2015), http://dx.doi.org/10.1016/j.mrgentox.2015.05.005

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Fig. 1. Cell viability of thymol (A) and carvacrol (B). Treatment lasted for 3–6 h in the presence of an S9 mix and for 24 h in the absence of the S9 mix. Results expressed as the mean ± SD. *Significant differences at P < 0.01.

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control (MMS 1 ␮g/mL), and six concentrations for each tested compound. RTG values were used to decide on the acceptability of the toxicity at each dose level. Thereby, the selected ranges for thymol and carvacrol, taking into account the obtained RTG values, were 0–250 ␮M and 0–1500 ␮M, respectively (Tables 5–7). Once the exposure concentrations were selected, cells were plated at a density of 104 cells/mL in 96-well plates (two replicates per experimental group) to evaluate the viability of cells after an incubation of 12 days at 37 ◦ C and 5% CO2 . After this time, viable colonies were counted. In addition, two more replicates per experimental group were exposed to TFT 4 ␮g/mL for the mutation analysis. Plates were also incubated for 12 days at 37 ◦ C and 5% CO2 . To assist the scoring of TFT mutation colonies, MTT 2.5 mg/mL was added to each well and the plates were incubated for 4 h. After this time, the mutant colonies of each plate were counted. Colony size was estimated in a similar manner to that described by Honma et al. [36].

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2.6. Statistical analysis

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The induced mutant frequency (IMF) was determined according to the formula IMF = MF − SMF, where MF is the test culture mutant frequency and SMF is the spontaneous mutant frequency. Positive

responses are determined as those that for any treatment meet or exceed the global evaluation factor (GEF, 126 for the microwell assay) and also when a positive trend test is obtained. The statistical approach included the one-way ANOVA followed by Dunnett’s test, which was used to evaluate the significance of the differences in micronucleated cells, NDI and MF between the control and treated cultures. Results were considered statistically significant when the P-value was