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Cheryl A. Hobbs, Sean Taylor, Carol Beevers, Melvyn Lloyd, Rachael Bowen, Lucinda Lillford, Robert Maronpot, and Shim-mo Hayashi
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Perillaldehyde, a natural monocyclic terpenoid found most abundantly in the herb perilla, has a long history of use as a flavouring ingredient to add spiciness and citrus taste to foods. Previously, it was judged to be safe by several international expert panels. To confirm the safety of flavourings placed on the European Union list of flavourings, perillaldehyde was selected by the European Food Safety Authority as a representative of a subgroup of alicyclic aldehyde flavouring substances to be evaluated for genotoxic potential. Perillaldehyde was tested in a bacterial reverse mutation assay, an in vitro micronucleus assay in human lymphocytes, an HPRT assay in mouse lymphoma cells, and a micronucleus/comet assay in Han Wistar rats. In contrast to previously published results, perillaldehyde induced mutation in Salmonella typhimurium strain TA98 in the absence of metabolic activation. The comet assay was negative for duodenum and weakly positive for liver but only at a hepatotoxic dose of perillaldehyde. All other genotoxicity assays were negative. These data do not provide an indication of any genotoxic potential for perillaldehyde, and they provide the primary basis for recent scientific opinions regarding perillaldehyde genotoxicity announced by several international organizations responsible for safety assessment of food additives and flavourings.

Key Words: genotoxicity, flavouring agent, perillaldehyde, p-mentha-1,8-dien-7-al, perilla aldehyde, DNA damage

Abbreviations: EFSA, European Food Safety Authority; FAO, Food and Agriculture Organization of the United Nations; FEMA, Flavor and Extract Manufacturers Association; GLP, Good Laboratory Practice; HPRT, hypoxanthine-guanine phosphoribosyl transferase; ICH, International Conference on Harmonisation of Technical Requirements for Pharmaceuticals for Human Use; JaCVAM, Japanese Center for the Validation of Alternative Methods; JECFA, FAO/WHO Joint Expert Committee on Food Additives; ln, natural log; MN, micronucleus or micronuclei; MN-PCE, micronucleated polychromatic erythrocytes(s); OECD, Organization for Economic Cooperation and Development; PCE, polychromatic erythrocytes; (Q)SAR, quantitative structure activity relationship; UKEMS, United Kingdom Environmental Mutagen Society; WHO, World Health Organization

1. Introduction

Perillaldehyde (also known as l-perillaldehyde, perilla aldehyde, l-perilla aldehyde, and p- mentha-1,8-dien-7-al) is a natural compound found abundantly in the annual herb perilla and the peel of citrus fruits. Perillaldehyde and volatile oils from perilla rich in perillaldehyde are used as flavouring agents to add spiciness and a woody, citrus taste to foods such as baked goods, puddings, meat products, salad dressing, sauces, pickled vegetables, and beverages. It is also used for its mint-like cinnamon odor in the perfume industry and is being investigated for potential hypolipidemic, anti-inflammatory, neuroprotective, antidepressant-like, and anti-fungal effects (Ji et al., 2014; Omari-Siaw et al., 2016; Tian et al., 2015; Xu et al., 2014). Perillaldehyde is considered as “generally recognized as safe” (GRAS) by the Expert Panel of the U.S. Flavor and Extract Manufacturers Association (FEMA) (Oser and Ford, 1978), was judged to be safe by the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Joint Expert Committee on Food Additives (JECFA) (JECFA, 2003), and has been designated unlikely to harm human health by the Japanese Ministry of Health, Labour and Welfare under the Food Sanitation Act ( since 1948.

Perillaldehyde is a monocyclic terpenoid containing an α,-unsaturated aldehyde functional group (Fig. 1). It is quickly metabolized, largely by oxidation of the side chain to a carboxylic acid, which is excreted unchanged and as conjugates (JECFA, 2003). In a safety assessment program for confirming the safety of flavourings listed on the European Union (EU) list, the European Food Safety Authority (EFSA) Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids requested additional data related to the possible genotoxic potential of flavouring substances containing α,β-unsaturated aldehyde and ketone structures, or their potential precursors

[Flavouring Group Evaluation 19 (FGE.19)]. These requests were driven by the structural alerts for potential genotoxicity associated with α,-unsaturated carbonyl compounds, which are believed to react with nucleophilic sites in DNA through a 1,4- nucleophilic addition (Michael reaction). To facilitate the data collection, the FGE.19 list of compounds was divided into 19 subgroups and representative substances were selected for each subgroup on the basis of chain length, branching, lipophilicity, possible additional functional groups, and consideration of the possible influence of substituents on the Michael reaction and (Q)SAR predictions (EFSA, 2008b). Perillaldehyde was selected as a chemical for which genotoxicity data could be considered representative of the other substances in subgroup 2.2, which included p-menth-1,8-dien-7-ol, myrtenol, myrtenal, 2,6,6-trimethyl-1-cyclohexen-1- carboxaldehyde, myrtenyl formate, p-mentha-1,8-dien-7-yl acetate, myrtenyl acetate, myrtenyl 2-methylbutyrate, and myrtenyl 3-methylbutyrate. Relevant industries were requested to submit genotoxicity study data for substances considered representative of a subgroup, including perillaldehyde.

In response to the request of EFSA for genotoxicity data for use in risk assessment, perillaldehyde was evaluated in a Good Laboratory Practice (GLP)-test battery compliant with EFSA and OECD guidance on genotoxicity testing (EFSA, 2008a, 2012; OECD, 1997a, b, c, 2008). Specifically, perillaldehyde was evaluated in a bacterial reverse mutation assay (OECD 471) using Salmonella and E. coli tester strains, an in vitro MN assay using human peripheral blood lymphocytes (OECD 487), and an in vitro HPRT mutation assay in mouse L5178Y lymphoma cells (OECD 476). In addition, a combined MN (OECD 474) and comet assay was conducted in male Han Wistar rats. Although an OECD test guideline for the in vivo comet assay did not exist at the time this study was conducted, the assay was performed in accordance with recommendations of EFSA and expert working groups (Burlinson et al., 2007; EFSA, 2012; Tice et al., 2000). The results of this comprehensive genotoxicity testing of perillaldehyde are reported.

fig 1

2. Material and methods

2.1. Chemicals

All genotoxicity assays were conducted according to OECD test guidelines or EFSA guidance (comet assay) and were GLP-compliant with the exception that dose formulations were not analyzed for achieved concentration. Perillaldehyde (91.9-94.2% pure; CAS No. 2111-75-3; Nippon Terpene Chemicals, Inc., Kobe, Japan) solutions were prepared by dissolving in anhydrous analytical grade dimethyl sulfoxide (DMSO) or suspending in corn oil under subdued light conditions with continual stirring before and during dosing. The test article solutions were protected from light and used within 2 (MN/comet and mouse lymphoma assays) or 5 (bacterial mutagenicity and in vitro MN assays) hours of initial formulation. Sodium azide, mitomycin C, vinblastine, and ethyl methanesulfonate were formulated in water; 2-nitrofluorene, 9- aminoacridine, benzo[a]pyrene, 2-aminoanthracene, and cyclophosphamide were formulated in DMSO. Aliquots of stock solutions were either prepared in advance and stored refrigerated (benzo[a]pyrene and mitomycin C for the bacterial reverse mutation test) or frozen at -80°C (2- nitrofluorene, 9-aminoacridine, sodium azide, and cyclophosphamide) in the dark, or they were prepared immediately prior to use (ethyl methanesulfonate, vinblastine, and mitomycin C for the in vitro MN assay). All chemicals and reagents were obtained from Sigma-Aldrich Chemical Co. (Poole, UK) or equivalent suppliers unless specifically stated otherwise.

2.2. Bacterial reverse mutation assay

The procedures used in this study were in accordance with OECD Guideline 471 (OECD, 1997a). Mutagenicity assays of perillaldehyde, with and without metabolic activation, were conducted using the following five strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537 and TA102). All the tester strains, with the exception of strain TA102, were originally obtained from the UK NCTC. Strain TA102 was derived from a culture obtained from Glaxo Group Research Limited. All strains were checked previously for the maintenance of genetic markers. Metabolic activation was provided by 10% liver post-mitochondrial fraction (S9) prepared from male Sprague Dawley rats induced with Aroclor 1254 (Moltox, Boone, NC). The composition of the S9 mix was: 10% S9, 8 mM MgCl2, 33 mM KCl, 1.5 mg/mL glucose-6- phosphate, 3.2 mg/mL -nicotinamide adenine dinucleotide phosphate (NADP), 0.1 M phosphate buffer, 40 μg/mL L-histidine HCl (in 250 mM MgCl2), and 49 μg/mL d-biotin. Strain specific positive controls tested without metabolic activation were 2-nitrofluorene (TA98; 5 μg/plate), sodium azide (TA100 and TA1535; 2 μg/plate), 9-aminoacridine (TA1537; 50 μg/plate), and mitomycin C (TA102; 0.2 μg/plate). Benzo[a]pyrene (10 μg/plate) and 2- aminoanthracene (TA100, TA1535, TA1537 at 5 μg/plate; TA102 at 20 μg/plate) were used as the positive controls for TA98, and all other strains, respectively, tested with metabolic activation. Bacteria were cultured at 37°C for 10 hours in nutrient broth containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate. Incubation was carried out with shaking in an anhydric incubator. All treatments were completed within 6 hours of the end of the incubation period. For plate incorporation assays, bacteria, control or test article formulation, and 10% S9 mix or buffer were added to molten agar at 46±1°C, mixed rapidly, and poured onto Vogel-Bonner E plates. For the pre-incubation assay, perillaldehyde or control formulation, bacteria, and 10% S9 mix were mixed and incubated at 37±1°C for 1 hour prior to the addition of molten agar and plating. Once set, triplicate plates per concentration (five plates for vehicle control) were incubated at 37°C in the dark for 3 days. Colonies were counted using the Sorcerer Colony Counter (Perceptive Instruments, Ltd., Suffolk, UK) or manually when confounding factors such as precipitation affected the accuracy of the automated counter. The background lawn was inspected for signs of toxicity. Data were confirmed to meet the following  acceptability criteria: 1) the mean vehicle control counts fell within the laboratory’s historical 99% confidence intervals for group means and/or 2) each vehicle control plate count fell within the historical 99% reference ranges, and 3) the positive control plate counts were comparable with the historical 99% reference ranges.

2.3. in vitro Micronucleus assay

The methodology used in this study was based on draft OECD Test Guideline 487 (OECD, 2008). An appropriate volume of whole blood from two healthy, non-smoking male volunteers was drawn from the peripheral circulation into heparinized tubes within two days of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled