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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 heparinized blood into 9 mL of HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated fetal calf serum and 50 μg/mL gentamycin. Phytohaemagglutinin (PHA, reagent grade, Gibco, Loughborough, UK) was included in the culture medium at a final concentration of approximately 2% to stimulate the lymphocytes to divide. Replicate cultures (four for vehicle controls, two for positive controls and each test article concentration) were established for each test condition. Cultures were incubated at 37°C for 48 hours and rocked continuously. Immediately prior to treatment, 0.1 mL of culture medium was removed from the 24 hour continuous cultures to achieve a final pre-treatment volume of 9.3 mL. The S9 mix used for metabolic activation was prepared by mixing glucose-6-phosphate (180 mg/mL), NADP (25 mg/mL), KCl (150 mM) and rat liver S9 in the ratio 1:1:1:2. The final concentration of the liver homogenate in the test system was 2%; an equivalent volume of 150 mM KCl was added to cultures treated in the absence of S9. S9 mix or KCl (0.5 mL per culture) was added and the cultures were treated with perillaldehyde or controls (0.1 mL per culture). Cyclophosphamide (CPA; 12.5 μg/mL), mitomycin C (MMC; 0.08 μg/mL), and vinblastine (VIN; 0.02 μg/mL) were used as indirect and direct-acting positive controls. Cytochalasin B in DMSO (0.1 mL) was added to the 24 hour continuous cultures at the time of treatment. Cultures were incubated at 37°C for 3 (±S9) or 24 (-S9) hours. Following exposure, the 3 hour (±S9) cultures were centrifuged at 300 g for 10 minutes, washed twice with sterile saline (pre-warmed to 37°C), and resuspended in fresh pre- warmed medium containing fetal calf serum, gentamycin and Cytochalasin B at 6 μg/mL. These cultures were allowed to recover at 37°C for 21 hours. At the defined sampling time, cultures were centrifuged at 300 g for 10 minutes. Pelleted cells were resuspended in 4 mL of 0.075 M KCl at 37°C for 4 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (300 g, 10 minutes) and resuspension and the process repeated as necessary (1250 g, 2-3 minutes) until the cell pellets were clean.

Lymphocytes were maintained refrigerated in fixative until at least the following day to ensure adequate fixation. Cells were centrifuged and resuspended in a minimal amount of fresh fixative as required to produce a milky suspension. Several drops of cell suspension were gently spread onto multiple clean microscope slides and allowed to dry. The slides were stained for 5 minutes in filtered 4% (v/v) Giemsa in Gurr’s phosphate buffer, pH 6.8, rinsed, dried, and mounted with coverslips. Slides were examined for proportions of mono-, bi-, and multinucleate cells (500 cells/culture). Slides from the concentration at which approximately 55% (typically 50%-60%) reduction in replication index (RI; [number binucleate cells + 2(number multinucleate cells)]/total number of cells in treated cultures) had occurred and at least two lower concentrations were selected for microscopic analysis, such that a range of cytotoxicity from maximum (not excessive) to little was covered. Coded slides were analyzed by scoring 1000 binucleate cells from each culture (2000 per concentration) for micronuclei.

2.4. in vitro HPRT mutation assay in L5178Y mouse lymphoma cells

The methodology and fluctuation protocol used in this study complies with OECD Test Guideline 476 (OECD, 1997b). The stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells were originally provided by Dr. Donald Clive (Burroughs Wellcome Co.). Cells were cultured in RPMI medium containing 100 U/mL penicillin and streptomycin, 2.5 μg/mL amphotericin B, 0.5 mg/mL Pluronic, and 5 or 10% heat inactivated horse serum. For 3 hour exposures, at least 107 cells in a volume of 18.8 mL of RPMI medium (5% serum) were seeded in sterile 50 mL centrifuge tubes. For 24 hour exposures, at least 4 x 106 cells in a volume of 19.8 mL RPMI medium (10% serum) were seeded in 75 cm2 tissue culture flasks. For all exposure conditions, 0.2 mL of vehicle, perillaldehyde, or positive control solutions was added to the culture. The S9 mix used for metabolic activation was prepared by mixing glucose-6-phosphate (180 mg/mL), NADP (25 mg/mL), KCl (150 mM) and Aroclor 1254-induced rat liver S9 (Moltox, Boone, NC) in the ratio 1:1:1:2. The final concentration of the liver homogenate in the test system was 2%; an equivalent volume of 150 mM KCl was added to cultures treated in the absence of S9. Duplicate (single for positive controls) exposures (±S9) were performed in a final volume of 20 mL. After incubation for 3 hours at 37oC with gentle agitation or stationary incubation at 37°C for 24 hours, cultures were centrifuged at 200 g for 5 minutes, washed, and resuspended in 20 mL RPMI medium (10% serum). Cell densities were adjusted to 2 x 105 cells/mL and cells were transferred to flasks for growth throughout the expression period or were diluted and plated for survival assessment. For survival measurements, 0.2 mL of cells diluted to 8 cells/mL was placed into each well of two 96-well microtiter plates. The plates were incubated at 37oC in a humidified incubator with 5% v/v CO2 in air for 7 days until scoreable. Wells containing viable clones were visually identified using background illumination and counted. For mutation frequency assessment, cultures were maintained in flasks for 7 days to allow for expression of Hprt- mutations. Sub-culturing was performed as necessary to avoid exceeding 1 x 106 cells/mL, retaining at least 6 x 106 cells/flask if possible.

Cultures to be plated for viability and 6-thioguanine resistance were selected on the basis of observations of recovery and growth of the cultures during the expression period. Following the 7 day expression period, cell densities in the selected cultures were adjusted to 1 x 105 cells/mL. For viability assessment, samples from these were further diluted to 8 cells/mL, plated, and scored after 7 to 8 days as described above. For mutation frequency assessment, 6-thioguanine was added to the cultures to give a final concentration of 15 μg/mL and 0.2 mL of each suspension was transferred to each well of four 96-well microtiter plates. Plates were incubated until scoreable (12 to 14 days), and wells containing clones were identified and counted.

From the zero term of the Poisson distribution, the probable number of clones/well (P) on microtiter plates is given by the formula: P = -ln(empty wells/total wells). Plating efficiency (PE) of each culture was calculated as PE = P/number of cells plated per well. Percentage relative survival (% RS) in each test culture was determined by comparing plating efficiencies in test and control cultures: % RS = [PE(test)/PE(control)] x 100. Any loss of cells during the 3 hour exposure period was accounted for by adjusting the % RS values for each test article concentration as follows: adjusted % RS = % RS x (post-treatment cell concentration for test article treatment/post-treatment cell concentration for vehicle control). Mutant frequency (MF) is usually expressed as “mutants/106 viable cells”: MF = [PE(mutant)/PE(viable)] x 106. Since 2 x 104 cells were plated/well for mutation to 6-thioguanine resistance and an average of 1.6 cells were plated/well on viability plates, MF = [P(mutant)/2 x 104]/[P(viable)/1.6] x 106 = (-ln[empty wells/total wells(mutant)]/-ln[empty wells/total wells(viable)]) x 80.

2.5. in vivo Micronucleus/comet assay

2.5.1 Animal husbandry

Male Han Wistar rats (Charles River (UK) Ltd., Margate, UK) were 8-10 weeks of age at the time of treatment. Animals were housed in wire topped, solid bottomed cages (three animals per cage) with European softwood bedding (Datesand Ltd., Manchester, UK), in a temperature and humidity controlled AAALAC accredited facility with a 12 hour light/12 hour dark cycle. SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd., Witham, UK) and water were provided ad libitum. Animals were provided wooden Aspen chew blocks and rodent retreats for environmental enrichment and were acclimatized for at least 5 days prior to initiation of dosing.

2.5.2. Experimental design

A combined micronucleus and comet assay was conducted in accordance with OECD test guideline 474 (OECD, 1997c) for the micronucleus assay and current recommendations for the comet assay at the time the study was conducted (Burlinson et al., 2007; EFSA, 2012; Tice et al., 2000). A dose range-finding study was conducted to estimate the maximum tolerated dose (MTD) of perillaldehyde in male and female rats. Since no substantial gender differences in toxicity were revealed in the range-finder experiment, male rats were selected for the MN/comet assay. Five groups of male rats (6 rats/group) were administered perillaldehyde at dose levels of 175, 350, and 700 mg/kg/day (estimated to be 25% MTD, 50% MTD, and MTD, respectively), vehicle (corn oil), or the positive control compound, ethyl methanesulfonate in purified water at 150 mg/kg/day, by oral gavage daily for three days (0, 24 and 45 hours). Three hours after the final dose the animals were euthanized and femurs were collected for analysis of micronuclei in the bone marrow, liver and duodenum tissues were collected for the comet assay and histopathology, and blood samples were collected for clinical chemistry as described below.

2.5.3. Erythrocyte micronucleus assay

Femurs were cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrow was flushed from each femur with 2 mL of fetal bovine serum (FBS). The samples were filtered through cellulose columns containing an equal mix of type 50 and α-cellulose at 50 mg/mL. Once the majority of the FBS had passed through the column an additional 4 mL of FBS were added to the sample tubes and loaded onto the columns. The filtered bone marrow cells were centrifuged at 200 g for 5 minutes at 15-25°C and the cell pellets were gently resuspended in 3 mL of FBS and pelleted again; the supernatant was aspirated to leave one or two drops used to resuspend the cell pellet using a Pasteur pipette. One drop of cell suspension was placed on the end of each slide and smeared by drawing the end of a clean slide along the sample slide. Slides were air-dried prior to fixing for 10 minutes in absolute methanol, rinsed several times in distilled water, and air-dried. One slide per animal was immediately stained for 5 minutes in 12.5 μg/mL acridine orange in 0.1 M phosphate buffer pH 7.4. Stained slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature. At least 500 erythrocytes were analyzed for the relative proportions of polychromatic erythrocytes (PCE), visible as bright orange enucleated cells, and normochromatic erythrocytes (NCE), visible as smaller dark green enucleated cells. At least 2000 PCE/animal were examined for the presence of micronuclei. All slides were coded to eliminate scorer bias.