2. Material and methods
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.