2 nitrofluorene

Rats were fed corn oil (Sigma-Aldrich, St. Louis, MO, USA) and laboratory autoclavable rodent diet 5010 (PMI® Nutrition International, Inc., St. Louis, MO, USA). For the Ame test, sodium azide, mitomycin C, cyclophosphamide, acridine mutagen ICR 191, 2-nitrofluorene, 2-aminofluorene, and 2-aminoanthracene were used as positive controls (Sigma-Aldrich), while the test sample were prepared with dimethyl sulfoxide (DMSO) (Merck, Hessen, Darmstadt, Germany).

Five characterized histidine-dependent strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535, TA1537; MFDS, Osong, Korea) were utilized for bacterial reverse mutation assay (Ames test) in accordance with OECD guideline 471 [27 ]. S. typhimurium strains were incubated with the AA extract with or without an S9 mix in the dark at 37℃ for 48 h. The standard mutagens (2-nitrofluorene, sodium azide, mitomycin C, 9-aminoacridine, and 2-aminoanthracene; Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls. The extract was considered to be positive if there was a two-fold increase relative to negative control or a dose-dependent increase in the number of revertant colonies.

Chemicals and reagents were obtained from the following suppliers: nutrient broth from Oxoid Ltd. (Basingstoke, UK), water (CAS No.7732-18-5) from Baxter (Newbury, UK), glucose (CAS No. 50-99-7), magnesium sulphate (CAS No. 7487-88-9), potassium chloride (CAS No. 7447-40-7) and sodium phosphate buffer from Fisher Scientific (Loughborough, UK), magnesium chloride (CAS No. 7786-30-3) from VWR (Radnor, PA, USA), citric acid (CAS No. 77-92-9), d-biotin (CAS No. 58-85-5), glucose-6-phosphate (CAS No. 3671-99-6), histidine (CAS No. 71-00-1) and sodium ammonium phosphate tetrahydrate (CAS No. 7783-13-3) from Sigma-Aldrich Co. Ltd. (Poole, UK), nicotinamide adenine dinucleotide phosphate (NADP) (CAS No. 698999-85-8) and rat liver S9 from Molecular Toxicology Inc. (Boone, NC, USA), Bactoagar from Becton Dickinson and Co. (Oxford, UK), dipotassium phosphate (CAS No. 7758-11-4) from Camlab (Cambridge, UK).
Positive control chemicals included 9-aminoacridine (CAS No. 90-45-9), 2-aminoanthracene (CAS No. 613-13-8), benzo[a]pyrene (CAS No. 50-32-8), mitomycin C (CAS No. 50-07-7), 2-nitrofluorene (CAS No. 607-57-8) and sodium azide (CAS No. 26628-22-8) all from Sigma-Aldrich (Poole, UK). Antibiotics comprised ampicillin (CAS No. 69-53-4) and tetracycline (CAS No. 60-54-8) from Sigma-Aldrich (Poole, UK).

The following chemicals—2-aminoanthracene (2-AA), benzo[a]pyrene (B[a]P), sodium azide (SA), 2-nitrofluorene (2-NF), 4-nitroquinoline-1-oxide (4NQO), acridine mutagen ICR 191 (ICR-191), cyclophosphamide monohydrate (CPA), acridine orange solution (AO), ethyl methanesulfonate (EMS), dimethylsulfoxide (DMSO), potassium chloride (KCl), magnesium sulfate, citric acid monohydrate, potassium phosphate dibasic anhydrous, sodium ammonium phosphate, glucose, sodium chloride, tryptophan, histidine, biotin, methyl alcohol, and glacial acetic acid—were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Oxoid Nutrient Broth No. 2 and Bacto agar were purchased from Thermo Scientific Inc. (Waltham, MA, USA) and BD (Franklin Lakes, NJ, USA), respectively. S9 mix (5% for bacterial reverse mutation assay and 30% for chromosomal aberration assay, v/v) was prepared using Aroclor 1254-induced rat liver S9 (Molecular Toxicology Inc., Boone, NC, USA) supplemented with cofactor-I (8 μM MgCl₂, 33 μM KCl, 5 μM glucose-6-phosphate, 4 μM nicotinamide adenine dinucleotide phosphate, 4 μM nicotinamide adenine dinucleotide, 100 μM sodium phosphate buffer, pH 7.4; Wako Pure Chem. Ind., Osaka, Japan). S9 mix was prepared fresh prior to use and kept on ice during the experiment. Information on chemicals and reagents not described here is supplied in the relevant sections in “Materials and methods” section.

TGAIs imidacloprid, imazalil and tebuconazole, separately and in combination, were studied using Ames test (Salmonella typhimurium) according to OECD Guideline № 471 [26 ]. The plate incorporation method both in the absence and in the presence of an exogenous metabolic activation system (S9) was used as described in one of our previous publications [27 (link)].
Bacteria were exposed to individual pesticides in concentrations up to 5.0 (imidacloprid), 1.25 (imazalil), 2.5 (tebuconazole) mg/plate and to their mixture in concentrations of 0.05, 0.16, 0.5, 1.6 and 2.5 mg/plate. Maximum concentrations were chosen based on the results of preliminary experiments for the cytotoxicity assessment on TA100 strain. 2-aminoantracene (Sigma-Aldrich), sodium azide (Sigma-Aldrich), 2-nitrofluorene (Sigma-Aldrich), 9-aminoacridine (Fluka), methyl methanesulfonate (Sigma-Aldrich) were taken as a positive control. The incubation of bacteria was conducted at 37 ± 2 °C for 48−72 h. For revertant counting SCAN® 500 Interscience was used.

Bacto agar was purchased from BD Chemicals; Nutrient broth No.: 2 was purchased from Oxoid Limited, UK; L-histidine, D-biotin, benzo(a)pyrene, mitomycin-c, 2-nitrofluorene, 9-aminoacredine, sodium azide was procured from Sigma Aldrich. Merck chemicals were supplied sodium chloride, sodium phosphate monobasic, sodium phosphate dibasic, and potassium chloride. D-glucose-6-phosphate and Nicotinamide Adenine Dinucleotide Phosphate were provided by HiMedia, a company based in India. Moltox USA delivered the Aroclor 1254-induced male Wistar rats' S9 liver homogenate.