Purities were stated if available. Formaldehyde solution (37% in water equal to 37 mg/mL, stabilized with 10% methanol), lysogenic broth (Lennox) powder (including 5 g/L sodium chloride), ampicillin sodium salt, D-(+)-glucose (99.5%), melamine (≥ 99%), aflatoxin B1 (AfB1, > 98%) and fluorescein-di-(β-D-galactopyranoside) (FDG) were bought from Sigma-Aldrich (Steinheim, Germany). Dimedone (≥ 99.0%), and HPTLC plates silica gel 60 with and without fluorescence indicator F254 were delivered by Merck (Darmstadt, Germany). The genetically modified strain Salmonella typhimurium TA1535/pSK1002 was obtained from Trinova Biochem (Giessen, Germany). The S9 rat liver enzymes (phenobarbital/ß-naphthoflavone-induced), nicotinamide adenine dinucleotide phosphate, D-glucopyranose 6-phosphate and buffer salt solution were purchased from Xenometrix (Allschwil, Switzerland). Methanol (gradient grade), dimethyl sulfoxide (≥ 99.5%), chloroform (≥ 99%), ethyl acetate (HPLC), dichloromethane (≥ 99.8) and sodium hydroxide (≥ 98%) were bought from Carl Roth (Karlsruhe, Germany). 4-Nitroquinoline-1-oxide (4-NQO, 98.0%) was purchased from TCI (Eschborn, Germany). Diethyl ether (≥ 99%, stabilized with 2,6-di-tert-butyl-4-methylphenol) and iso-propanol were delivered by Honeywell Riedel-de-Haën (Seelze, Germany). Purified water was prepared by a Destamat Bi 18E (Heraeus, Hanau, Germany). Bamboo tableware was from La Playa Outdoor products & Sporting Goods Company (Swinoujscie, Poland).

Culture medium was prepared by solving lysogenic broth (20 g) in 1 L water, followed by autoclavation. Ampicillin sodium salt (106 mg) and D-(+)-glucose (1 g) were dissolved in 5 mL water and afterwards transferred into the sterile lysogenic broth via a sterilizing syringe filter. An overnight Salmonella culture was prepared by inoculating 20 µL cell cryostock in culture medium (35 mL), which was then incubated (16 h, 37 °C, 100 rpm). Salmonella assay suspension was prepared by diluting the overnight culture of Salmonella cells using fresh culture medium to reach an optical density of 0.2 measured at 660 nm. The FDG substrate solution was prepared by solving 5 mg FDG in 1 mL dimethyl sulfoxide. An aliquot of this solution (25 µL) was transferred into buffer salt solution (2.5 mL). Melamine was dissolved in methanol (1 mg/mL) and further diluted to 10 ng/µL with methanol. Dimedone (2 mg/mL food simulant) and formaldehyde (2 mg/mL food simulant; 5.4 µL of 37% formaldehyde solution dissolved in 995 µL food simulant containing 10% methanol) solutions were prepared, and an aliquot of each standard solution was also neutralized with solid sodium hydroxide. Positive control solutions of 4-NQO and AfB1 (1 mg/mL each) were prepared in dimethyl sulfoxide and diluted with methanol to 1 µg/mL and 0.5 µg/mL, respectively.

A representative 250-mL mug of the bamboo-melamine–formaldehyde resin tableware was filled up to the edge with food simulant simulating hot beverages (250 mL of 3% aqueous acetic acid [7]) and closed with aluminum foil (50 μm thickness, Korff, Oberbipp, Switzerland). After extraction with this food simulant at elevated temperature (70 °C, clean heating chamber) for a prolonged time (2 h), the extract was cooled down to room temperature. An aliquot (30 mL) was neutralized with solid sodium hydroxide (2 small spoon spatulas) to pH 6.8. As blank, the mere food simulant was used and also neutralized in the same way.

For all following HPTLC analyses, instrumentation (Automated TLC Sampler 4, Twin Trough Chamber, Derivatizer, Visualizer 2 and TLC Scanner 3, visionCATS software version 3.0) was used from CAMAG, Muttenz, Switzerland. The extract (30 µL/band) was applied next to the melamine solution (30 ng/band, 3 µL of 10 ng/µL solution) and food simulant blank (30 µL/band, 3% aqueous acetic acid) on the HPTLC plate silica gel 60 F254, developed up to 70 mm using iso-propanol–ethyl acetate–water 10:5:6, V/VV (according to [3] but dichloromethane was substituted with ethyl acetate), documented at FLD 254 nm and densitometrically detected via absorption measurement at 202 nm. On each track at the analogous melamine position (hRF 50), the respective UV spectrum (190−400 nm) was recorded.

Eight acidic and eight neutralized solutions were applied analogously on two HPTLC plates silica gel 60 F254, separated with chloroform–dichloromethane–diethyl ether 4:5:6, V/V/V up to 70 mm and detected at UV 254 nm [4]. The following solutions were applied (mixtures were obtained by overspraying), i.e., of formaldehyde (F, 20 µg/band, 10 µL of 2 µg/µL solution), formaldehyde−dimedone mixture (FD, 20 µg/band each, 10 µL of each 2 µg/µL solution), dimedone (D, 20 µg/band, 10 µL of 2 µg/µL solution), dimedone−mug extract sample mixture (DS, 10 µL/band each), mug extract sample (S, 10 µL/band), mug extract sample−formaldehyde mixture (SF, 10 µL/band, 2 µg/µL formaldehyde in extract), mug extract sample−formaldehyde−dimedone mixture (SFD, 20 µL/band, plus 10 µL of each 1 µg/µL solution of formaldehyde and dimedone in extract), and the food simulant blank, which is 3% aqueous acetic acid used for extraction (B, 10 µL/band).

The acidic and neutralized mug extract sample (each 50, 30, and 10 µL) and corresponding blank solutions (50 µL each) were applied on the HPTLC plate silica gel 60, developed up to 70 mm using chloroform–dichloromethane–diethyl ether 4:5:6, V/V/V [4]. Thereafter positive control solutions (1 µL/band 4-NQO, and for metabolization control, 0.5 µL/band AfB1) were applied. The planar SOS-Umu-C bioassay was performed as described elsewhere [4, 5]. Briefly, Salmonella assay suspension (2.8 mL, yellow nozzle, level 4) were sprayed onto the chromatogram, incubated (3 h, 37 °C) in a humid polypropylene box (26.5 cm × 16 cm × 10 cm, KIS, ABM, Wolframs-Eschenbach, Germany), and dried in a cold airstream (4 min). Then, FDG substrate solution was applied (2.5 mL, yellow nozzle, level 4). After incubation (15 min, 37 °C) in a humid box, the plate was dried in a cold airstream (4 min) and documented at FLD 254 nm. The whole experiment was repeated using a Salmonella suspension containing the S9 mixture system (2.8 mL of a mixture of 3334 µL Salmonella assay suspension 500 µL S9 liver enzyme mixture, 162 µL nicotinamide adenine dinucleotide phosphate, 42 µL D-glucopyranose 6-phosphate and 953 µL buffer salt solution).