2.1 Chemicals and materials

Bidistilled water was prepared with a Destamat Bi18E (Heraeus, Hanau, Germany). Salmonella enterica subspecies enterica Typhimurium bacteria strain TA1535 cryostock containing the plasmid pSK1002 (PTM Salmonella Typhimurium TA1535/pSK1002; DSM no. 9274) was purchased from DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. Sodium dihydrogenphosphate monohydrate (> 98%), magnesium sulfate heptahydrate (99.5%), d-glucose, lysogeny broth (Lennox) powder including 5 g/L sodium chloride, and resorufin-β-d-galactopyranoside, all from Sigma Aldrich, and HPTLC plates, 20 cm × 10 cm, as well as TLC aluminum foils, 20 cm × 20 cm, silica gel 60 with and without F254 were delivered by Merck, Darmstadt, Germany. Cyclohexane, ethanol, and methanol, all Chromasolv, were bought from Fisher Scientific, Seelze, Germany. Potassium dihydrogen phosphate (≥ 99%), potassium chloride (≥ 99.5%), sodium hydroxide (> 99%), and ampicillin sodium salt (> 99%) were purchased from Carl Roth, Karlsruhe, Germany. The 4-nitroquinoline 1-oxide (≥ 98%) was delivered by TCI, Eschborn, Germany. One perfume sample (ID 1) was purchased from a local discounter, and three packaging products (ID 2: packaging material, ID 3: recycled packaging material, and ID 4: raw material for packaging) were obtained for research purposes. Mineral oil saturated/aromatic hydrocarbons (ID 5, 1 mL/ampule MOSH/MOAH mixture consisting of n-undecane, n-tridecane, bicyclohexyl, 5-α-cholestane, 1-methylnaphthalene, 2-methylnaphthalene, n-pentylbenzene, perylene, and 1,3,5-tri-tert-butylbenzene; 150–600 µg/mL each in toluene) were supplied by Restek, Bad Homburg, Germany.

2.2 Sample solutions, enzyme substrate solution, and positive control solution

The perfume was used directly, without the least sample preparation. Two packaging products and one raw material for packaging sample (10 g each cut to 1–2 cm2 pieces) were extracted with 200 mL ethanol for 6 h and concentrated to 5 mL (TurboVap 500, Zymark, Hopkiton, MA, USA) to obtain 2 g/mL sample solutions. From the resorufin-β-d-galactopyranoside substrate stock solution (20 mg/mL in dimethyl sulfoxide), 12.5 μL was dissolved in 2.5 mL phosphate buffer, prepared from disodium phosphate (4.3 g), potassium dihydrogen phosphate (4.1 g), potassium chloride (0.37 g), and magnesium sulfate heptahydrate (0.12 g) in 100 mL bidistilled water, adjusted to pH 7 with solid sodium hydroxide. For use as positive control standard for the bioassay, 4-nitroquinoline 1-oxide was dissolved in methanol (10 ng/µL).

2.3 Preparation of Salmonella cell suspension

As culture medium, lysogeny broth dissolved in bidistilled water (2 g/100 mL) was autoclaved at 120 °C for 20 min. Then, 1 mL each of aqueous glucose (100 mg/mL) and aqueous ampicillin (10 mg/mL) solution was added via a sterilizing 0.2 μm polytetrafluoroethylene syringe filter (VWR, Darmstadt, Germany). For a 16-h overnight culture, 25 μL Salmonella Typhimurium TA1535/pSK1002 cryostock (cell pellet of 10 mL 16-h Salmonella culture, in 10 mL fresh culture medium containing 10% glycerol, and frozen as 0.5 mL cryostock portions) was suspended in 30 mL culture medium in a 100-mL culture flask, and cultivated at 37 °C and 75 rpm in a mini-incubator (Cultura M, Carl Roth) positioned on a Miniature Shaker KM CO2-FL (Edmund Bühler, Bodelshausen, Germany). To adjust an optical density at 600 nm (OD600 [18]) of 0.2, the 16 h overnight culture was 1:10 diluted in fresh culture medium.

2.4 Simple performance of the planar SOS-Umu-C genotoxicity bioassay

If necessary, TLC/HPTLC layers were cut to 5 cm × 10 cm and prewashed with methanol and dried for 10 min in an oven at 110 °C. Sample solutions were manually applied in an interrupted dosing mode using a 1-µL or 2-µL capillary, as spots with a track distance of 6 or 9 mm, distance from the lower edge 8 mm and left edge 10 mm. The plate was developed with 3 mL cyclohexane–ethanol 17:3 or toluene–ethyl acetate 2:3, all V/V, up to 70 mm, taking about 25 min (10 cm × 10 cm twin-trough chamber or similar glass vessel). During experiments, the relative humidity of the surrounding air was 57 ± 3%. After plate drying for 4 min (hairdryer), chromatograms were detected at 254 nm, 366 nm, and white light illumination (reflectance mode).

If necessary, the pH of the plate was controlled to be neutral (pH 7.0 ± 0.4) before bioassay application using a WTW SenTix Sur pH surface measurement electrode (Xylem Analytics, Weilheim, Germany). As positive control for the genotoxicity bioassay, 4-nitroquinoline-1-oxide solution (10 ng/µL) was applied as 0.2-µL and 1-µL spot or band on the upper plate part above the solvent front. The plate was manually immersed into 40 mL Salmonella Typhimurium suspension of OD660 0.2 filled in a small glass dipping chamber (biostep, Burkhardtsdorf, Germany or similar small glass vessel), or it was manually sprayed with about 0.8 mL (2.5 mL for four plate pieces of 5 cm × 10 cm; 200 mL high-density polyethylene bottle with mist spray pump 24 mm neck; IndiaMART InterMESH, Uttar Pradesh, India or Glass Laboratory Sprayer, Macherey–Nagel, Düren, Germany) until visual plate wetness using a simple manual pump sprayer filled with sufficient Salmonella Typhimurium suspension. The wet plate was placed horizontally in a humid box with moistened filter paper lining (KIS 26.5 cm × 16 cm × 10 cm, ABM, Wolframs-Eschenbach, Germany), and the closed box was positioned in an incubator at 37 °C (Memmert, Schwabach, Germany). After incubation at 37 °C for 3 h and plate drying for 5 min, the plate was manually immersed in 40 mL or sprayed with about 0.8 mL (until visual plate wetness) resorufin-β-d-galactopyranoside substrate solution and incubated at 37 °C for 45 min, followed by plate drying and detection under white light illumination (reflectance mode) and FLD 366 nm, optionally also at FLD 254 nm (simple self-made 3D-printed box with LEDs, unpublished device, with image taken by a smartphone camera, or TLC Visualizer, CAMAG, Muttenz).

2.5 Comparison with performance using HPTLC instrumentation

Instrumentation from CAMAG, controlled by visionCATS software version 3.2 SP2, consisted of Automatic TLC Sampler 4, twin-trough chamber 10 cm × 10 cm, Derivatizer, TLC Visualizer 2, and TLC Plate Heater III. Sample solutions were applied analogously to manual application, but using the 100-µL syringe, 6-mm bands, track distance of 9 mm, and “fill only programmed volume” as setting, since only 1 mL of each sample solution was available. Cell suspension as well as substrate solution were piezoelectrically sprayed (2.5 mL, Derivatizer, level 4, red nozzle for cell suspension, yellow nozzle for substrate solution).