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The rat alveolar macrophage cell line NR8383 was cultured at 37 °C and 5% CO₂ in 175 cm² culture flasks in F-12K medium prepared from powder medium (Sigma-Aldrich, N3520, Germany), supplemented with 15% heat-inactivated fetal calf serum, glutamine (2 mM), and 100 U penicillin and 10 mg/mL streptomycin. Experiments were performed as described with minor modifications [21]. In brief, cells were incubated with increasing concentrations of particles in serum-free F-12K medium. Therefore, the aqueous particle stock suspensions were diluted to a working concentration of 360 μg/mL. These aqueous suspensions were further diluted in an equal volume of double-concentrated F-12K cell culture medium or double-concentrated Krebs–Ringer buffer containing 2 mmol/L glucose (KRPG), to achieve cell compatibility of the suspensions. F-12K medium or KRPG were used for all further dilution steps. NR8383 cells (3 × 10⁵ per well of a 96-well plate) were incubated with increasing concentrations of particle suspensions prepared in serum-free F-12K medium for 16 h. Supernatants were retrieved and centrifuged at 200× g for 10 min. Each supernatant was analyzed for lactate dehydrogenase activity (LDH), glucuronidase activity (GLU), and tumor necrosis factor α (TNFα) using a specific enzyme-linked immunosorbent assay (ELISA) for rat TNF-α (bio-techne, Wiesbaden, Germany). LDH was measured with the Cytotoxicity Detection Kit (Merck KGaA, Darmstadt, Germany) and GLU was measured photometrically using p-nitrophenyl-β-D-glucuronide as a substrate. Both enzyme activities were expressed as a % of the positive control value, which was obtained by adding 0.1% triton X-100 to an equal number of non-particle-treated cells. Bioactive TNF-α was measured indirectly via induction of apoptosis/necrosis in L-929 fibroblasts in the presence of actinomycin D and was expressed as % killing activity [22]. The release of H₂O₂ was measured 90 min after the addition of particles, which were suspended in KRPG. H₂O₂ concentration was determined quantitatively using resorufin as a detection reagent in the presence of horseradish peroxidase. All assays were run in 96-well plates and repeated three times. Vehicle-treated cells were used as negative controls. Cell-free wells were processed in the same way and used to exclude particle interference.