For the investigations in the cutting mill SM300 (Retsch GmbH, Germany) equipped with a 10 mm screen is used while the rotational speed, and the feedstock size are varied. The organic sheets are cut into pieces of 30 × 30 × 1 mm or 15 × 15 × 1 mm size to analyze the effect of different feedstock sizes. Samples of 50 g each are prepared with the respective size and are then cut with the cutting mill for 1 minute each. The rotational speed is varied from 800 rpm, 1500 rpm to 2500 rpm. The ground material is sieved for 20 minutes to receive the particle size distribution and to separate the fiber dust from the rest of the material. The matrix of the fraction 0.125 mm is ashed to retain the pure fibers. The length of the fibers is analyzed using the scanner Epson Perfection V750 Pro (EPSON Deutschland GmbH, Germany) and the software FiVer (SKZ-KFE gGmbH, Germany). The results are shown in the particle size distribution (Figure 1) and the fiber length distribution (Figure 2).

The investigations with the single screw shredder WSC 250-400 (Weima Maschinenbau GmbH, Germany) focus on the analysis of the airborne fiber dust. Therefore, a feedstock size of the organic sheets of 250 × 250 × 1 mm is determined. The rotational speed is varied from 66 rpm to 120 rpm and the screen size from 10 mm to 20 mm. A Scanning Mobility Particle Sizer (SMPS, Modell 3080, TSI Inc., USA) counts and measures particles on a nanometer scale (9 - 414 nm) whereas an Aerodynamic Particle Sizer (APS, Modell 3321, TSI Inc., USA) counts and measures particles in micrometer scale (0.5 - 20 µm). The inhalable fiber fraction (E-dust) was collected on a cellulose nitrate filter by using a VC25-Sampling device (former, Ströhlein GmbH, Germany). The filter was weighed before and after the shredding process and the fiber mass was standardized by dividing the collected fiber mass through the feedstock mass and the filtered air.

The fiber suspensions were prepared as follows: 20 mg of the fibers were filled in 4 mL brown glass vials and sterilized for 4 hours at 220˚C. The fibers were then transferred in 5 mL Eppendorf vials and suspended in 2 mL PBS by ultrasonication using a Bandelin Sonopuls HD4050 Sonifier at 30 watt for 4 minutes. The used ultrasonic tip had a diameter of 2 mm and cooling was done using a salt/ice bath. Preparation of the stock solution for the milled matrix components was done by suspending 10 mg/mL of the particles in PBS.

NR8383 cells were purchased from ATCC via LGC Standards GmbH (Germany) and cultivated at 37˚C, 100% humidity and 5% CO₂ in Ham’s F12 + 15% FCS (fetal calf serum, PAN-Biotech GmbH, Germany), 2 mM L-glutamine, 100 µg/mL penicillin, and 100 U/mL streptomycin. Approximately 3 × 10⁶ cells were seeded in 35 mL (175 cm²) medium each.

HL-60 cells were purchased from DSMZ (Germany). For the investigation of the chemotaxis we used trans-retinal differentiated HL-60 cells (dHL-60): The HL-60 cells were cultivated in RPMI 1640 medium (PAN-Biotech GmbH, Germany), 10% FSC, 2 mM L-glutamine, 100 μg/mL penicillin, 100 U/mL streptomycin and 1 μM trans-retinal at 37˚C, 100% humidity and 5% CO₂, for three days [19].

Measurement of cytotoxicity was performed by using the AlamarBlue Assay (Invitrogen, Life Technologies Corporation, USA). The assay measures the energy charge of the cells using the redox indicator. Resazurin enters the live cells as a blue, non-fluorescent compound and gets reduced to red-colored fluorescing Resorufin by any enzyme activity that is involved in the generation of NADH or NADPH, such as NADPH and NADH dehydrogenases or diaphorase. 10,000 NR8383 rat macrophages in 100 µL full growth medium (Ham’s F12 medium containing 15% FCS, 2 mM L-glutamine, 100 µg/mL penicillin, and 100 U/mL streptomycin) were seeded into the cavities of a 96-well cell fluorescence culture plate (Corning Incorporated, USA). The cells were incubated at 37˚C, 100% humidity and 5% CO₂ for 24 hours. The fibers were suspended in 100 µL full growth medium to a concentration of 512 µg/cm², diluted and pipetted into the wells, followed by another incubation time of 24 hours. Then 22 µl of the cell viability reagent was added to the plate and after another 2 hours of incubation measurement was done with a fluorescence plate reader at 560/590nm (SpectraMax M3, Molecular Devices, USA).

NR8383 rat macrophages (3 × 10⁶ cells/mL) were suspended with a vortex in 1 mL Ham’s F12 medium containing 15% FCS, 2 mM L-glutamine, 100 µg/mL penicillin, and 100 U/mL streptomycin and culture medium was added to a final volume of 3 mL. The surface area of the cell culture flasks was 12.5 cm², hence 240,000 cells/cm² cell culture dishes were seeded. The fibers were suspended in 1 mL culture medium Ham’s F12 containing 15% FCS, 2 mM L-glutamine, 100 µg/mL penicillin, and 100 U/mL streptomycin and added to the cell culture flasks. The assay can be performed in a smaller volume at constant surface volume ratio. A sample in which cells without particles were incubated served as negative control. The subsequent experiments were repeated up to concentrations which gave the maximum induction of chemotaxis. Incubation of the cells with the fibers and particles was performed at 37˚C, 100% humidity and 5% CO₂ for 16 hours. Thereafter, the cells were transferred to 5 mL Eppendorf vials. Cells were removed by centrifugation with 400 g for 5 minutes and particles were removed by centrifugation with 15,000 g for 10 minutes at room temperature. The supernatants were immediately used for the migration tests.

Cell migration was investigated according to Boyden [20], with the modifications described previously [17] [18]: 200,000 unchallenged dHL-60 cells were F12 or RPMI 1640, respectively without FCS and seeded in each plate well insert (THINCERT, 3 µm pore size, Greiner bio-one, Frickenhausen, Germany) that were placed in the cavities of 24 black well plates (Krystal, Duna Labortechnik, Asbach, Germany). 500 µL of the supernatants of the particle-challenged NR8383 cells were added to the lower chamber. Migration of dHL-60 cells across the membrane was performed at 37˚C, 100% humidity and 5% CO₂ for 24 hours. For calibration, 0 to 100,000 HL-60 cells were seeded directly into four plate wells that were left without inserts.

Staining of migrated cells and of the cell calibration was performed with Calcein-AM for 60 minutes at 37˚C, 5% CO₂ and 100% humidity by adding 500 µL Calcein-AM in the plate wells (>90% HPLC, Sigma-Aldrich, Steinheim, Germany). Calcein-AM was delivered as 4 mM solution in DMSO, stored in aliquots at −18˚C and diluted to a final concentration of 4 µM in PBS.

The cell suspensions were removed from the plate wells and collected with 400 g for 5 minutes at room temperature. 850 µL of the supernatant were discarded while the cells were re-suspended in the remaining volume of 150 µL. In addition, the adherent cells at the outside of the inserts were detached by adding 500 µL trypsin/EDTA (ethylene diamine tetraacetic acid) (0.05%/0.02%, PAN-Biotech GmbH, Aidenbach, Germany) for 10 minutes at 37˚C, 5% CO₂ and 100% humidity. Subsequently, the inserts were removed from the plate wells. The 150 µL of the collected cells were added into the plate wells that contained the 500 µL of the trypsine/EDTA detached cells. The cell count was determined by fluorescence measurement at 490/520nm and calculated from the cell calibration (SpectraMax M3, Molecular Devices, USA).

In order to determine the number of airborne fibers that meet the WHO convention in terms of particularly hazardous fibers, air samples have been collected by using a FAP fiber sampling device (GSA Messgerätebau GmbH, Germany) at a distance of approximately 35 cm from the feeding shaft of the shredder. A gold-coated polycarbonate filter with a diameter of 37 mm and a pore size of 0.8 µm has been used for these investigations. The air flow is 2.0 L/minute and the collection time is 15 minutes in each case; only for the tests with high rotational speed and small screen size has the sampling time been reduced to 10 minutes. The fibers on the gold-coated filters have then been counted by using a scanning electron microscope (SEM) S-2300 (Hitachi, Ltd., Tokyo, Japan) including an energy dispersive X-ray analysis (EDX) and the resulting fiber concentration has been calculated according to the guideline of the DGUV [21]. For this purpose, a Kevex Si (Li) detector system (eumeX Instrumentebau GmbH, Heidenrod, Germany) at 1000× magnification was used.

The AlamarBlue test was used to determine cytotoxic effects of the particles as an indicative measurement. The test measures the cells’ energy charge as a measure of toxicity using the fluorescent dye and redox indicator resazurin. A decrease in fluorescence indicates cytotoxicity. Fluorescence is plotted against the concentration of particles added to the cells.

Initial investigations were carried out with the cutting mill (Figure 2) in order to determine the conditions for the large-scale test with the single-shaft shredder (Table 1). Increasing the rotational speed and feedstock size did not lead to any changes in the toxicity using the cutting mill (Figure 4).

Since cytotoxicity remains unchanged for all examined parameters and rotational speed, feedstock size and screen size were the main influencing factors concerning the particle quantity, these were varied for the large-scale investigation using the single-shaft shredder. Even under these conditions, the AlamarBlue test showed no increased cell toxicity, even at very high dust concentrations (Figure 5).

Rotational speed, feedstock size and screen size were the main influencing factors concerning the particle quantity and the AlamarBlue did not show any significant cytotoxic effects, regardless of how they were obtained. Particle induced cell migration was therefore investigated for particles that were obtained for different speed and feedstock size in the large-scale test with the single-shaft shredder (Table 1). Particle induced cell migration was similar for all examined parameters. The PICMA, therefore, shows no increased effects, even at very high dust concentrations as well as the AlamarBlue test (Figure 6).

Comparison with the historical control (Figure 6 and Figure 7) shows that the induction of cell migration by carbon fibers is very weak and comparable to that of inert particles such as barium sulfate. Consistent with the results from the cell toxicity test, the migration test provides no evidence that the inflammatory or toxic effects of carbon fibers are significantly influenced by the type of shredding.

High fiber concentrations have been counted for all shredding parameters, as well as a high proportion of WHO fibers (Table 2). Figure 8 presents an example of the collected fibers on the gold-coated core pore filter after shredding with different parameters.

EDX analysis of the collected fibers detected only the element carbon in addition to the sputtering material gold (Figure 9).

The results support the findings from the investigations in the micro- and nanoscale range that higher dust concentrations are produced during shredding at high rotational speed. The additional reduction in screen size significantly increases the amount of fibers. Furthermore, it is shown that the number of WHO fibers is not affected by the shredding parameters. This is in accordance with the results from the toxicological studies (Figures 4-6). Despite the different total fiber concentrations, no influence of the shredding parameters on toxicity can be detected here, since the potentially toxic WHO fiber fragments do not differ significantly in number.

Formally, the detected carbon fiber fragments meet the fiber definition of the legislator. However, carbon fibers currently have no specific limit value. Prevention is based on the provisions of TRGS 521 [26]. Thus, a protective measure concept takes effect that is based on the decision concentrations of 50,000 and 250,000 F/m³ (no limit values). In the future, the adaptation to this protection concept should be based on the actual health risk from carbon fiber dust.