Characterization of test materials

The combination of morphological imaging by scanning electron microscopy (SEM) (Figure 1) with quantified porosimetry by Hg intrusion highlights the drastically different mesoscopic structures. The specific MWCNT investigated here revealed the expected primary structure of 15 nm outer diameter fibers, but showed the formation of fibrilles at the intermediate structural range between 100 nm and 1 μm (Figure 2), and large agglomerates of tens to hundreds of μm (Figures 1 and 2). The fibrillar intermediate structure makes the agglomerates rather spongy in comparison to other MWCNT materials such as NM400 from the OECD sponsorship program, which has a similar outer diameter of 9.5 nm. In NM400 this intermediate structure is absent, and agglomerates are formed from the individual nanotubes. Carbon Black consisted of globular shaped particles with a primary size of 50 to 100 nm, no intermediates structure up to agglomerates of tens to hundreds of μm (Figures 1 and 2). The expected three-dimensional nanostructure of Carbon Black is clearly confirmed by the SEM images, as well as the one-dimensional primary structure of MWCNT. The two other nanomaterials are obtained by (partial) exfoliation of graphite and are nominally two-dimensional Carbon, but differ drastically at all length scales. Even the material that we designate as graphene does not deserve this name in a strict sense, because it consists of thin multilayers, not monolayers. The smallest observable structural peak in the pore size distribution at 9 nm may be indicative of the dominating thickness range (Figure 2), with lateral sheet dimensions of many μm (Figure 1). The contribution of pore sizes below 100 nm evidences the contrasting structure of graphite nanoplatelets, which is clearly two-dimensional (Figure 1C) but has only a vanishing contribution of pores below 100 nm (Figure 2), and instead has a primary structure of platelets around and above 100 nm (Figure 1G). The Xray diffraction with sharp peaks (Rietveld-Scherrer evaluation of 19 nm crystallite size) further supports the existence of comparably thick platelets. The agglomerate density was read from the cumulative volume of pores with sizes below the approximate diameter of agglomerates (taking 1 μm as cut-off), and is an indicator for the volume occupied at equal mass after lung deposition.

Figure 1 Scanning electron microscopy images of the test materials. A-D: agglomerate scale, E-H: primary structure scale. A, E: MWCNT, B, F: graphene, C, G: graphite nanoplatelets, D, H: Carbon Black. Note the same scale for MWCNT and Carbon Black, and slightly different set of scales for graphite nanoplatelets and graphene, in order to point to their individual characteristics. Full size image

Figure 2 Diagrams of mercury intrusion porosimetry experiments with MWCNT, graphene, graphite nanoplatelets and carbon black. Full size image

Carbon Black and MWCNT were of rather high carbon purity above 98%. By X-ray photoelectron spectroscopy (XPS) with its information depth of 10 nm (enough to reach the core of the MWCNT), remaining metals from the catalyst used for MWCNT production were not observed.

Graphite nanoplatelets and graphene were of lower carbon purity: Sulfur impurities at different oxidation states from the intercalating acids used for exfoliation, and oxygen from partial oxidation of the carbon material. The structural purity was further assessed by Raman spectroscopy [31]. The Raman spectrum of Carbon Black matches the soot reference, whereas the Raman spectrum of graphite nanoplatelets matches the bulk graphite reference. The G-band is shifted to 1590 cm-1 only for the Carbon Black, which also has a very low Raman intensity, confirming a dominantly amorphous material. Although both two-dimensional carbon materials share a dominance of the G band over the G’ band, the G’ band is found at 2663 cm-1 for graphene, but at 2702 cm-1 for graphite nanoplatelets, and the relative intensity of D-band versus G-band decreases from 1.40 ± 0.05 (graphene) to 0.01 ± 0.01 (graphite nanoplatelets). Together with the SEM results (Figure 1), the Raman results thus confirm the strong structural difference of thicker low-defect graphite nanoplatelets against few-layer graphene with vanishing monolayer content and only little nm average distance between two defects [31]. Apart from sulfur and oxygen, the content of thicker three-dimensional carbon structures must also be considered an impurity for a nanomaterial that is engineered to be two-dimensional. The purity of graphite nanoplatelets and graphene is thus at about 85%.

A comprehensive list of physico-chemical characteristics is given in Table 1.

Table 1 Physico-chemical parameters of the test materials Full size table

Characterization of the test atmosphere

Results of gravimetric determination of aerosol concentrations and particle size distribution are summarized in Table 2.

Table 2 Results of gravimetric determination of test atmosphere concentration and particle size distribution Full size table

Measured concentrations of aerosols were close to the target concentrations.

The mass median aerodynamic diameters (MMAD) of the particles in the aerosols were small enough to reach the lower respiratory tract of rats (the particles were respirable for rats).

Scanning electron microscopic evaluation of the test atmospheres collected on gold-coated capillary filters is shown in Figure 3. MWCNTs showed a wool-like structure, which was comparable to the appearance of the material before aerosol generation. Graphene particles were considerably smaller than those of graphite nanoplatelets, which matched with the results obtained with the raw materials.

Figure 3 Scanning electron images of representative test atmosphere samples. A, B: MWCNT, C: graphene, D: graphite nanoplatelets. Full size image

Clinical observations

Inhalation of MWCNT, graphene, graphite nanoplatelets, or Carbon Black did not cause any adverse clinical signs. The body weight development of exposed rats was comparable to control animals.

Hematology and acute phase proteins in serum

In the studies of all four compounds no toxicological relevant effects were observed regarding hematology and the α 2 -macroglobulin as well as haptoglobin levels. If inflammatory response occurred (with MWCNT and graphene) they were restricted to the lungs.

Broncho-alveolar lavage and lung tissue homogenates

In case of mediators, only parameters which were altered by exposure to the test substances are shown in the tables.

MWCNT

Differences of parameters measured in BALF and lung tissue homogenates when compared to the controls are shown in Tables 3, 4, 5 and 6.

Table 3 Cytology parameters in BALF after exposure to MWCNT Full size table

Table 4 Protein concentration and enzyme activities in BALF after exposure to MWCNT Full size table

Table 5 Mediators in BALF after exposure to MWCNT Full size table

Table 6 Mediator levels in lung tissue after exposure to MWCNT Full size table

On study day 7, a significant increase of the total cell counts was found at 2.5 mg/m3. This increase was mainly due to high polymorphonuclear neutrophil counts and to a lesser degree by an increase of the lymphocyte counts. In contrast, macrophage counts were significantly decreased at 2.5 mg/m3. Significantly increased polymorphonuclear neutrophil counts were also present at 0.5 mg/m3.

On study day 28, cell counts were lower, but total cell count as well as polymorphonuclear neutrophil and lymphocyte counts were still increased at 2.5 mg/m3.

On study day 7, total protein levels and enzyme activities were markedly increased at 2.5 mg/m3. γ-glutamyltranspeptidase (GGT), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities were also dose-dependently increased at 0.5 mg/m3.

On study day 28, total protein levels at 2.5 mg/m3 were higher, but no longer statistically significantly increased. However, activities of LDH, GGT and ALP were still significantly increased. The higher GGT activity at 0.5 mg/m3 was in the range of the historical controls, and therefore not considered to be toxicologically relevant.

Levels of 13 of the 69 measured mediators listed in Table 5 were above the detection limit and statistically significantly increased at 2.5 mg/m3. On study day 7, most of the parameters were also increased at 0.5 mg/m3.

On study day 28, mediator concentrations were generally lower compared to study day 7 with the exception of the granulocyte chemotactic peptide (GCP)-2 level, but were still statistically significantly increased compared to the controls at 2.5 mg/m3. Slightly increased levels of GCP-2, MCP-1, MCP-3, macrophage colony stimulating factor (M-CSF) and MIP-2 were present also at 0.5 mg/m3.

On study day 7, levels of 10 mediators listed in Table 6 were above the detection limit and were significantly increased at 2.5 mg/m3. Most mediator levels, which were increased in the lung tissue homogenates at 2.5 mg/m3 were also increased in BALF. Increases in the BALF were higher compared to those in lung tissue homogenates. MCP-1, MCP-3 and myeloperoxidase (MPO) concentrations were statistically significantly increased in the BALF at 0.5 mg/m3, but not in lung tissue samples. The levels of β 2 -Microglobulin, Clusterin, GCP-2, MIP-1β and vascular endothelial growth factor (VEGF) were only increased in BALF samples at 2.5 mg/m3. However, two mediator levels (neutrophil gelatinase-associated lipocalin (NGAL) and Osteopontin) were increased in lung tissue homogenates only.

On study day 28, mediator concentrations in the lung tissue homogenates at 2.5 mg/m3 were smaller, except growth-related oncogene (KC/GROα) and Osteopontin, but were still significantly increased compared to the controls. Slight increases of the cytokine-induced neutrophil chemoattractant (CINC)-1/IL-8, KC/GROα, M-CSF and MIP-2 levels were already found at 0.5 mg/m3.

Graphene

Changes in BALF and lung tissue parameters of exposed animals compared to control animals were observed and are summarized in Tables 7, 8, 9, 10.

Table 7 Cytology parameters in BALF after exposure to graphene Full size table

Table 8 Protein concentration and enzyme activities in BALF after exposure to graphene Full size table

Table 9 Mediator levels in BALF after exposure to graphene Full size table

Table 10 Mediator levels in lung tissue after exposure to graphene Full size table

Total cell counts were higher at 10 mg/m3 at both time points. Lymphocyte and polymorphonuclear neutrophil counts were increased at 10 mg/m3 at both time points, but the changes were considerably more pronounced at study day 7. Additionally, eosinophil counts were increased at 10 mg/m3 on study day 7.

Protein levels were increased at 10 mg/m3 at both time points. Additionally, increased activities of GGT, LDH, and ALP were observed. N-Acetyl-β-Glucosaminidase (NAG) activities were comparable among groups. Slightly higher, but statistically significant changes in ALP activities occurred at 2.5 mg/m3 on study day 7, and in total protein concentration and LDH activities at 2.5 mg/m3 on study day 28. However, these effects were minor and therefore not considered to be of toxicological relevance.

Changes of mediators below factor 2 were not considered to be toxicologically relevant. At 2.5 and 10 mg/m3, CINC-1/IL-8, and MCP-1 levels were dose-dependently increased at both time points. Osteopontin concentrations were increased at 10 mg/m3 on day 7 and day 28 and at 2.5 mg/m3 on study day 28.

Increased levels of IL-1α were present on study day 7 in lung tissue homogenates of rats exposed to 10 mg/m3.

A comparison of cytology and enzyme changes in BALF after exposure to MWCNT and graphene is shown in Figure 4.

Figure 4 Comparison of changes in BALF parameters after exposure to MWCNT and graphene. Changes are shown as x-fold differences compared to controls using a logarithmic scaling. Full size image

Graphite nanoplatelets

On study day 7, approximately 3-fold higher GGT activity was measured in BALF of animals exposed to 10 mg/m3 compared to that of control animals. There was no change in any other enzyme activity, and total protein content.

Carbon Black

No compound-related effects were observed in rats exposed to Carbon Black.

Organ weights

Absolute and relative lung weights were increased at 0.5 and 2.5 mg/m3 MWCNT on study day 4 by approximately 12%. No compound-related effects on organ weights were observed after inhalation of graphene, graphite nanoplatelets and Carbon Black.

Macroscopic and microscopic examination

No compound-related macroscopically visible findings were noted at necropsy after exposure to any of the compounds. Microscopically, compound-related adverse effects were observed in rats exposed to MWCNT and graphene but not graphite nanoplatelets and Carbon Black.

MWCNT

On study day 4, the lungs of one animal exposed to 2.5 mg/m3 revealed intra-septally located microgranulomas (diagnosed as granulomatous inflammation) which were composed mainly of alveolar macrophages. All animals exposed to 2.5 mg/m3 showed a diffuse alveolar histiocytosis (single macrophages within the alveolar space distributed all over the lung not forming aggregates) in contrast to the other test groups and the control animals which occasionally had (multi)focal aggregates of macrophages. Alveolar macrophages of all treated animals had black, fibrous structures within their cytoplasm (regarded to be carbon nanotubes) (Figure 5B). On study day 25, this material was moved to the draining lymph node by alveolar macrophages (Figure 5F). The material was present intracellular (in macrophages) in 5 animals at 2.5 mg/m3 and in 2 animals exposed to 0.5 mg/m3.

Figure 5 Microscopic appearance of lungs (A-E) and lymph node (F). A – Control. B – MWCNT, 2.5 mg/m3, study day 4: Microgranuloma, particles in the lesion and inside of alveolar macrophages. C – Graphene, 10 mg/m3, study day 7: Microgranuloma, particles within the lesion. D – Graphite nanoplatelets, 10 mg/m3, study day 4: Particles inside of alveolar macrophages. E – Carbon Black, 10 mg/m3, study day 4: Particles inside of alveolar macrophages. F – MWCNT, 2.5 mg/m3, study day 25: Mediastinal lymph nodes with macrophages containing black particles (arrow). Full size image

Graphene

On study day 7, single macrophages or small aggregates of alveolar macrophages were observed in the lungs of all treated animals. Most of them were loaded with black variable sized particles (regarded as graphene). Most of these macrophages were located in the lumen of alveoli, few occurred in the alveolar wall, the alveolar ducts, and in terminal bronchioles. The accumulation of macrophages increased with the exposure concentration. In addition, single ‘microgranulomas’ were observed in animals exposed to 2.5 and 10 mg/m3 (Figure 5C). Microgranulomas were characterized by small particle-loaded aggregates of macrophages that partly appeared fused or showed single giant cells and were connected to the alveolar septum. They were not associated with any other inflammatory response and there were no further alterations of the lung parenchyma.

On study day 28, the occurrence of particle-loaded macrophages or aggregates of macrophages was still concentration-dependently increased. Incidence and severity were comparable to the groups examined on day 7. In addition, single or few microgranulomas were observed in animals exposed to 2.5 and 10 mg/m3. Two animals exposed to 10 mg/m3 showed single small particles in the mediastinal lymph nodes without any histopathological findings. One animal exposed to 0.5 mg/m3 showed macroscopically enlarged mediastinal lymph nodes with a moderate lympho-reticular cell hyperplasia. This was considered to be incidental.

Graphite nanoplatelets

On study day 4, the lungs of one animal exposed to 10 mg/m3 showed few intra-alveolar located multifocal aggregates of alveolar macrophages. All animals exposed to this concentration (10 mg/m3) showed black, irregularly shaped particles within the cytoplasm of single intra-alveolar macrophages (regarded as graphite nanoplatelets) (Figure 5D).

On study day 25, as well as on study day 95, particles were still observed in alveolar macrophages. Few intracellular particles in the mediastinal lymph node (within macrophages) were observed in one animal exposed to 10 mg/m3 on study day 95.

No compound-related microscopic alterations were observed after exposure to 0.5 and 2.5 mg/m3 Graphite nanoplatelets.

Carbon Black

On study day 4, black particles (regarded as Carbon Black) were observed within alveolar macrophages in animals exposed to 10 mg/m3. In three of the six treated animals there was a minimal increase in numbers of alveolar macrophages (Figure 5E). There were no other findings.

Among the four tested carbon-based nanomaterials, MWCNT caused the strongest effects in the lower respiratory tract (granulomas, inflammation). Most prominent changes in BALF cellularity comprised marked increases in polymorphonuclear neutrophils and lymphocytes, which decreased but were still present after 28 days. Markedly increased activities of MPO are in line with the observed increased presence of polymorphonuclear neutrophils.

Changes observed after inhalation of graphene were less pronounced. Like MWCNT granuloma formation was observed in the lower respiratory tract. Increases of polymorphonuclear neutrophils and lymphocytes in BALF were less pronounced when compared to MWCNT. The increase in osteopontin concentrations was higher compared to MWCNT exposure, probably indicating macrophage and lymphocyte activation. Increased osteopontin concentrations were still observed on study day 28.

Only local inflammation and no further alterations were observed after exposure to MWCNT or graphene.

Macrophage aggregates and phagocytized particles were the only microscopically visible alteration observed after inhalation of graphite nanoplatelets. Merely a slight, transient increase of GGT activity was present in BALF.

Neither adverse microscopic effects nor changes in broncho-alveolar parameters were observed after inhalation of Carbon Black.