In cell culture systems, the addition of medium doses of Carbon Black (CB) induces the formation of reactive oxygen species (ROS) while high doses decrease the viability of the cells.

 

Different studies conducted in the recent years have shown that certain doses of nanoscale carbon black (primary particle size approx. 14 nm), also referred to as ultra-fine carbon black in previous investigations, cause more oxidative stress [1,13] and are more toxic to cells than coarser particles (> 100 nm) [2,3]. Dose-dependent cytotoxicity, secretion of inflammatory markers, and reactive oxygen species were detected for different cell types [1,4,5,6]. Pulskamp et al. have shown that in macrophages of the rat and in human epithelial lung cells, defined doses of 14 nm carbon nanoparticles can cause formation of dose-dependent reactive oxygen species (ROS) and that, in both cell lines, cell activity decreases accordingly [5]. Other studies show that, depending on their type and origin, different cell types can react differently on the treatment with such particles [2].

Yet a further study observed an increase in apoptotic cells followed by apoptosis – the programmed death of cells - in cell layers treated with high concentrations of 20 µg of carbon black per cm2  [3]. During apoptosis, the damaged cells start shrinking, the DNA decomposes, and the cell destroys itself.

 

It has been reported by several authors that carbonic particles or fibers can cause problems in cell culture test systems [5,7,8,9]. The false-positive, invalid results obtained due to interferences of the particles with pigments or dyes prevent one from making explicit statements on the toxicity of carbonic particles.

 

TEM image of Carbon Black agglomerates. © NanoCare Final Scientific ReportTEM image of Carbon Black agglomerates. © NanoCare Final Scientific Report

With this in view, the MTT assay was replaced by the WST test (water soluble tetrazolium) within the NanoCare project and at least one additional vitality test was carried out to obtain valid results.

Neither did any of the eleven cell lines of different origins tested in layers containing up to 10 µg of carbon black particles per cm2 exhibit stress symptoms nor did carbon black cause any cell-damaging effects. Moreover, a test for cell culture apoptosis proved negative. As shown in other studies, carbon black caused the formation of reactive oxygen species. Further in vitro experiments on human lung cells proved that the latter do not get stressed before being exposed to high doses of 25 µg particles per cm2 cell layer. The cell vitality was observed to decrease strongly at and above such particle concentrations.

 

Within NanoCare complex, so-called co-culture systems were used in addition to simple culture systems with only one cell line. By simulating the interaction of the cells, such systems allow a better representation of the in vivo situation in the body. The carbon black particles were found to trigger small biological effects in the respective systems [10].

 

In general, it could be shown that carbon black tends to agglomerate considerably. The agglomerates can be detected in the cells. Medium doses of carbon black cause the formation of ROS [11] while high doses can damage cells in in vitro experiments. In addition, a genotoxic effect is described for high concentrations [12] of carbon black contained in toners together with several other components (e.g. artificial resins, magnetizable metal oxides, dyes, and other additives).

 

Literature arrow down

  1. Stone, V et al. (1998), Toxicol In Vitro, 12(6): 649-659.
  2. L'Azou, B et al. (2008), Part Fibre Toxicol, 5 22.
  3. Hussain, S et al. (2010), Part Fibre Toxicol, 7 10.
  4. Val, S et al. (2009), Inhal Toxicol, 21 Suppl 1 115-122.
  5. Pulskamp, K et al. (2007), Toxicol Lett, 168(1): 58-74.
  6. Barlow, PG et al. (2005), Part Fibre Toxicol, 2(1): 11.
  7. Monteiro-Riviere, NA et al. (2006), Carbon, 44(6): 1070-1078.
  8. Monteiro-Riviere, NA et al. (2009), Toxicol Appl Pharmacol, 234(2): 222-235.
  9. Woerle-Knirsch, JM et al. (2006), Nano Lett, 6(6): 1261-1268.
  10. NanoCare 2009, Final Scientific Report, ISBN 978-3-89746-108-6. (PDF-Document, 19 MB).
  11. Foucaud, L et al. (2010), Toxicol In Vitro, 24(6): 1512-1520.
  12. Gminski, R et al. (2011), Environ Mol Mutagen, 52(4): 296-309.
  13. Barlow, PG et al. (2005), Toxicol Lett, 155(3): 397-401.

 

 

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