ICONS - Integrated testing strategy for mechanistically assessing the respiratory toxicity of functionalized MWCNTs - Comparative in vitro investigations

Multiwalled carbon nanotubes (MWCNTs) represent tube-like structures with nano-sized diameters, made of multiple carbon layers. MWCNTs exhibit promising properties, including electrical/thermal conductivity and high tensile strength, thus practically pointing to a wide range of applications in the electronics and composite materials sectors. However, hazard characterization of MWCNTs, highly needed for work place and consumers' safety, is still incomplete. Besides morphology, surface functionalization of MWCNTs may have marked impact on adverse biological activity. The ERA-NET SIINN project ICONS (""International Collaboration On Nanotube Safety"") thus aimed at mechanistically evaluating and ranking the pro-fibrotic and genotoxic potential of eight differentially purified (chemically or thermally) and functionalized (-COOH and -NH2) MWCNT samples, made from one batch of industrially relevant Nanocyl NC7000 (tangled morphology). One focus of the subproject, carrried out by Fraunhofer ITEM (BMBF-funded: FKZ: 03XP0063), deals with the comparative in vitro (geno)toxicity testing of the eight samples, using primary human lung fibroblast MRC-5 and primary human peritoneal mesothelial LP9 cells. MWCNTs were intially tested for sterility and endotoxin, followed by development of an ultrasound-based dispersion protocol. Scanning electron microscopy indicated presence of both free fibers and agglomerates, and turbidity measurements pointed to differential sedimentation and thus perhaps unequal in vitro doses. Subsequent in vitro testing revealed concentration-dependent membrane damage (LDH release), differential inhibition of proliferation (RICC, mitotic index), slight induction of micronuclei (1 mg/cm2, 24 h), and loss of chromosomes in MRC-5 cells. In LP9 cells (5 mg/cm2, 24 h), induction of LDH release, differential accumulation of MWCNTs around the cell nucleus and impairment of the cytoskeleton (a-tubulin immunofluorescence) were noted. But, no direct DNA damage (gH2A.X or 53BPI foci) was evident, and only sporadic gH2A.X panstaining occurred to account for induction of apoptosis or cellular senescence. Our results point so far to a rather mechanical than clastogenic adverse activity of the MWCNTs. Two candidate MWCNTs will be selected for a 28-day inhalation test in rats.