Toxicological Effects of Metal Oxide Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 160

Cultured cells may respond differentially compared to whole organs or organisms.
For instance, it appears not justified to assume that an in vitro effect of one cell
type fully reflects a pathophysiological outcome or toxic response in vivo . Effects
observed in vitro may for instance not reflect adaptation or clearance processes, thus
leading to an overestimation of adverse effects. On the other side, in vitro mod-
els consisting of one cell type may lack important metabolic properties and/or the
interplay between different cells typically occurring in organized tissue such as the
liver. This in turn may lead to false negative results. In general, it is therefore recom-
mended to use a number of different cell lines and various assay readouts to collect
more relevant data (Kroll et al. 2011). Many of the established in vitro test systems
were initially designed for testing soluble chemical substances and are often not
suitable to assess the toxic potential of nanomaterials since these can interfere with
assay reagents or detection systems thereby influencing the toxicological outcome
(Kroll et al. 2012).
In a previous German project (NanoCare), a set of metal oxide nanoparticles were
used to identify those particles that are most influential in driving nanomaterial tox-
icity. During the project, an in vitro test matrix initially comprising 10 standardized
cell lines of diverse tissue origin and a set of standardized assays for different end
points was used (Kroll et al. 2011). Due to the combination of several cell lines from
different tissues and assays reporting on the impairment of various cell functions
this approach largely increases the reliability and predictability of an in vitro test
and overcomes the problem that nanomaterials are heterogeneous in cell type and
end point activation. The combination of highly sensitive cytotoxicity test systems
(see Kroll et al. 2009) may therefore be used to prescreen nanomaterials for their
potential toxicity and to identify candidates that require more detailed testing and
thereby reducing the number of animal studies. The test matrix was shown to func-
tion as an effective prescreening for the selection of nanomaterial candidates for an
in vivo assessment and may allow an extrapolation of results to the in vivo situation
to inform about potential adverse health effects in humans.
In the following German project nanoGEM, the in vitro test matrix was contin-
ued using a selection of the most responsive cell lines. Here, the test matrix includes
human A549 and rat RLE-6TN cells, which display features of lung alveolar epithe-
lial cells as in vitro models for inhalation. HaCaT keratinocytes were derived from
normal human skin and have retained the full epidermal differentiation capacity.
As cellular models to assess toxic effects of metal oxide nanoparticles that have
been distributed via the blood system to secondary target organs, kidney cell lines
(MDCK, NRK-52E) as well as fibroblasts (NIH-3T3) and macrophages (RAW264.7)
were used (for further details see Table 8.1).
In both projects, NanoCare and nanoGEM, the physicochemical properties of
the nanoscaled metal oxide dispersions were thoroughly characterized, and stan-
dardized protocols as well as reference materials were used to improve the compa-
rability between in vivo and in vitro . The nanoGEM project focused on the impact
of different surface coatings and included four types of SiO 2 nanoparticles (SiO 2 .
naked, SiO 2 .PEG, SiO 2 .amino, or SiO 2 .phosphate), and four types of ZrO 2 nanopar-
ticles (ZrO 2 .TODS, Z r O 2 .PEG, ZrO 2 .amino, or ZrO 2 .acryl) (see Chapter 1, Table 1.2
for particle details). This chapter will provide an overview of the physicochemical