Challenges of Regulation and Risk Assessment of Nanomaterials

genotoxicity data, since the experimental conditions might have been not optimal for ENM uptake by
cells. Moreover, the selection of the dose metric is fundamental, since no effect was observed by
using mass, but a statistical difference with the control was clear if measured by using particle
number or surface area, and a dose-response curve was obtained. It was observed that ENM entered
the cells as single particles, and that the mode of action of insoluble silica nanoparticles was due to
interference with microtubules in the absence of ROS induction and without crossing the nuclear
membrane. The fact that absence of serum increased the cytotoxic and genotoxic effects could be an
indication that under physiological conditions in a body the protein corona would have a protective
effect.
Within the ENPRA project, in vitro and in vivo models are developed to investigate dose-response
relationships for pulmonary, hepatic, renal, cardio-vascular (including blood) and developmental
endpoints. The result should provide further information on the relationship between
physicochemical characteristics and toxicity, on the predictive value of in vitro tests, on the biological
mechanisms of NM toxicity and on the sensitivity of target organs. Further they should allow
establishing dose-response relationships for a number of endpoints and target organs and to derive
benchmark doses to be used (as alternative to NOAEL) in the risk assessment process. The first results
of studies carried out with a panel of selected ENPRA nanomaterials were presented. The studies
were focusing on both, acute toxic effects in vivo following instillation of NM into the mouse lung,
and in vitro testing in a human hepatic cell line. The investigated in vivo endpoints included: lung
inflammation and cell damage and systemic toxicity (inflammation, effects on bodyweight), while in
vitro endpoints included cytotoxicity, pro-inflammatory mediators (IL8, TNF-α, il6 CRP), ROS and GSH
depletion and cell function. ZnO was identified as the most toxic particles for both the in vivo and in
vitro studies. In in vivo studies, TiO2 NP and nano-Ag were both not acutely toxic at the tested dose
ranges (0-128 μg/mouse), while MWCNTs showed some lung inflammation at the highest dose (128
μg/mouse). The nano-TiO2 testing was more problematic, because the nano-TiO2 chosen as a positive
reference at the beginning of the experiments was not toxic at all, and thus it was not a good choice.
In general, to fully interpret the toxicity data there is the need to measure the physical-chemical
properties in the exposure media.
The in vitro investigations in liver cells showed that nano-Ag and nano-ZnO were constantly more
toxic than MWCNT and nano-TiO2 with respect to cytotoxicity and cytokine production, as the latter
ones only induced cytokine production at very high exposure concentrations which was associated
with oxidative stress. The total levels of the antioxidant glutathione GSH decreased dose-dependently
in all tested nanoparticles with greater effects for nano-Ag and nano-ZnO, confirming the production
of oxidative stress. The need to investigate effects at sub-lethal concentrations was raised as it has
been shown that cytokine levels were decreasing when cells were dying and this may lead to an
underestimation of effects. The pre-treatment of cells with an antioxidant resulted in general in
decreased particle induced cytotoxicity and decreased IL8 secretion. Nano-TiO2 was able to induce
DNA-damage at sub-lethal concentrations as shown by comet assays. Transmission electron
microscopy pictures identified all particles in compartments within hepatocytes in vitro, however
only one type (MWCNT) was found within the nucleus. The ENPRA project is further analysing the
data with an aim to try understanding why nano-Ag appears very toxic in vitro but low toxic in the
applied in vivo system.
Overall, from all presentations in that session it was concluded that it is of greatest importance to
correlate all results from the different studies to all other data, that is currently generated, e.g. under
ENPRA but also under the OECD test program, to gain most benefit out of the studies and to improve
the overall knowledge about nanomaterials toxicity and the best ways to test it.
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