Concern-Driven Safety Assessment of Nanomaterials - Safety of Nanomaterials along Their Lifecycle - page 364

and assessment of NMs have begun to evolve to the stage of providing quantitative
estimations of the relationship of specific NM properties with mechanistic outcomes.
Based on this, early attempts at NM grouping are progressing that can already be
used to facilitate testing of large batches of NMs (Nel et al. 2013a), although until
now this is only considered for mode-of-action.
Already in 1999, Rehn and coworkers described a multiparametric in vitro testing
battery using isolated alveolar macrophages for the screening of pulmonary effects of
dust aerosols. By combining the results obtained for a set of independent end points
(release of glucuronidase and lactate dehydrogenase, esterase activation, release of
H 2 O 2 , induction of tumor necrosis factor alpha, and ROS generation), assay sensitiv-
ity and specificity could be improved in comparison to single end point cytotoxicity
assays, and distinct toxicity patterns of different dusts could be discerned (Rehn
et al. 1999). These mentioned end points covered the four basic principles of (nano)
dust toxicity: ROS generation, release of inflammatory mediators, impairment of
cellular function, and cytotoxicity.
The University of California Center for the Environmental Implications of
Nanotechnology has developed a predictive toxicological approach using in vitro
mechanism-based assays for high-throughput screening (HTS) (Nel et al. 2013b).
Data obtained with the HTS assays are evaluated to make predictions about those
physicochemical properties of NMs that may lead to the generation of adverse effects
in vivo. Thereby, multiparametric HTS platforms foster the grouping of NMs (Nel
et al. 2013b).
The HTS platform described by Nel and coworkers utilizes a series of compat-
ible fluorescent dyes to simultaneously measure a set of sublethal and lethal cellular
end points in 384-well plates. The investigated end points reflect important toxicity
pathways of NMs, that is, ROS production, intracellular calcium flux, mitochon-
drial membrane depolarization, and membrane disruption (Damoiseaux et al. 2011;
George et al. 2011; Thomas et al. 2011; Xia et al. 2012; Nel et al. 2013b).
The molecular initiating events and toxicological pathways of NMs can be inves-
tigated in vitro. To be of value for risk assessment, in a predictive toxicological
approach the results of the in vitro screening assays are used to quantitatively assess
dose- and time-dependent molecular initiating events and toxicity pathways that are
predictive of in vivo adverse outcomes (which would then have to be confirmed in
limited in vitro studies) (Nel et al. 2013b).
Data from the HTS screening platforms are provided in a multivariate context
(e.g., concentration, exposure times, sublethal, and lethal biological responses). Due
to the complexity of the data, feature-extraction methods are applied for visual data
interpretation. In this respect, so-called “heat-map clusters” provide ordered repre-
sentations of data facilitating the identification of similarity patterns of large datasets
based on homologous biological responses or linkage to physicochemical properties.
Thereby, multiparametric mechanism-based HTS approaches combined with heat-
map clustering enable NM grouping and the development of nanostructure-activity
relationships (Damoiseaux et al. 2009; George et al. 2011; Liu et al. 2011; Rallo et al.
2011; Thomas et al. 2011; Nel et al. 2013b).
The United States National Institute for Occupational Safety and Health has
presented a strategy to assign science-based hazard and risk categories for NMs that