Expert consensus on an in vitro approach to assess pulmonary fibrogenic potential of aerosolized nanomaterials.

The increasing use of multi-walled carbon nanotubes (MWCNTs) in consumer products and their potential to induce adverse lung effects following inhalation has lead to much interest in better understanding the hazard associated with these nanomaterials (NMs). While the current regulatory requirement for substances of concern, such as MWCNTs, in many jurisdictions is a 90-day rodent inhalation test, the monetary, ethical, and scientific concerns associated with this test led an international expert group to convene in Washington, DC, USA, to discuss alternative approaches to evaluate the inhalation toxicity of MWCNTs. Pulmonary fibrosis was identified as a key adverse outcome linked to MWCNT exposure, and recommendations were made on the design of an in vitro assay that is predictive of the fibrotic potential of MWCNTs. While fibrosis takes weeks or months to develop in vivo, an in vitro test system may more rapidly predict fibrogenic potential by monitoring pro-fibrotic mediators (e.g., cytokines and growth factors). Therefore, the workshop discussions focused on the necessary specifications related to the development and evaluation of such an in vitro system. Recommendations were made for designing a system using lung-relevant cells co-cultured at the air-liquid interface to assess the pro-fibrogenic potential of aerosolized MWCNTs, while considering human-relevant dosimetry and NM life cycle transformations. The workshop discussions provided the fundamental design components of an air-liquid interface in vitro test system that will be subsequently expanded to the development of an alternative testing strategy to predict pulmonary toxicity and to generate data that will enable effective risk assessment of NMs.

Case studies putting the decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping) into practice.

Case studies covering carbonaceous nanomaterials, metal oxide and metal sulphate nanomaterials, amorphous silica and organic pigments were performed to assess the Decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping). The usefulness of the DF4nanoGrouping for nanomaterial hazard assessment was confirmed. In two tiers that rely exclusively on non-animal test methods followed by a third tier, if necessary, in which data from rat short-term inhalation studies are evaluated, nanomaterials are assigned to one of four main groups (MGs). The DF4nanoGrouping proved efficient in sorting out nanomaterials that could undergo hazard assessment without further testing. These are soluble nanomaterials (MG1) whose further hazard assessment should rely on read-across to the dissolved materials, high aspect-ratio nanomaterials (MG2) which could be assessed according to their potential fibre toxicity and passive nanomaterials (MG3) that only elicit effects under pulmonary overload conditions. Thereby, the DF4nanoGrouping allows identifying active nanomaterials (MG4) that merit in-depth investigations, and it provides a solid rationale for their sub-grouping to specify the further information needs. Finally, the evaluated case study materials may be used as source nanomaterials in future read-across applications. Overall, the DF4nanoGrouping is a hazard assessment strategy that strictly uses animals as a last resort.

A decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping).

The European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) 'Nano Task Force' proposes a Decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping) that consists of 3 tiers to assign nanomaterials to 4 main groups, to perform sub-grouping within the main groups and to determine and refine specific information needs. The DF4nanoGrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways, i.e. intrinsic material and system-dependent properties, biopersistence, uptake and biodistribution, cellular and apical toxic effects. Use (including manufacture), release and route of exposure are applied as 'qualifiers' within the DF4nanoGrouping to determine if, e.g. nanomaterials cannot be released from a product matrix, which may justify the waiving of testing. The four main groups encompass (1) soluble nanomaterials, (2) biopersistent high aspect ratio nanomaterials, (3) passive nanomaterials, and (4) active nanomaterials. The DF4nanoGrouping aims to group nanomaterials by their specific mode-of-action that results in an apical toxic effect. This is eventually directed by a nanomaterial's intrinsic properties. However, since the exact correlation of intrinsic material properties and apical toxic effect is not yet established, the DF4nanoGrouping uses the 'functionality' of nanomaterials for grouping rather than relying on intrinsic material properties alone. Such functionalities include system-dependent material properties (such as dissolution rate in biologically relevant media), bio-physical interactions, in vitro effects and release and exposure. The DF4nanoGrouping is a hazard and risk assessment tool that applies modern toxicology and contributes to the sustainable development of nanotechnological products. It ensures that no studies are performed that do not provide crucial data and therefore saves animals and resources.

A critical appraisal of existing concepts for the grouping of nanomaterials.

The grouping of substances serves to streamline testing for regulatory purposes. General grouping approaches for chemicals have been implemented in, e.g., the EU chemicals regulation. While specific regulatory frameworks for the grouping of nanomaterials are unavailable, this topic is addressed in different publications, and preliminary guidance is provided in the context of substance-related legislation or the occupational setting. The European Centre for Ecotoxicology and Toxicology of Chemicals Task Force on the Grouping of Nanomaterials reviewed available concepts for the grouping of nanomaterials for human health risk assessment. In their broad conceptual design, the evaluated approaches are consistent or complement each other. All go beyond the determination of mere structure-activity relationships and are founded on different aspects of the nanomaterial life cycle. These include the NM's material properties and biophysical interactions, specific types of use and exposure, uptake and kinetics, and possible early and apical biological effects. None of the evaluated grouping concepts fully take into account all of these aspects. Subsequent work of the Task Force will aim at combining the available concepts into a comprehensive 'multiple perspective' framework for the grouping of nanomaterials that will address all of the mentioned aspects of their life cycles.

Consistency and variation in phenotypic selection exerted by a community of seed predators.

Phenotypic selection that is sustained over time underlies both anagenesis and cladogenesis, but the conditions that lead to such selection and what causes variation in selection are not well known. We measured the selection exerted by three species of predispersal seed predators of lodgepole pine (Pinus contorta latifolia) in the South Hills, Idaho, and found that net selection on different cone and seed traits exerted by red crossbills (Loxia curvirostra) and cone borer moths (Eucosma recissoriana) over 10 years of seed crops was similar to that measured in another mountain range. We also found that the strength of selection increased as seed predation increased, which provides a mechanism for the correlation between the escalation of seed defenses and the density of seed predators. Red crossbills consume the most seeds and selection they exert accounts for much of the selection experienced by lodgepole pine, providing additional support for a coevolutionary arms race between crossbills and lodgepole pine in the South Hills. The third seed predator, hairy woodpeckers (Picoides villosus), consumed less than one-sixth as many seeds as crossbills. Across the northern Rocky Mountains, woodpecker abundance and therefore selective impact appears limited by the elevated seed defenses of lodgepole pine.

An investigation of the photo-clastogenic potential of ultrafine titanium dioxide particles.

Ultrafine titanium dioxide is widely used in a number of commercial products including sunscreens and cosmetics. There is extensive evidence on the safety of ultrafine titanium dioxide. However, there are some published studies indicating that some forms at least may be photogenotoxic, photocatalytic and/or carcinogenic. In order to clarify the conflicting opinions on the safety of ultrafine titanium dioxide particles, the current studies were performed to investigate the photo-clastogenic potential of eight different classes of ultrafine titanium dioxide particles. The photo-clastogenicity of titanium dioxide was measured in Chinese hamster ovary (CHO) cells in the absence and presence of UV light at a dose of 750 mJ/cm(2). The treatments were short (3 h) followed by a 17-h recovery and achieved concentrations that either induced approximately 50% cytotoxicity or reached 5000 microg/ml if non-cytotoxic. None of the titanium dioxide particles tested induced any increase in chromosomal aberration frequencies either in the absence or presence of UV. These studies show that ultrafine titanium dioxide particles do not exhibit photochemical genotoxicity in the model system used.

Does lung surfactant promote disaggregation of nanostructured titanium dioxide?

OBJECTIVE: Nanostructured titanium dioxide (TiO2) is highly aggregated and agglomerated when inhaled. There are discussions regarding whether lung surfactant may promote the disaggregation of TiO2 particles. We investigated whether dipalmitoyl phosphatidyl-choline (DPPC), the main component of lung surfactant, can split the bonds between TiO2 aggregates and agglomerates. METHODS: We calculated the energy required to split aggregates into primary particles and agglomerates into aggregates as well the energy of the interaction of a TiO2 surface with a DPPC bilayer. To test the calculations, we measured the particle size distribution of TiO2 suspensions in a pulmonary liquid model. RESULTS: Calculated splitting energy between TiO2 aggregates was 1 J/m2 and 10 J/m2 between primary particles. The calculated interaction between DPPC and TiO2 was significantly weaker (0.05 J/m2). Calculations were shown to be in accordance with the measured particle size distribution of TiO2 suspensions in the pulmonary liquid model. CONCLUSION: We conclude that lung surfactant does not promote the disaggregation of TiO2 agglomerates and aggregates.