Probabilistic environmental risk assessment of five nanomaterials (nano-TiO2, nano-Ag, nano-ZnO, CNT, and fullerenes).

The environmental risks of five engineered nanomaterials (nano-TiO2, nano-Ag, nano-ZnO, CNT, and fullerenes) were quantified in water, soils, and sediments using probabilistic Species Sensitivity Distributions (pSSDs) and probabilistic predicted environmental concentrations (PECs). For water and soil, enough ecotoxicological endpoints were found for a full risk characterization (between 17 and 73 data points per nanomaterial for water and between 4 and 20 for soil) whereas for sediments, the data availability was not sufficient. Predicted No Effect Concentrations (PNECs) were obtained from the pSSD and used to calculate risk characterization ratios (PEC/PNEC). For most materials and environmental compartments, exposure and effect concentrations were separated by several orders of magnitude. Nano-ZnO in freshwaters and nano-TiO2 in soils were the combinations where the risk characterization ratio was closest to one, meaning that these are compartment/ENM combinations to be studied in more depth with the highest priority. The probabilistic risk quantification allows us to consider the large variability of observed effects in different ecotoxicological studies and the uncertainty in modeled exposure concentrations. The risk characterization results presented in this work allows for a more focused investigation of environmental risks of nanomaterials by consideration of material/compartment combinations where the highest probability for effects with predicted environmental concentrations is likely.

Nano-QSAR: Model of mutagenicity of fullerene as a mathematical function of different conditions.

The experimental data on the bacterial reverse mutation test (under various conditions) on C60 nanoparticles for the cases (i) TA100, and (ii) WP2uvrA/pkM101 are examined as endpoints. By means of the optimal descriptors calculated with the Monte Carlo method a mathematical model of these endpoints has been built up. The models are a mathematical function of eclectic data such as (i) dose (g/plate); (ii) metabolic activation (i.e. with mix S9 or without mix S9); and (iii) illumination (i.e. darkness or irradiation). The eclectic data on different conditions were represented by so-called quasi-SMILES. In contrast to the traditional SMILES which are representation of molecular structure, the quasi-SMILES are representation of conditions by sequence of symbols. The calculations were carried out with the CORAL software, available on the Internet at http://www.insilico.eu/coral. The main idea of the suggested descriptors is the accumulation of all available eclectic information in the role of logical and digital basis for building up a model. The computational experiments have shown that the described approach can be a tool to build up models of mutagenicity of fullerene under different conditions.

Quasi-SMILES and nano-QFAR: united model for mutagenicity of fullerene and MWCNT under different conditions.

Simplified molecular input-line entry system (SMILES) are a tool to represent molecular features of various compounds. Quasi-SMILES is a tool to represent various eclectic features of interaction between complex substances and bio targets (cells, organs, organisms). The construction and the application of quasi-SMILES in order to build up a model for prediction of mutagenicity of fullerene and multi-walled carbon-nanotubes (MWCNTs) are described in this work: instead of paradigm "endpoint is a mathematical function of molecular structure", the paradigm "endpoint is a mathematical function of eclectic data (features)" is used.

In silico assessment of the acute toxicity of chemicals: recent advances and new model for multitasking prediction of toxic effect.

The assessment of acute toxicity is one of the most important stages to ensure the safety of chemicals with potential applications in pharmaceutical sciences, biomedical research, or any other industrial branch. A huge and indiscriminate number of toxicity assays have been carried out on laboratory animals. In this sense, computational approaches involving models based on quantitative-structure activity/toxicity relationships (QSAR/QSTR) can help to rationalize time and financial costs. Here, we discuss the most significant advances in the last 6 years focused on the use of QSAR/QSTR models to predict acute toxicity of drugs/chemicals in laboratory animals, employing large and heterogeneous datasets. The advantages and drawbacks of the different QSAR/QSTR models are analyzed. As a contribution to the field, we introduce the first multitasking (mtk) QSTR model for simultaneous prediction of acute toxicity of compounds by considering different routes of administration, diverse breeds of laboratory animals, and the reliability of the experimental conditions. The mtk-QSTR model was based on artificial neural networks (ANN), allowing the classification of compounds as toxic or non-toxic. This model correctly classified more than 94% of the 1646 cases present in the whole dataset, and its applicability was demonstrated by performing predictions of different chemicals such as drugs, dietary supplements, and molecules which could serve as nanocarriers for drug delivery. The predictions given by the mtk-QSTR model are in very good agreement with the experimental results.

Comparative and mechanistic genotoxicity assessment of nanomaterials via a quantitative toxicogenomics approach across multiple species.

This study reports a comparative and mechanistic genotoxicity assessment of four engineered nanomaterials (ENMs) across three species, including E. coli, yeast, and human cells, with the aim to reveal the distinct potential genotoxicity mechanisms among the different nanomaterials and their association with physiochemical features. Both the conventional phenotypic alkaline comet test and the newly developed quantitative toxicogenomics assay, that detects and quantifies molecular level changes in the regulation of six DNA damage repair pathways, were employed. The proposed molecular endpoints derived from the toxicogenomics assays, namely TELI (Transcriptional Effect Level Index) and PELI (Protein Effect Level Index), correlated well with the phenotypic DNA damage endpoints from comet tests, suggesting that the molecular genotoxicity assay is suitable for genotoxicity detection. Temporal altered gene or protein expression profiles revealed various potential DNA damage types and relevant genotoxic mechanisms induced by the tested ENMs. nTiO2_a induced a wide spectrum of DNA damage consistently across three species. Three carbon-based ENMs, namely carbon black, single wall carbon nanotube (SWCNT) and fullerene, exhibited distinct, species and ENM property-dependent DNA damage mechanisms. All carbon based ENMs induced relatively weak DNA damage repair response in E. coli, but more severe DNA double strand break in eukaryotes. The differences in cellular structure and defense systems among prokaryotic and eukaryotic species lead to distinct susceptibility and mechanisms for ENM uptake and, thus, varying DNA damages and repair responses. The observation suggested that eukaryotes, especially mammalian cells, are likely more susceptible to genotoxicity than prokaryotes in the ecosystem when exposed to these ENMs.

Optimal descriptor as a translator of eclectic data into endpoint prediction: mutagenicity of fullerene as a mathematical function of conditions.

The experimental data on the bacterial reverse mutation test on C60 nanoparticles (TA100) is examined as an endpoint. By means of the optimal descriptors calculated with the Monte Carlo method a mathematical model of the endpoint has been built up. The model is the mathematical function of (i) dose (g/plate); (ii) metabolic activation (i.e. with S9 mix or without S9 mix); and (iii) illumination (i.e. dark or irradiation). The statistical quality of the model is the following: n=10, r(2)=0.7549, q(2)=0.5709, s=7.67, F=25 (Training set); n=5, r(2)=0.8987, s=18.4 (Calibration set); and n=5, r(2)=0.6968, s=10.9 (Validation set).

In vitro toxicity assessment of three hydroxylated fullerenes in human skin cells.

Carbon fullerenes possess unique properties and their interactions with biomolecules have widespread applications. Functionalization of fullerenes with hydroxyl groups (fullerenols) can increase the solubility and potential for cellular interaction, but the health and safety effects of varying degrees of fullerene hydroxylation in biological systems is poorly understood. Existing reports regarding the toxicity and inflammatory potential of fullerenols give conflicting conclusions. To further elucidate the potential for toxicity of fullerenols, human epidermal keratinocytes (HEK) were exposed to fullerenols (low (C60(OH)20), medium (C60(OH)24), and high (C60(OH)32)) at concentrations ranging from 0.000544-42.5 µg/ml for 24 and 48 h. A statistically significant (p<0.05) decrease in viability with alamar Blue (aB) was noted only with C60(OH)32 at 42.5 µg/ml after 24 h. Nanoparticle (NP) controls showed minimal NP/assay interference of the three fullerenols with the aB viability assay. Normalized IL-8 concentration for C60(OH)20 was not significantly different from control, while C60(OH)24 and C60(OH)32 showed a significant decrease at 24 and 48 h. These results suggest that different hydroxylation of fullerenes caused no cytotoxicity or inflammation up to 8.55 µg/ml. These findings suggest that extrapolation across similar NP will be dependent upon surface chemistry and concentration which may affect the degree of agglomeration and thus biological effects.

Fullerene c60: inhalation hazard assessment and derivation of a period-limited acceptable exposure level.

Fullerene C(60) has great potential for use in many industry and medical nanotechnology applications. Although the use of nanomaterials has been increasing in the recent years, limited information about its potential hazardous effects is available. Therefore, safety of nanomaterials is a world concern. Before health effects arise in workers and the general population, development and use under appropriate management are desirable. Therefore, we aimed to determine an acceptable exposure level for humans by reviewing the limited animal toxicity data available. Here, we present an initial hazard assessment, including a review of the available toxicity information of the effects of C(60) on the lungs. We then estimated the no-observed-adverse-effect level (NOAEL) of C(60) on rat lung toxicity by using lung retention of C(60) in inhalation exposure and intratracheal instillation tests. The NOAEL of C(60) on rat lung toxicity was estimated to be 3.1 mg/m(3). Because this is the NOAEL for subchronic toxicity, a period-limited acceptable exposure level (AEL(PL)) for humans was proposed, which assumed 15 years of exposure and modification within the next 10 years since more knowledge will be gained in the future. The AEL(PL) of C(60) particles with a geometric mean of 96 nm and a geometric standard deviation (GSD) of 2.0 was estimated to be 0.39 mg/m(3) for healthy workers and 1.4 × 10(-2) mg/m(3) for the general human population. The AEL(PL) of C(60) particles with different sizes was estimated to be for healthy workers and for the general human population.

Conceptual modeling for identification of worst case conditions in environmental risk assessment of nanomaterials using nZVI and C60 as case studies.

Conducting environmental risk assessment of engineered nanomaterials has been an extremely challenging endeavor thus far. Moreover, recent findings from the nano-risk scientific community indicate that it is unlikely that many of these challenges will be easily resolved in the near future, especially given the vast variety and complexity of nanomaterials and their applications. As an approach to help optimize environmental risk assessments of nanomaterials, we apply the Worst-Case Definition (WCD) model to identify best estimates for worst-case conditions of environmental risks of two case studies which use engineered nanoparticles, namely nZVI in soil and groundwater remediation and C(60) in an engine oil lubricant. Results generated from this analysis may ultimately help prioritize research areas for environmental risk assessments of nZVI and C(60) in these applications as well as demonstrate the use of worst-case conditions to optimize future research efforts for other nanomaterials. Through the application of the WCD model, we find that the most probable worst-case conditions for both case studies include i) active uptake mechanisms, ii) accumulation in organisms, iii) ecotoxicological response mechanisms such as reactive oxygen species (ROS) production and cell membrane damage or disruption, iv) surface properties of nZVI and C(60), and v) acute exposure tolerance of organisms. Additional estimates of worst-case conditions for C(60) also include the physical location of C(60) in the environment from surface run-off, cellular exposure routes for heterotrophic organisms, and the presence of light to amplify adverse effects. Based on results of this analysis, we recommend the prioritization of research for the selected applications within the following areas: organism active uptake ability of nZVI and C(60) and ecotoxicological response end-points and response mechanisms including ROS production and cell membrane damage, full nanomaterial characterization taking into account detailed information on nanomaterial surface properties, and investigations of dose-response relationships for a variety of organisms.

Biosafety assessment of Gd@C82(OH)22 nanoparticles on Caenorhabditis elegans.

Gd@C(82)(OH)(22), a water-soluble endohedral metallofullerene derivative, has been proven to possess significant antineoplastic activity in mice. Toxicity studies of the nanoparticle have shown some evidence of low or non toxicity in mice and cell models. Here we employed Caenorhabditis elegans (C. elegans) as a model organism to further evaluate the short- and long-term toxicity of Gd@C(82)(OH)(22) and possible behavior changes under normal and stress culture conditions. With treatment of Gd@C(82)(OH)(22) at 0.01, 0.1, 1.0 and 10 µg ml(-1) within one generation (short-term), C. elegans showed no significant decrease in longevity or thermotolerance compared to the controls. Furthermore, when Gd@C(82)(OH)(22) treatment was extended up to six generations (long-term), non-toxic effects to the nematodes were found. In addition, data from body length measurement, feeding rate and egg-laying assays with short-term treatment demonstrated that the nanoparticles have no significant impact on the individual growth, feeding behavior and reproductive ability, respectively. In summary, this work has shown that Gd@C(82)(OH)(22) is tolerated well by worms and it has no apparent toxic effects on longevity, stress resistance, growth and behaviors that were observed in both adult and young worms. Our work lays the foundations for further developments of this anti-neoplastic agent for clinical applications.

Review of fullerene toxicity and exposure--appraisal of a human health risk assessment, based on open literature.

Fullerenes have gained considerable attention due to their anti-oxidant and radical scavenging properties. Their current applications include targeted drug delivery, energy application, polymer modifications and cosmetic products. The production of fullerenes and their use in consumer products is expected to increase in future. This study aims to investigate the feasibility and challenges associated with conducting a human health risk assessment for fullerenes based on the open literature, utilising an approach similar to that of a classical regulatory risk assessment. Available data relates to different types of fullerenes (with varying size, surface chemistry, solubility, aggregation/agglomeration) and care should therefore be taken when drawing general conclusions across the parameters. Pristine fullerenes have shown low toxicity and there is probably no risks expected for humans exposed to fullerenes in the workplace under good hygiene conditions. The main concern for consumers is exposure via direct dermal application of fullerenes present in cosmetics. Available studies do not indicate a short term risk from the tested fullerene types, however no extrapolation to all fullerene types and to chronic exposure can be made. In conclusion, the current dataset on fullerenes in relation to both, human exposure and hazard is limited and does not allow reaching any definite conclusions suitable for regulatory decision making. Main future work should focus on generating occupational and consumer exposure data, as well as suitable data on toxicokinetics and potential toxic effects following repeated inhalation and dermal exposure allowing to determine a NOAEL. It seems also relevant to clarify whether certain fullerene types may potentially induce genotoxic and/or carcinogenic effects via physiologically relevant routes.

Attributing effects of aqueous C60 nano-aggregates to tetrahydrofuran decomposition products in larval zebrafish by assessment of gene expression.

BACKGROUND: C(60) is a highly insoluble nanoparticle that can form colloidal suspended aggregates in water, which may lead to environmental exposure in aquatic organisms. Previous research has indicated toxicity from C(60) aggregate; however, effects could be because of tetrahydrofuran (THF) vehicle used to prepare aggregates. OBJECTIVE: Our goal was to investigate changes in survival and gene expression in larval zebrafish Danio rerio after exposure to aggregates of C(60) prepared by two methods: a) stirring and sonication of C(60) in water (C(60)-water); and b) suspension of C(60) in THF followed by rotovaping, resuspension in water, and sparging with nitrogen gas (THF-C(60)). RESULTS: Survival of larval zebrafish was reduced in THF-C(60) and THF-water but not in C(60)-water. The greatest differences in gene expression were observed in fish exposed to THF-C(60) and most (182) of these genes were similarly expressed in fish exposed to THF-water. Significant up-regulation (3- to 7-fold) of genes involved in controlling oxidative damage was observed after exposure to THF-C(60) and THF-water. Analyses of THF-C(60) and THF-water by gas chromatography-mass spectrometry did not detect THF but found THF oxidation products gamma-butyrolactone and tetrahydro-2-furanol. Toxicity of gamma-butyrolactone (72-hr lethal concentration predicted to kill 50% was 47 ppm) indicated effects in THF treatments can result from gamma-butyrolactone toxicity. CONCLUSION: This research is the first to link toxic effects directly to a THF degradation product (gamma-butyrolactone) rather than to C(60) and may explain toxicity attributed to C(60) in other investigations. The present work was first presented at the meeting "Overcoming Obstacles to Effective Research Design in Nanotoxicology" held 24-26 April 2006 in Cambridge, Massachusetts, USA.