Risk Assessment Of Nanomaterials Research Articles

Metallic Fingerprints of Carbon: Label-Free Tracking and Imaging of Graphene in Plants.

Detection and quantification of carbon nanomaterials are extremely challenging, especially under the background interference of carbon. Here, we propose a new label-free method to quantify, track, and in situ image graphene and graphene oxide (GO) in plants based on their inherent metallic impurities as fingerprints. We show the ubiquity and high stability of inherent metallic fingerprints of graphene and GO obtained from different exposure routes under the natural environments, which enables the materials to be easily quantified and in situ imaged by high-sensitivity (laser ablation) inductively coupled plasma mass spectrometry. The method was applied to investigate the uptake and spatial distribution of graphene and GO in soybean plants. The plants were cultivated in graphene or GO solutions for 7 days, and the indicative elements (Ni or Mn) in different parts of plants were monitored and imaged. We found that graphene and GO showed different distribution patterns in plants (the highest uptake percentages in root up to 14.4% for graphene and 47.8% for GO), and high concentration of material exposure might cause excessive accumulation of materials in roots which blocked their further transport to the other parts of plants. The present method is more straightforward, accessible, and economical than normally used isotopic or metal-labeling methods. It also avoids the uncertainties or alterations of properties caused by the labeling process and thus has great promise in analysis and risk assessment of carbon nanomaterials.

Nanoparticle induced barrier function assessment at liquid-liquid and air-liquid interface in novel human lung epithelia cell lines.

Inhalation is the most relevant entry point for nanoparticles (NPs) into the human body. To date, toxicity testing of nanomaterials in respect to oral, dermal and inhalative application is mainly based on animal experiments. The development of alternative test methods is the subject of current research. In vitro models can help to investigate mechanistic aspects, as e.g. cellular uptake or genotoxicity and might help to reduce in vivo testing. Lung cell lines are proper in vitro tools to assess NP toxicity. In respect to this, various cell models have been developed during the recent years, but often lack in a proper intact barrier function. However, besides other important in vivo criteria which are still missing like e.g. circulation, this is one basic prerequisite to come closer to the in vivo situation in certain mechanistic aspects such as particle translocation which is an important task for risk assessment of nanomaterials. Novel developed in vitro models may help to investigate the translocation of nanomaterials from the lung. We investigated the barrier function of the recently developed human lung cell lines CI-hAELVi and CI-huAEC. The cells were further exposed to CeO2 NPs and ZnO NPs, and their suitability as in vitro models for toxicological investigations was proven. The obtained data were compared with data generated with the A549 cell line. Measurement of transepithelial resistance and immunohistochemical examination of tight junctions confirmed the formation of a functional barrier for both cell lines for submerged and air-liquid cultivation. For particle exposure, hAELVi and huAEC cells showed comparable results to A549 cells without losing the barrier function. CeO2 NP exposure revealed no toxicity for all cell lines. In contrast, ZnO NPs was toxic for all cell lines at a concentration between 10-50 μg ml-1. Due to the comparable results to A549 cells CI-hAELVi and CI-huAEC offer new opportunities to investigate nanoparticle cell interactions more realistic than recent 2D cell models.

Risk Governance of Nanomaterials: Review of Criteria and Tools for Risk Communication, Evaluation, and Mitigation.

Nanotechnologies have been increasingly used in industrial applications and consumer products across several sectors, including construction, transportation, energy, and healthcare. The widespread application of these technologies has raised concerns regarding their environmental, health, societal, and economic impacts. This has led to the investment of enormous resources in Europe and beyond into the development of tools to facilitate the risk assessment and management of nanomaterials, and to inform more robust risk governance process. In this context, several risk governance frameworks have been developed. In our study, we present and review those, and identify a set of criteria and tools for risk evaluation, mitigation, and communication, the implementation of which can inform better risk management decision-making by various stakeholders from e.g., industry, regulators, and the civil society. Based on our analysis, we recommend specific methods from decision science and information technologies that can improve the existing risk governance tools so that they can communicate, evaluate, and mitigate risks more transparently, taking stakeholder perspectives and expert opinion into account, and considering all relevant criteria in establishing the risk-benefit balance of these emerging technologies to enable more robust decisions about the governance of their risks.

In Vivo-In Vitro Comparative Toxicology of Cadmium Sulphide Quantum Dots in the Model Organism Saccharomyces cerevisiae.

The aim of this work was to use the yeast Saccharomyces cerevisiae as a tool for toxicogenomic studies of Engineered Nanomaterials (ENMs) risk assessment, in particular focusing on cadmium based quantum dots (CdS QDs). This model has been exploited for its peculiar features: a short replication time, growth on both fermentable and oxidizable carbon sources, and for the contextual availability of genome wide information in the form of genetic maps, DNA microarray, and collections of barcoded mutants. The comparison of the whole genome analysis with the microarray experiments (99.9% coverage) and with the phenotypic analysis of 4688 barcoded haploid mutants (80.2% coverage), shed light on the genes involved in the response to CdS QDs, both in vivo and in vitro. The results have clarified the mechanisms involved in the exposure to CdS QDs, and whether these ENMs and Cd2+ exploited different pathways of response, in particular related to oxidative stress and to the maintenance of mitochondrial integrity and function. Saccharomyces cerevisiae remains a versatile and robust alternative for organismal toxicological studies, with a high level of heuristic insights into the toxicology of more complex eukaryotes, including mammals.

Sampling and pre-treatment effects on the quantification of (nano)silver and selected trace elements in surface water - Application in a Dutch case study

Detection and quantification of trace elements in aqueous samples is crucial in terms of environmental monitoring and risk assessment for (heavy) metals in the environment. Silver (Ag) in its nanoparticulate form is commonly used as antimicrobial additive in consumer products and pharmaceuticals. Since released dissolved Ag species act as the actual antimicrobial agent, Ag nanomaterials are supposed to pose risks to the environment by a release of dissolved species. Unfortunately, no standard protocols exist yet to gain reliable information about the presence and distribution of nanomaterials in the environment. Therefore, we present an interlaboratory collaboration involving three laboratories to quantify silver, silver based nanoparticles (Ag-b-NPs) and a wide range of relevant trace elements after different sample pre-treatments for profiling surface water of a Dutch channel. Besides quantification of the elements, different sample pretreatments like acidification, with or without filtration, and their effect on the measurable elemental content were studied. Total Ag and Ag-b-NPs were quantified at lower ng L−1 range in the channel water whereas reasonable differences depending on the pre-treatment were identified; Ba, As, Pb, Co, Cr, Cu, Ni and Zn were detected at μg L−1 range and Na, K, Mg, Ca and Fe at mg L−1 range. Significant sample pre-treatment effects were observed for the elements Cr, Cu, Fe, Pb and Zn, which is very likely due to the existence of particulate species. Measured concentrations were well comparable among the three laboratories underpinning method validity and correctness allowing for a comprehensive, reliable risk assessment for nanomaterials in the environment.

Systematic selection of a dose metric for metal-based nanoparticles

Mass is traditionally the unique measure of the administered dose in toxicity studies with conventional chemical substances. Because of the variety of specific physical properties of nanoparticles, other dose metrics such as the number of particles, their size, shape, surface area or volume may be more appropriate. Here we applied a systematic, unbiased approach to derive the most appropriate dose metric for nanoparticles from experimental data. The approach was exemplified for copper, zinc oxide, and silver nanoparticles with different diameters, coatings and shapes, combining experiments with six aquatic organisms, two mammalian and two piscine liver cell lines from different research groups. The nanoparticle diameter appeared to be a powerful estimator of metal oxide nanoparticle effects. Since effect concentrations were related to size to the power 3, it is indicated that volume (mass) is the appropriate dose metric for all tested species and toxicological endpoints and all tested metal oxide nanoparticles within the tested size range (25–500 nm). The new method enables extrapolation of test results from one type of metal oxide nanomaterial to another, thereby offering a powerful tool to improved efficiency in risk research and risk assessment of nanomaterials.

Contribution of mesocosm testing to a single-step and exposure-driven environmental risk assessment of engineered nanomaterials

Environmental risk assessment of nanomaterials generally relies on a decision-tree based strategy which provides guidance and protocols for the determination of a collection of hazard end-points. Mesocosm testing is based on a different approach. This method consists in monitoring the evolution of a re-created miniature ecosystem subsequent to a nanomaterial contamination. The only decision in this risk assessment strategy is the definition of an environmentally relevant exposure scenario (incl. dose), which, given current analytical capabilities, may unfavorably affect the nature and precision of parameters and endpoints to be determined. Despite these limitations, mesocosm testing bears clear advantages for the determination of both exposure and hazard in a single experiment, and for producing dependable and intercomparable data.

Introducing WikiPathways as a Data-Source to Support Adverse Outcome Pathways for Regulatory Risk Assessment of Chemicals and Nanomaterials.

A paradigm shift is taking place in risk assessment to replace animal models, reduce the number of economic resources, and refine the methodologies to test the growing number of chemicals and nanomaterials. Therefore, approaches such as transcriptomics, proteomics, and metabolomics have become valuable tools in toxicological research, and are finding their way into regulatory toxicity. One promising framework to bridge the gap between the molecular-level measurements and risk assessment is the concept of adverse outcome pathways (AOPs). These pathways comprise mechanistic knowledge and connect biological events from a molecular level toward an adverse effect outcome after exposure to a chemical. However, the implementation of omics-based approaches in the AOPs and their acceptance by the risk assessment community is still a challenge. Because the existing modules in the main repository for AOPs, the AOP Knowledge Base (AOP-KB), do not currently allow the integration of omics technologies, additional tools are required for omics-based data analysis and visualization. Here we show how WikiPathways can serve as a supportive tool to make omics data interoperable with the AOP-Wiki, part of the AOP-KB. Manual matching of key events (KEs) indicated that 67% could be linked with molecular pathways. Automatic connection through linkage of identifiers between the databases showed that only 30% of AOP-Wiki chemicals were found on WikiPathways. More loose linkage through gene names in KE and Key Event Relationships descriptions gave an overlap of 70 and 71%, respectively. This shows many opportunities to create more direct connections, for example with extended ontology annotations, improving its interoperability. This interoperability allows the needed integration of omics data linked to the molecular pathways with AOPs. A new AOP Portal on WikiPathways is presented to allow the community of AOP developers to collaborate and populate the molecular pathways that underlie the KEs of AOP-Wiki. We conclude that the integration of WikiPathways and AOP-Wiki will improve risk assessment because omics data will be linked directly to KEs and therefore allow the comprehensive understanding and description of AOPs. To make this assessment reproducible and valid, major changes are needed in both WikiPathways and AOP-Wiki.

Probabilistic approach for the risk assessment of nanomaterials: A case study for graphene nanoplatelets.

An experimental probabilistic approach for health risk assessment was applied for graphene nanoplatelets (GNPs). The hazard assessment indicated a low level of toxicity for the GNPs. The benchmark dose method, based on sub-chronic and chronic inhalation exposure studies, was used to quantify a guidance value (BMCh) for occupational inhalation exposure to GNPs, expressed as a lognormal distribution with a geometric mean ± geometric standard deviation of 0.212 ± 7.79 mg/m3 and 9.37 × 104 ± 7.6 particle/cm3. Exposure scenarios (ES) were defined based on the scientific literature for large-scale production (ES1) and manufacturing (ES2) of GNPs; a third ES, concerning in-lab handling of GNPs (ES3) was based on results of experiments performed for this study. A probability distribution function was then assumed for each ES. The risk magnitude was calculated using a risk characterization ratio (RCR), defined as the ratio of the exposure distributions and the BMCh distribution. All three ES resulted in RCR distributions ≥1 (i.e. risk present); however, none of the ES had a statistically significant level of risk at a 95% confidence interval. A sensitivity analysis indicated that ∼75% of the variation in the RCR distributions was due to uncertainties in the BMCh calculation.

The uncertainty with nanosafety: Validity and reliability of published data.

Use and production of chemicals and new materials are always reasons for concern especially with regard to human health and the environmental impacts. Over the past few decades occupational safety is a greater focus for toxicologists and of national and international registration programs for new products. Thus, the careful investigation of the biological effects of new chemicals and materials is critical. However, the hype around "The Nanotechnology" has boosted a competition for public funds and thereby the number of publications on this "nanotoxicology" topic has exploded. For more than two decades the public discussion around the special effects of nanomaterials or nanoparticles is ongoing without a final conclusion regarding an existing issue of a "nano-specific effect". Facing the situation of a dramatic increase in the number of publications (>4400 PubMed references in 2017 alone!); the quality of the findings appears to be questionable, particularly with regard to the implementation of risk assessment for nanomaterials. Most of the published nanotoxicology studies are associated with fundamental deficiencies in the experimental design of these investigations, including 1) a lack of rigorous and adequate physicochemical characterization of the test materials; 2) the absence of adequate particle controls; and 3) the implementation of high dose experiments, designed to produce toxicological effects - which are publishable (and sensational). As a consequence, the "toxicology" results have limited utility, and therefore must be critically (re)evaluated. This service is provided by the internet knowledge base DaNa (www.nanoobjects.info). On this website a criteria catalogue for the re-evaluation of scientific publications has been published and if these criteria are utilized > 60 70% of reported study findings are not acceptable and cannot be taken into consideration for risk assessment criteria.

Estimation of Inhalation Exposure on the Basis of Airborne Nanomaterial Release Data and Propagation Modeling

The lack of data for inhalation exposure concerning the handling of nanostructured materials is limiting the current nanomaterial risk assessment. However, there are numerous studies dealing with the origin of exposure, i.e., the release. The link between release and exposure are transport and transformation phenomena. Thus, propagation modeling was used to simulate transport from source to breathing zone in order to estimate quantitative exposure levels from measured release data for selected scenarios. On the basis of three ventilation and five release scenarios, exposure levels were calculated with and without human thermal plumes for both the nearfield and the farfield. Results show that the often neglected human-induced convective airflow can have a strong impact on the level of exposure, especially for natural ventilation or non-well-designed technical ventilation systems as typical for nonindustrial consumers or handicraft businesses.

Nanoecotoxicity assessment of graphene oxide and its relationship with humic acid.

The risk assessment of nanomaterials is essential for regulatory purposes and for sustainable nanotechnological development. Although the application of graphene oxide has been widely exploited, its environmental risk is not well understood because several environmental conditions can affect its behavior and toxicity. In the present study, the graphene oxide effect from aquatic ecosystems was assessed considering the interaction with humic acid on 9 organisms: Raphidocelis subcapitata (green algae), Lemna minor (aquatic plant), Lactuca sativa (lettuce), Daphnia magna (planktonic microcrustacean), Artemia salina (brine shrimp), Chironomus sancticaroli (Chironomidae), Hydra attenuata (freshwater polyp), and Caenorhabditis elegans and Panagrolaimus sp. (nematodes). The no-observed-effect concentration (NOEC) was calculated for each organism. The different criteria used to calculate NOEC values were transformed and plotted as a log-logistic function. The hypothetical 5 to 50% hazardous concentration values were, respectively, 0.023 (0.005-0.056) and 0.10 (0.031-0.31) mg L-1 for graphene oxide with and without humic acid, respectively. The safest scenario associated with the predicted no-effect concentration values for graphene oxide in the aquatic compartment were estimated as 20 to 100 μg L-1 (in the absence of humic acid) and 5 to 23 μg L-1 (in the presence of humic acid). Finally, the present approach contributed to the risk assessment of graphene oxide-based nanomaterials and the establishment of nano-regulations. Environ Toxicol Chem 2018;37:1998-2012. © 2018 SETAC.

Directions in QPPR development to complement the predictive models used in risk assessment of nanomaterials

There is an increasing need for predictive risk assessment of nanomaterials (NMs) using methods that are rapid, accurate and resource efficient. To fulfill this need, the development and use of Quantitative Property Property Relationships (QPPRs) for estimating the hazard of NMs and NM-related parameters in exposure modelling seems eminent. In this study, we analyze a selection of models used for hazard and/or exposure assessment of NMs. This analysis was done by identifying all the NM-related properties used in these models related to three categories of data: (i) Intrinsic properties specific to the NM, matrix or experimental conditions, (ii) Extrinsic NM properties related to interaction between the intrinsic properties and (iii) Measured hazard or exposure data. This analysis is combined with the current state of QPPR development to recommend further development of QPPRs for predictive risk assessment of NMs. In particular, the use of descriptors related to the interaction between a NM and its surroundings, e.g. the attachment efficiency is proposed.

The significance of nanomaterial post-exposure responses in Daphnia magna standard acute immobilisation assay: Example with testing TiO2 nanoparticles

One of the most widely used aquatic standarized tests for the toxicity screening of chemicals is the acute toxicity test with the freshwater crustacean Daphnia magna, which has also been applied in the toxicity screening of manufactured nanoparticles (NPs). However, in the case of non-soluble NPs most of the results of this test have showed no effect. The aim of the work presented here was to modify the standardized test by the least possible extent to make it more sensitive for non-soluble particles. The standard acute immobilisation assay with daphnids was modified by prolonging the exposure period and by measuring additional endpoints. Daphnids were exposed to TiO2 NPs in a standard acute test (48h of exposure), a standard acute test (48h of exposure) followed by 24h recovery period in clean medium or a prolonged exposure in the NPs solutions totaling 72h. Together with immobility, the adsorption of NPs to body surfaces was also observed as an alternative measure of the NPs effects. Our results showed almost no effect of TiO2 NPs on D. magna after the 48h standard acute test, while immobility was increased when the exposure period to TiO2 NPs was prolonged from 48h to 72h. Even when daphnids were transferred to clean medium for additional 24h after 48h of exposure to TiO2 NPs the immobility increased. We conclude that by transferring the daphnids to clean medium at the end of the 48h exposure to TiO2 NPs, the delayed effects of the tested material can be seen. This methodological step could improve the sensitivity of D. magna test as a model in nanomaterial environmental risk assessment.

Interdisciplinary Research Brought to School-Connecting Chemistry and Biology through Nanotechnology.

Nanoscience is a cutting edge and highly interdisciplinary field of research. The experiment presented here is a good example of bringing this interdisciplinary research to school by working with biological and chemical methods. Students first synthesize silver nanoparticles and then test them for their antimicrobial effect in a yeast culture. By observing the yeast growth and the nanosilver-induced suppression over 72 hours, students not only learn about nanoscience but also get an insight into research methods commonly used in the field of toxicology – which is highly relevant for the risk assessment of nanomaterials. Further, attention was given to develop a teaching unit that has close connections to real research, i.e. highlighting typical features of current research like interdisciplinarity, but also standard research procedures like multiple measurements and systematic test analysis to gain more reliable results. Detailed illustrated instructions for all parts of the experiment are included in the Appendix, as well as a brief background on nanosilver, an Excel sheet for the test evaluation and the description of a setup to take time-lapse videos of the experiment.

Bio-distribution and bio-availability of silver and gold in rat tissues with silver/gold nanorod administration.

Along with the increasing applications of nanomaterials in medical fields, to know the systemic distribution of nanomaterials in the body through a precise method is required for the biosafety assessment of nanomaterials. In this study, we firstly have established a reliable inductively coupled plasma mass spectrometry (ICP-MS) method for concentration measurement of silver (Ag) and gold (Au) in biological tissues. Then, based on this method, the Ag or Au distribution in rat blood and almost all of the organs were analyzed after an i.v. or s.c. administration of Au@Ag NRs. Both the time-dependent contents of Ag and Au in blood and two pharmacokinetic models confirmed the rapid clearance of Ag from blood. At 24 h after i.v. injection, there was the highest level of Ag in liver, followed by portal nodes, spleen, lung, bone marrow and pancreas. In addition, we also found there were gender-related distributions of Ag and Au in some organs, especially after s.c. injection. Therefore, these more comprehensive and important results would give fundamental information for the biological risk assessment of nanomaterials.

Decision tree models to classify nanomaterials according to the DF4nanoGrouping scheme

To keep pace with its rapid development an efficient approach for the risk assessment of nanomaterials is needed. Grouping concepts as developed for chemicals are now being explored for its applicability to nanomaterials. One of the recently proposed grouping systems is DF4nanoGrouping scheme. In this study, we have developed three structure-activity relationship classification tree models to be used for supporting this system by identifying structural features of nanomaterials mainly responsible for the surface activity. We used data from 19 nanomaterials that were synthesized and characterized extensively in previous studies. Subsets of these materials have been used in other studies (short-term inhalation, protein carbonylation, and intrinsic oxidative potential), resulting in a unique data set for modeling. Out of a large set of 285 possible descriptors, we have demonstrated that only three descriptors (size, specific surface area, and the quantum-mechanical calculated property ‘lowest unoccupied molecular orbital’) need to be used to predict the endpoints investigated. The maximum number of descriptors that were finally selected by the classification trees (CT) was very low– one for intrinsic oxidative potential, two for protein carbonylation, and three for NOAEC. This suggests that the models were well-constructed and not over-fitted. The outcome of various statistical measures and the applicability domains of our models further indicate their robustness. Therefore, we conclude that CT can be a useful tool within the DF4nanoGrouping scheme that has been proposed before.

Use of metal/metal oxide spherical cluster and hydroxyl metal coordination complex for descriptor calculation in development of nanoparticle cytotoxicity classification model

Computational approaches have been suggested as an informative tool for risk assessment of nanomaterials. Nano (quantitative) structure-activity relationship, nano-(Q)SAR, models have been developed to predict toxicity of metal oxide (MOx) nanoparticles (NPs); however, the packing structure and cluster of nanoparticle have been included for the descriptor calculation in only two studies. This study proposed spherical cluster and hydroxyl metal coordination complex to calculate descriptors for development of nanoparticle cytotoxicity classification model. The model cluster was generated from metal (M) or MOx crystal structure to calculate physicochemical properties of M/MOx NPs and the hydroxyl metal coordination complex was used to calculate the properties of the metal cation in an aqueous environment. Data were collected for 2 M and 19 MOx NPs in human bronchial epithelial cell lines and murine myeloid cell lines at 100 μg/ml after 24 hours exposure. The model was developed with scaled HOMO energy of the model cluster and polarizability of the hydroxyl metal coordination complex, as reactivity of the particles and the cations explained cause of cytotoxic action by M/MOx NPs. As the developed model achieved 90.31% accuracy, the classification model in this work can be used for virtual screening of toxic action of M/MOx NPs.

Salinity-Based Toxicity of CuO Nanoparticles, CuO-Bulk and Cu Ion to Vibrio anguillarum.

Bacteria are used in ecotoxicology for their important role in marine ecosystems and their quick, reproducible responses. Here we applied a recently proposed method to assess the ecotoxicity of nanomaterials on the ubiquitous marine bacterium Vibrio anguillarum, as representative of brackish and marine ecosystems. The test allows the determination of 6-h EC50 in a wide range of salinity, by assessing the reduction of bacteria actively replicating and forming colonies. The toxicity of copper oxide nanoparticles (CuO NPs) at different salinities (5-20-35 ‰) was evaluated. CuSO4 5H2O and CuO bulk were used as reference toxicants (solubility and size control, respectively). Aggregation and stability of CuO NP in final testing dispersions were characterized; Cu2+ dissolution and the physical interactions between Vibrio and CuO NPs were also investigated. All the chemical forms of copper showed a clear dose-response relationship, although their toxicity was different. The order of decreasing toxicity was: CuSO4 5H2O > CuO NP > CuO bulk. As expected, the size of CuO NP aggregates increased with salinity and, concurrently, their toxicity decreased. Results confirmed the intrinsic toxicity of CuO NPs, showing modest Cu2+ dissolution and no evidence of CuO NP internalization or induction of bacterial morphological alterations. This study showed the V. anguillarum bioassay as an effective tool for the risk assessment of nanomaterials in marine and brackish environments.

Environmental Risk Assessment Strategy for Nanomaterials.

An Environmental Risk Assessment (ERA) for nanomaterials (NMs) is outlined in this paper. Contrary to other recent papers on the subject, the main data requirements, models and advancement within each of the four risk assessment domains are described, i.e., in the: (i) materials, (ii) release, fate and exposure, (iii) hazard and (iv) risk characterisation domains. The material, which is obviously the foundation for any risk assessment, should be described according to the legislatively required characterisation data. Characterisation data will also be used at various levels within the ERA, e.g., exposure modelling. The release, fate and exposure data and models cover the input for environmental distribution models in order to identify the potential (PES) and relevant exposure scenarios (RES) and, subsequently, the possible release routes, both with regard to which compartment(s) NMs are distributed in line with the factors determining the fate within environmental compartment. The initial outcome in the risk characterisation will be a generic Predicted Environmental Concentration (PEC), but a refined PEC can be obtained by applying specific exposure models for relevant media. The hazard information covers a variety of representative, relevant and reliable organisms and/or functions, relevant for the RES and enabling a hazard characterisation. The initial outcome will be hazard characterisation in test systems allowing estimating a Predicted No-Effect concentration (PNEC), either based on uncertainty factors or on a NM adapted version of the Species Sensitivity Distributions approach. The risk characterisation will either be based on a deterministic risk ratio approach (i.e., PEC/PNEC) or an overlay of probability distributions, i.e., exposure and hazard distributions, using the nano relevant models.