Grouping of orally ingested silica nanomaterials via use of an integrated approach to testing and assessment to streamline risk assessment.

BACKGROUND: Nanomaterials can exist in different nanoforms (NFs). Their grouping may be supported by the formulation of hypotheses which can be interrogated via integrated approaches to testing and assessment (IATA). IATAs are decision trees that guide the user through tiered testing strategies (TTS) to collect the required evidence needed to accept or reject a grouping hypothesis. In the present paper, we investigated the applicability of IATAs for ingested NFs using a case study that includes different silicon dioxide, SiO2 NFs. Two oral grouping hypotheses addressing local and systemic toxicity were identified relevant for the grouping of these NFs and verified through the application of oral IATAs. Following different Tier 1 and/or Tier 2 in vitro methods of the TTS (i.e., in vitro dissolution, barrier integrity and inflammation assays), we generated the NF datasets. Furthermore, similarity algorithms (e.g., Bayesian method and Cluster analysis) were utilized to identify similarities among the NFs and establish a provisional group(s). The grouping based on Tier 1 and/or Tier 2 testing was analyzed in relation to available Tier 3 in vivo data in order to verify if the read-across was possible and therefore support a grouping decision. RESULTS: The measurement of the dissolution rate of the silica NFs in the oro-gastrointestinal tract and in the lysosome identified them as gradually dissolving and biopersistent NFs. For the local toxicity to intestinal epithelium (e.g. cytotoxicity, membrane integrity and inflammation), the biological results of the gastrointestinal tract models indicate that all of the silica NFs were similar with respect to the lack of local toxicity and, therefore, belong to the same group; in vivo data (although limited) confirmed the lack of local toxicity of NFs. For systemic toxicity, Tier 1 data did not identify similarity across the NFs, with results across different decision nodes being inconsistent in providing homogeneous group(s). Moreover, the available Tier 3 in vivo data were also insufficient to support decisions based upon the obtained in vitro results and relating to the toxicity of the tested NFs. CONCLUSIONS: The information generated by the tested oral IATAs can be effectively used for similarity assessment to support a grouping decision upon the application of a hypothesis related to toxicity in the gastrointestinal tract. The IATAs facilitated a structured data analysis and, by means of the expert's interpretation, supported read-across with the available in vivo data. The IATAs also supported the users in decision making, for example, reducing the testing when the grouping was well supported by the evidence and/or moving forward to advanced testing (e.g., the use of more suitable cellular models or chronic exposure) to improve the confidence level of the data and obtain more focused information.

Integrated approaches to testing and assessment for grouping nanomaterials following dermal exposure.

Exposure to different nanoforms (NFs) via the dermal route is expected in occupational and consumer settings and thus it is important to assess their dermal toxicity and the contribution of dermal exposure to systemic bioavailability. We have formulated four grouping hypotheses for dermal toxicity endpoints which allow NFs to be grouped to streamline and facilitate risk assessment. The grouping hypotheses are developed based on insight into how physicochemical properties of NFs (i.e. composition, dissolution kinetics, size, and flexibility) influence their fate and hazard following dermal exposure. Each hypothesis is accompanied by a tailored Integrated Approach to Testing and Assessment (IATA) that is structured as a decision tree and tiered testing strategies (TTS) for each relevant question (at decision nodes) that indicate what information is needed to guide the user to accept or reject the grouping hypothesis. To develop these hypotheses and IATAs, we gathered and analyzed existing information on skin irritation, skin sensitization, and dermal penetration of NFs from the published literature and performed experimental work to generate data on NF dissolution in sweat simulant fluids. We investigated the dissolution of zinc oxide and silicon dioxide NFs in different artificial sweat fluids, demonstrating the importance of using physiologically relevant conditions for dermal exposure. All existing and generated data informed the formulation of the grouping hypotheses, the IATAs, and the design of the TTS. It is expected that the presented IATAs will accelerate the NF risk assessment for dermal toxicity via the application of read-across.

An Integrated Approach to Testing and Assessment to Support Grouping and Read-Across of Nanomaterials After Inhalation Exposure.

Introduction: Here, we describe the generation of hypotheses for grouping nanoforms (NFs) after inhalation exposure and the tailored Integrated Approaches to Testing and Assessment (IATA) with which each specific hypothesis can be tested. This is part of a state-of-the-art framework to support the hypothesis-driven grouping and read-across of NFs, as developed by the EU-funded Horizon 2020 project GRACIOUS. Development of Grouping Hypotheses and IATA: Respirable NFs, depending on their physicochemical properties, may dissolve either in lung lining fluid or in acidic lysosomal fluid after uptake by cells. Alternatively, NFs may also persist in particulate form. Dissolution in the lung is, therefore, a decisive factor for the toxicokinetics of NFs. This has led to the development of four hypotheses, broadly grouping NFs as instantaneous, quickly, gradually, and very slowly dissolving NFs. For instantaneously dissolving NFs, hazard information can be derived by read-across from the ions. For quickly dissolving particles, as accumulation of particles is not expected, ion toxicity will drive the toxic profile. However, the particle aspect influences the location of the ion release. For gradually dissolving and very slowly dissolving NFs, particle-driven toxicity is of concern. These NFs may be grouped by their reactivity and inflammation potency. The hypotheses are substantiated by a tailored IATA, which describes the minimum information and laboratory assessments of NFs under investigation required to justify grouping. Conclusion: The GRACIOUS hypotheses and tailored IATA for respiratory toxicity of inhaled NFs can be used to support decision making regarding Safe(r)-by-Design product development or adoption of precautionary measures to mitigate potential risks. It can also be used to support read-across of adverse effects such as pulmonary inflammation and subsequent downstream effects such as lung fibrosis and lung tumor formation after long-term exposure.

Grouping Hypotheses and an Integrated Approach to Testing and Assessment of Nanomaterials Following Oral Ingestion.

The risk assessment of ingested nanomaterials (NMs) is an important issue. Here we present nine integrated approaches to testing and assessment (IATAs) to group ingested NMs following predefined hypotheses. The IATAs are structured as decision trees and tiered testing strategies for each decision node to support a grouping decision. Implications (e.g., regulatory or precautionary) per group are indicated. IATAs integrate information on durability and biopersistence (dissolution kinetics) to specific hazard endpoints, e.g., inflammation and genotoxicity, which are possibly indicative of toxicity. Based on IATAs, groups of similar nanoforms (NFs) of a NM can be formed, such as very slow dissolving, highly biopersistent and systemically toxic NFs. Reference NMs (ZnO, SiO2 and TiO2) along with related NFs are applied as case studies to testing the oral IATAs. Results based on the Tier 1 level suggest a hierarchy of biodurability and biopersistence of TiO2 > SiO2 > ZnO, and are confirmed by in vivo data (Tier 3 level). Interestingly, our analysis suggests that TiO2 and SiO2 NFs are able to induce both local and systemic toxicity along with microbiota dysbiosis and can be grouped according to the tested fate and hazard descriptors. This supports that the decision nodes of the oral IATAs are suitable for classification and assessment of the toxicity of NFs.

Guidance on risk assessment of nanomaterials to be applied in the food and feed chain: human and animal health.

The EFSA has updated the Guidance on risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain, human and animal health. It covers the application areas within EFSA's remit, including novel foods, food contact materials, food/feed additives and pesticides. The updated guidance, now Scientific Committee Guidance on nano risk assessment (SC Guidance on Nano-RA), has taken account of relevant scientific studies that provide insights to physico-chemical properties, exposure assessment and hazard characterisation of nanomaterials and areas of applicability. Together with the accompanying Guidance on Technical requirements for regulated food and feed product applications to establish the presence of small particles including nanoparticles (Guidance on Particle-TR), the SC Guidance on Nano-RA specifically elaborates on physico-chemical characterisation, key parameters that should be measured, methods and techniques that can be used for characterisation of nanomaterials and their determination in complex matrices. The SC Guidance on Nano-RA also details aspects relating to exposure assessment and hazard identification and characterisation. In particular, nanospecific considerations relating to in vitro/in vivo toxicological studies are discussed and a tiered framework for toxicological testing is outlined. Furthermore, in vitro degradation, toxicokinetics, genotoxicity, local and systemic toxicity as well as general issues relating to testing of nanomaterials are described. Depending on the initial tier results, additional studies may be needed to investigate reproductive and developmental toxicity, chronic toxicity and carcinogenicity, immunotoxicity and allergenicity, neurotoxicity, effects on gut microbiome and endocrine activity. The possible use of read-across to fill data gaps as well as the potential use of integrated testing strategies and the knowledge of modes or mechanisms of action are also discussed. The Guidance proposes approaches to risk characterisation and uncertainty analysis.

Issues currently complicating the risk assessment of synthetic amorphous silica (SAS) nanoparticles after oral exposure.

Synthetic amorphous silica (SAS) is applied in food products as food additive E 551. It consists of constituent amorphous silicon dioxide (SiO2) nanoparticles that form aggregates and agglomerates. We reviewed recent oral toxicity studies with SAS. Some of those report tissue concentrations of silicon (Si). The results of those studies were compared with recently determined tissue concentrations of Si (and Si-particles) in human postmortem tissues. We noticed inconsistent results of the various toxicity studies regarding toxicity and reported tissue concentrations, which hamper the risk assessment of SAS. A broad range of Si concentrations is reported in control animals in toxicity studies. The Si concentrations found in human postmortem tissues fall within this range. On the other hand, the mean concentration found in human liver is higher than the reported concentrations causing liver effects in some animal toxicity studies after oral exposure to SAS. Also higher liver concentrations are observed in other, negative animal studies. Those inconsistencies could be caused by the presence of other Si-containing chemical substances or particles (which potentially also includes background SAS) and/or different sample preparation and analytical techniques that were used. Other factors which could explain the inconsistencies in outcome between the toxicity studies are the distinct SAS used and different dosing regimes, such as way of administration (dietary, via drinking water, oral gavage), dispersion of SAS and dose. More research is needed to address these issues and to perform a proper risk assessment for SAS in food. The current review will help to progress research on the toxicity of SAS and the associated risk assessment.

An integrated approach to testing and assessment of high aspect ratio nanomaterials and its application for grouping based on a common mesothelioma hazard.

Here we describe the development of an Integrated Approach to Testing and Assessment (IATA) to support the grouping of different types (nanoforms; NFs) of High Aspect Ratio Nanomaterials (HARNs), based on their potential to cause mesothelioma. Hazards posed by the inhalation of HARNs are of particular concern as they exhibit physical characteristics similar to pathogenic asbestos fibres. The approach for grouping HARNs presented here is part of a framework to provide guidance and tools to group similar NFs and aims to reduce the need to assess toxicity on a case-by-case basis. The approach to grouping is hypothesis-driven, in which the hypothesis is based on scientific evidence linking critical physicochemical descriptors for NFs to defined fate/toxicokinetic and hazard outcomes. The HARN IATA prompts users to address relevant questions (at decision nodes; DNs) regarding the morphology, biopersistence and inflammatory potential of the HARNs under investigation to provide the necessary evidence to accept or reject the grouping hypothesis. Each DN in the IATA is addressed in a tiered manner, using data from simple in vitro or in silico methods in the lowest tier or from in vivo approaches in the highest tier. For these proposed methods we provide justification for the critical descriptors and thresholds that allow grouping decisions to be made. Application of the IATA allows the user to selectively identify HARNs which may pose a mesothelioma hazard, as demonstrated through a literature-based case study. By promoting the use of alternative, non-rodent approaches such as in silico modelling, in vitro and cell-free tests in the initial tiers, the IATA testing strategy streamlines information gathering at all stages of innovation through to regulatory risk assessment while reducing the ethical, time and economic burden of testing.

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.

Risk assessment of titanium dioxide nanoparticles via oral exposure, including toxicokinetic considerations.

Titanium dioxide white pigment consists of particles of various sizes, from which a fraction is in the nano range (<100 nm). It is applied in food as additive E 171 as well as in other products, such as food supplements and toothpaste. Here, we assessed whether a human health risk can be expected from oral ingestion of these titanium dioxide nanoparticles (TiO2 NPs), based on currently available information. Human health risks were assessed using two different approaches: Approach 1, based on intake, i.e. external doses, and Approach 2, based on internal organ concentrations using a kinetic model in order to account for accumulation over time (the preferred approach). Results showed that with Approach 1, a human health risk is not expected for effects in liver and spleen, but a human health risk cannot be excluded for effects on the ovaries. When based on organ concentrations by including the toxicokinetics of TiO2 NPs (Approach 2), a potential risk for liver, ovaries and testes is found. This difference between the two approaches shows the importance of including toxicokinetic information. The currently estimated risk can be influenced by factors such as absorption, form of TiO2, particle fraction, particle size and physico-chemical properties in relation to toxicity, among others. Analysis of actual particle concentrations in human organs, as well as organ concentrations and effects in liver and the reproductive system after chronic exposure to well-characterized TiO2 (NPs) in animals are recommended to refine this assessment.

Towards a nanospecific approach for risk assessment.

In the current paper, a new strategy for risk assessment of nanomaterials is described, which builds upon previous project outcomes and is developed within the FP7 NANoREG project. NANoREG has the aim to develop, for the long term, new testing strategies adapted to a high number of nanomaterials where many factors can affect their environmental and health impact. In the proposed risk assessment strategy, approaches for (Quantitative) Structure Activity Relationships ((Q)SARs), grouping and read-across are integrated and expanded to guide the user how to prioritise those nanomaterial applications that may lead to high risks for human health. Furthermore, those aspects of exposure, kinetics and hazard assessment that are most likely to be influenced by the nanospecific properties of the material under assessment are identified. These aspects are summarised in six elements, which play a key role in the strategy: exposure potential, dissolution, nanomaterial transformation, accumulation, genotoxicity and immunotoxicity. With the current approach it is possible to identify those situations where the use of nanospecific grouping, read-across and (Q)SAR tools is likely to become feasible in the future, and to point towards the generation of the type of data that is needed for scientific justification, which may lead to regulatory acceptance of nanospecific applications of these tools.

The MARINA Risk Assessment Strategy: A Flexible Strategy for Efficient Information Collection and Risk Assessment of Nanomaterials.

An engineered nanomaterial (ENM) may actually consist of a population of primary particles, aggregates and agglomerates of various sizes. Furthermore, their physico-chemical characteristics may change during the various life-cycle stages. It will probably not be feasible to test all varieties of all ENMs for possible health and environmental risks. There is therefore a need to further develop the approaches for risk assessment of ENMs. Within the EU FP7 project Managing Risks of Nanoparticles (MARINA) a two-phase risk assessment strategy has been developed. In Phase 1 (Problem framing) a base set of information is considered, relevant exposure scenarios (RESs) are identified and the scope for Phase 2 (Risk assessment) is established. The relevance of an RES is indicated by information on exposure, fate/kinetics and/or hazard; these three domains are included as separate pillars that contain specific tools. Phase 2 consists of an iterative process of risk characterization, identification of data needs and integrated collection and evaluation of data on the three domains, until sufficient information is obtained to conclude on possible risks in a RES. Only data are generated that are considered to be needed for the purpose of risk assessment. A fourth pillar, risk characterization, is defined and it contains risk assessment tools. This strategy describes a flexible and efficient approach for data collection and risk assessment which is essential to ensure safety of ENMs. Further developments are needed to provide guidance and make the MARINA Risk Assessment Strategy operational. Case studies will be needed to refine the strategy.

Grouping and Read-Across Approaches for Risk Assessment of Nanomaterials.

Physicochemical properties of chemicals affect their exposure, toxicokinetics/fate and hazard, and for nanomaterials, the variation of these properties results in a wide variety of materials with potentially different risks. To limit the amount of testing for risk assessment, the information gathering process for nanomaterials needs to be efficient. At the same time, sufficient information to assess the safety of human health and the environment should be available for each nanomaterial. Grouping and read-across approaches can be utilised to meet these goals. This article presents different possible applications of grouping and read-across for nanomaterials within the broader perspective of the MARINA Risk Assessment Strategy (RAS), as developed in the EU FP7 project MARINA. Firstly, nanomaterials can be grouped based on limited variation in physicochemical properties to subsequently design an efficient testing strategy that covers the entire group. Secondly, knowledge about exposure, toxicokinetics/fate or hazard, for example via properties such as dissolution rate, aspect ratio, chemical (non-)activity, can be used to organise similar materials in generic groups to frame issues that need further attention, or potentially to read-across. Thirdly, when data related to specific endpoints is required, read-across can be considered, using data from a source material for the target nanomaterial. Read-across could be based on a scientifically sound justification that exposure, distribution to the target (fate/toxicokinetics) and hazard of the target material are similar to, or less than, the source material. These grouping and read-across approaches pave the way for better use of available information on nanomaterials and are flexible enough to allow future adaptations related to scientific developments.

Novel insights into the risk assessment of the nanomaterial synthetic amorphous silica, additive E551, in food.

This study presents novel insights in the risk assessment of synthetic amorphous silica (SAS) in food. SAS is a nanostructured material consisting of aggregates and agglomerates of primary particles in the nanorange (<100 nm). Depending on the production process, SAS exists in four main forms, and each form comprises various types with different physicochemical characteristics. SAS is widely used in foods as additive E551. The novel insights from other studies relate to low gastrointestinal absorption of SAS that decreases with increasing dose, and the potential for accumulation in tissues with daily consumption. To accommodate these insights, we focused our risk assessment on internal exposure in the target organ (liver). Based on blood and tissue concentrations in time of two different SAS types that were orally and intravenously administered, a kinetic model is developed to estimate the silicon concentration in liver in (1) humans for average-to-worst-case dietary exposure at steady state and (2) rats and mice in key toxicity studies. The estimated liver concentration in humans is at a similar level as the measured or estimated liver concentrations in animal studies in which adverse effects were found. Hence, this assessment suggests that SAS in food may pose a health risk. Yet, for this risk assessment, we had to make assumptions and deal with several sources of uncertainty that make it difficult to draw firm conclusions. Recommendations to fill in the remaining data gaps are discussed. More insight in the health risk of SAS in food is warranted considering the wide applications and these findings.

Concern-driven integrated approaches to nanomaterial testing and assessment--report of the NanoSafety Cluster Working Group 10.

Bringing together topic-related European Union (EU)-funded projects, the so-called "NanoSafety Cluster" aims at identifying key areas for further research on risk assessment procedures for nanomaterials (NM). The outcome of NanoSafety Cluster Working Group 10, this commentary presents a vision for concern-driven integrated approaches for the (eco-)toxicological testing and assessment (IATA) of NM. Such approaches should start out by determining concerns, i.e., specific information needs for a given NM based on realistic exposure scenarios. Recognised concerns can be addressed in a set of tiers using standardised protocols for NM preparation and testing. Tier 1 includes determining physico-chemical properties, non-testing (e.g., structure-activity relationships) and evaluating existing data. In tier 2, a limited set of in vitro and in vivo tests are performed that can either indicate that the risk of the specific concern is sufficiently known or indicate the need for further testing, including details for such testing. Ecotoxicological testing begins with representative test organisms followed by complex test systems. After each tier, it is evaluated whether the information gained permits assessing the safety of the NM so that further testing can be waived. By effectively exploiting all available information, IATA allow accelerating the risk assessment process and reducing testing costs and animal use (in line with the 3Rs principle implemented in EU Directive 2010/63/EU). Combining material properties, exposure, biokinetics and hazard data, information gained with IATA can be used to recognise groups of NM based upon similar modes of action. Grouping of substances in return should form integral part of the IATA themselves.

Knowledge gaps in risk assessment of nanosilica in food: evaluation of the dissolution and toxicity of different forms of silica.

This manuscript describes the follow-up study of our previous publication on the presence and risks of nanosilica in food. New information on the presence of nanosilica in the gastrointestinal tract is evaluated and information on nanosilica and synthetic amorphous silica (SAS) is compared to assess its relevance for risk assessment of nanosilica in food. Irrespective of whether SAS should be regarded as a nanomaterial or a non-nanoform of silica, a comparison to nanosilica is relevant to determine whether there are differences in physicochemical properties, which may lead to differences in toxicity. Based on this comparison, knowledge gaps are identified and recommendations for a targeted approach to facilitate risk assessment of nanosilica in food are given. Considering the discussion to which extent nanomaterials with (slightly) different physicochemical characteristics can be grouped for risk assessment--the sameness issue--actual exercises as presented in this manuscript are highly relevant for bringing this discussion forward.

Considerations on the EU definition of a nanomaterial: science to support policy making.

In recent years, an increasing number of applications and products containing or using nanomaterials have become available. This has raised concerns that some of these materials may introduce new risks for humans or the environment. A clear definition to discriminate nanomaterials from other materials is prerequisite to include provisions for nanomaterials in legislation. In October 2011 the European Commission published the 'Recommendation on the definition of a nanomaterial', primarily intended to provide unambiguous criteria to identify materials for which special regulatory provisions might apply, but also to promote consistency on the interpretation of the term 'nanomaterial'. In this paper, the current status of various regulatory frameworks of the European Union with regard to nanomaterials is described, and major issues relevant for regulation of nanomaterials are discussed. This will contribute to better understanding the implications of the choices policy makers have to make in further regulation of nanomaterials. Potential issues that need to be addressed and areas of research in which science can contribute are indicated. These issues include awareness on situations in which nano-related risks may occur for materials that fall outside the definition, guidance and further development of measurement techniques, and dealing with changes during the life cycle.

Consumer product in vitro digestion model: Bioaccessibility of contaminants and its application in risk assessment.

This paper describes the applicability of in vitro digestion models as a tool for consumer products in (ad hoc) risk assessment. In current risk assessment, oral bioavailability from a specific product is considered to be equal to bioavailability found in toxicity studies in which contaminants are usually ingested via liquids or food matrices. To become bioavailable, contaminants must first be released from the product during the digestion process (i.e. become bioaccessible). Contaminants in consumer products may be less bioaccessible than contaminants in liquid or food. Therefore, the actual risk after oral exposure could be overestimated. This paper describes the applicability of a simple, reliable, fast and relatively inexpensive in vitro method for determining the bioaccessibility of a contaminant from a consumer product. Different models, representing sucking and/or swallowing were developed. The experimental design of each model can be adjusted to the appropriate exposure scenarios as determined by the risk assessor. Several contaminated consumer products were tested in the various models. Although relevant in vivo data are scare, we succeeded to preliminary validate the model for one case. This case showed good correlation and never underestimated the bioavailability. However, validation check needs to be continued.