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.

Limiting dysfunctional neutrophilic inflammation while preserving effective immunity requires a better understanding of the processes that dictate neutrophil function in the tissues. Quantitative mass-spectrometry identified how inflammatory murine neutrophils regulated expression of cell surface receptors, signal transduction networks, and metabolic machinery to shape neutrophil phenotypes in response to hypoxia. Through the tracing of labeled amino acids into metabolic enzymes, proinflammatory mediators, and granule proteins, we demonstrated that ongoing protein synthesis shapes the neutrophil proteome. To maintain energy supplies in the tissues, neutrophils consumed extracellular proteins to fuel central carbon metabolism. The physiological stresses of hypoxia and hypoglycemia, characteristic of inflamed tissues, promoted this extracellular protein scavenging with activation of the lysosomal compartment, further driving exploitation of the protein-rich inflammatory milieu. This study provides a comprehensive map of neutrophil proteomes, analysis of which has led to the identification of active catabolic and anabolic pathways that enable neutrophils to sustain synthetic and effector functions in the tissues.

Neutrophilic inflammation is central to disease pathogenesis, for example, in chronic obstructive pulmonary disease, yet the mechanisms that retain neutrophils within tissues remain poorly understood. With emerging evidence that axon guidance factors can regulate myeloid recruitment and that neutrophils can regulate expression of a class 3 semaphorin, SEMA3F, we investigated the role of SEMA3F in inflammatory cell retention within inflamed tissues. We observed that neutrophils upregulate SEMA3F in response to proinflammatory mediators and following neutrophil recruitment to the inflamed lung. In both zebrafish tail injury and murine acute lung injury models of neutrophilic inflammation, overexpression of SEMA3F delayed inflammation resolution with slower neutrophil migratory speeds and retention of neutrophils within the tissues. Conversely, constitutive loss of sema3f accelerated egress of neutrophils from the tail injury site in fish, whereas neutrophil-specific deletion of Sema3f in mice resulted in more rapid neutrophil transit through the airways, and significantly reduced time to resolution of the neutrophilic response. Study of filamentous-actin (F-actin) subsequently showed that SEMA3F-mediated retention is associated with F-actin disassembly. In conclusion, SEMA3F signaling actively regulates neutrophil retention within the injured tissues with consequences for neutrophil clearance and inflammation resolution.

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.

Background: The EU-project GRACIOUS developed an Integrated Approach to Testing and Assessment (IATA) to support grouping high aspect ratio nanomaterials (HARNs) presenting a similar inhalation hazard. Application of grouping reduces the need to assess toxicity on a case-by-case basis and supports read-across of hazard data from substances that have the data required for risk assessment (source) to those that lack such data (target). The HARN IATA, based on the fibre paradigm for pathogenic fibres, facilitates structured data gathering to propose groups of similar HARN and to support read-across by prompting users to address relevant questions regarding HARN morphology, biopersistence and inflammatory potential. The IATA is structured in tiers, allowing grouping decisions to be made using simple in vitro or in silico methods in Tier1 progressing to in vivo approaches at the highest Tier3. Here we present a case-study testing the applicability of GRACIOUS IATA to form an evidence-based group of multiwalled carbon nanotubes (MWCNT) posing a similar predicted fibre-hazard, to support read-across and reduce the burden of toxicity testing. Results: The case-study uses data on 15 different MWCNT, obtained from the published literature. By following the IATA, a group of 2 MWCNT was identified (NRCWE006 and NM-401) based on a high degree of similarity. A pairwise similarity assessment was subsequently conducted between the grouped MWCNT to evaluate the potential to conduct read-across and fill data gaps required for regulatory hazard assessment. The similarity assessment, based on expert judgement of Tier 1 assay results, predicts both MWCNT are likely to cause a similar acute in vivo hazard. This result supports the possibility for read-across of sub-chronic and chronic hazard endpoint data for lung fibrosis and carcinogenicity between the 2 grouped MWCNT. The implications of accepting the similarity assessment based on expert judgement of the MWCNT group are considered to stimulate future discussion on the level of similarity between group members considered sufficient to allow regulatory acceptance of a read-across argument. Conclusion: This proof-of-concept case-study demonstrates how a grouping hypothesis and IATA may be used to support a nuanced and evidence-based grouping of ‘similar’ MWCNT and the subsequent interpolation of data between group members to streamline the hazard assessment process.

The reactivity of particle surfaces can be used as a criterion to group nanoforms (NFs) based on similar potential hazard. Since NFs may partially or completely dissolve over the duration of the assays, with the ions themselves inducing a response, reactivity assays commonly measure the additive reactivity of the particles and ions combined. Here, we determine the concentration of ions released over the course of particle testing, and determine the relative contributions of the released ions to the total reactivity measured. We differentiate three classes of reactivity, defined as being (A) dominated by particles, (B) additive of particles and ions, or (C) dominated by ions. We provide examples for each class by analyzing the NF reactivity of Fe₂O₃, ZnO, CuO, Ag using the ferric reduction ability of serum (FRAS) assay. Furthermore, another two reactivity tests were performed: Dichlorodihydrofluorescin diacetate (DCFH₂-DA) assay and electron paramagnetic resonance (EPR) spectroscopy. We compare assays and demonstrate that the dose-response may be almost entirely assigned to ions in one assay (CuO in DCFH₂-DA), but to particles in others (CuO in EPR and FRAS). When considering this data, we conclude that one cannot specify the contribution of ions to NF toxicity for a certain NF, but only for a certain NF in a specific assay, medium and dose. The extent of dissolution depends on the buffer used, particle concentration applied, and duration of exposure. This culminates in the DCFH₂-DA, EPR, FRAS assays being performed under different ion-to-particle ratios, and differing in their sensitivity towards reactions induced by either ions or particles. If applied for grouping, read-across, or other concepts based on the similarity of partially soluble NFs, results on reactivity should only be compared if measured by the same assay, incubation time, and dose range.