Establishing relationships between particle-induced in vitro and in vivo inflammation endpoints to better extrapolate between in vitro markers and in vivo fibrosis.

BACKGROUND: Toxicity assessment for regulatory purposes is starting to move away from traditional in vivo methods and towards new approach methodologies (NAM) such as high-throughput in vitro models and computational tools. For materials with limited hazard information, utilising quantitative Adverse Outcome Pathways (AOPs) in a testing strategy involving NAM can produce information relevant for risk assessment. The aim of this work was to determine the feasibility of linking in vitro endpoints to in vivo events, and moreover to key events associated with the onset of a chosen adverse outcome to aid in the development of NAM testing strategies. To do this, we focussed on the adverse outcome pathway (AOP) relating to the onset of pulmonary fibrosis. RESULTS: We extracted in vivo and in vitro dose-response information for particles known to induce this pulmonary fibrosis (crystalline silica, specifically α-quartz). To test the in vivo-in vitro extrapolation (IVIVE) determined for crystalline silica, cerium dioxide nanoparticles (nano-CeO2) were used as a case study allowing us to evaluate our findings with a less studied substance. The IVIVE methodology outlined in this paper is formed of five steps, which can be more generally summarised into two categories (i) aligning the in vivo and in vitro dosimetry, (ii) comparing the dose-response curves and derivation of conversion factors. CONCLUSION: Our analysis shows promising results with regards to correlation of in vitro cytokine secretion to in vivo acute pulmonary inflammation assessed by polymorphonuclear leukocyte influx, most notable is the potential of using IL-6 and IL-1ß cytokine secretion from simple in vitro submerged models as a screening tool to assess the likelihood of lung inflammation at an early stage in product development, hence allowing a more targeted investigation using either a smaller, more targeted in vivo study or in the future a more complex in vitro protocol. This paper also highlights the strengths and limitations as well as the current difficulties in performing IVIVE assessment and suggestions for overcoming these issues.

Simulated biological fluids - a systematic review of their biological relevance and use in relation to inhalation toxicology of particles and fibres.

The use of simulated biological fluids (SBFs) is a promising in vitro technique to better understand the release mechanisms and possible in vivo behaviour of materials, including fibres, metal-containing particles and nanomaterials. Applications of SBFs in dissolution tests allow a measure of material biopersistence or, conversely, bioaccessibility that in turn can provide a useful inference of a materials biodistribution, its acute and long-term toxicity, as well as its pathogenicity. Given the wide range of SBFs reported in the literature, a review was conducted, with a focus on fluids used to replicate environments that may be encountered upon material inhalation, including extracellular and intracellular compartments. The review aims to identify when a fluid design can replicate realistic biological conditions, demonstrate operation validation, and/or provide robustness and reproducibility. The studies examined highlight simulated lung fluids (SLFs) that have been shown to suitably replicate physiological conditions, and identify specific components that play a pivotal role in dissolution mechanisms and biological activity; including organic molecules, redox-active species and chelating agents. Material dissolution was not always driven by pH, and likewise not only driven by SLF composition; specific materials and formulations correspond to specific dissolution mechanisms. It is recommended that SLF developments focus on biological predictivity and if not practical, on better biological mimicry, as such an approach ensures results are more likely to reflect in vivo behaviour regardless of the material under investigation.

A comparison of dermal toxicity models; assessing suitability for safe(r)-by-design decision-making and for screening nanomaterial hazards.

The objective of Safe-by-Design (SbD) is to support the development of safer products and production processes, and enable safe use throughout a materials' life cycle; an intervention at an early stage of innovation can greatly benefit industry by reducing costs associated with the development of products later found to elicit harmful effects. Early hazard screening can support this process, and is needed for all of the expected nanomaterial exposure routes, including inhalation, ingestion and dermal. In this study, we compare in vitro and ex vivo cell models that represent dermal exposures (including HaCaT cells, primary keratinocytes, and reconstructed human epidermis (RhE)), and when possible consider these in the context of regulatory accepted OECD TG for in vitro dermal irritation. Various benchmark nanomaterials were used to assess markers of cell stress in each cell model. In addition, we evaluated different dosing strategies that have been used when applying the OECD TG for dermal irritation in assessment of nanomaterials, and how inconsistencies in the approach used can have considerable impact of the conclusions made. Although we could not demonstrate alignment of all models used, there was an indication that the simpler in vitro cell model aligned more closely with RhE tissue than ex vivo primary keratinocytes, supporting the use of HaCaT cells for screening of dermal toxicity of nanomaterials and in early-stage SbD decision-making.

Safe(r)-by-design principles in the thermoplastics industry: guidance on release assessment during manufacture of nano-enabled products.

Background: The application of nanomaterials (NMs) and nano-enabled products (NEPs) across many industries has been extensive and is still expanding decades after first being identified as an emerging technology. Additive manufacturing has been greatly impacted and has seen the benefits of integrating NMs within products. With the expansion of nanotechnology, there has been a need to develop more adaptive and responsive methods to ascertain risks and ensure technology is developed safely. The Safe(r)-by-Design (SbD) concept can be used to establish safe parameters and minimise risks during the materials' lifecycle, including the early stages of the supply chain. Exposure monitoring has advanced in recent years with the creation of standardised protocols for occupational exposure assessment of nano-objects and their aggregates and agglomerates (NOAA). Methods: To aid in the development of an online SbD-supporting platform by the EU-funded project SAbyNA, we adopt a Europe Standard for monitoring release of NOAA to identify if a greater release of NOAA is associated with incorporation of NMs within NEPs compared to a polymer matrix alone. Case studies included filaments of polypropylene (PP) with nano-Ag or polycarbonate (PC) with single-walled carbon nanotubes (SWCNTs). NMs were received in masterbatch, and therefore previously modified to align with SbD interventions. Results were collected in line with European Standard recommendations: monitoring particle concentrations using direct reading instruments (DRI), sampling for offline chemical and morphological analysis, and collecting contextual information. Results and discussion: Based on the criteria described in the European standard (BS EN 17058), data from both case studies identified that inhalation exposure relating to NM was "unlikely". Despite this, during the production of the SWCNT-PC filaments, some noteworthy observations were made, including several DRI activity measurements shown to be higher than background levels, and material morphologically similar to the reference SWCNT/polymeric masterbatch observed in offline analysis. The data collected during this campaign were used to discuss choices available for data interpretation and decision-making in the European Standard for monitoring release of NOAA and also to facilitate the development of SAbyNA's user-friendly industry platform for the SbD of NMs and NEPs.

The State of the Art and Challenges of In Vitro Methods for Human Hazard Assessment of Nanomaterials in the Context of Safe-by-Design.

The Safe-by-Design (SbD) concept aims to facilitate the development of safer materials/products, safer production, and safer use and end-of-life by performing timely SbD interventions to reduce hazard, exposure, or both. Early hazard screening is a crucial first step in this process. In this review, for the first time, commonly used in vitro assays are evaluated for their suitability for SbD hazard testing of nanomaterials (NMs). The goal of SbD hazard testing is identifying hazard warnings in the early stages of innovation. For this purpose, assays should be simple, cost-effective, predictive, robust, and compatible. For several toxicological endpoints, there are indications that commonly used in vitro assays are able to predict hazard warnings. In addition to the evaluation of assays, this review provides insights into the effects of the choice of cell type, exposure and dispersion protocol, and the (in)accurate determination of dose delivered to cells on predictivity. Furthermore, compatibility of assays with challenging advanced materials and NMs released from nano-enabled products (NEPs) during the lifecycle is assessed, as these aspects are crucial for SbD hazard testing. To conclude, hazard screening of NMs is complex and joint efforts between innovators, scientists, and regulators are needed to further improve SbD hazard testing.