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Abstract Micro- and nanoplastics (MNPs) are ubiquitous environmental pollutants representing a concern for human health. MNPs have been detected in human placentas, indicating that during pregnancy maternal exposure may lead to placental transfer and foetal exposure, with potential for adverse effects on early-life development. However, a comprehensive risk assessment (RA) framework, specific to early-life is lacking. Here, we propose a novel roadmap to assist the development of an early-life health RA of MNPs. This roadmap is designed based on established chemical, mixture, particle, and MNP assessment strategies aligned with standard RA components (problem formulation, hazard identification, hazard characterisation, exposure assessment, risk characterisation). We systematically work through these stages to identify what is needed to progress a RA for the early-life impacts of MNPs, including what information is missing, and what may be used in the interim. While challenges such as complex physicochemical properties of MNPs, limited toxicity data at relevant exposure levels, and uncertainties related to characterising complex exposures have been described elsewhere, our work discusses how these challenges specifically impact early-life stages such as the significance of MNP presence in biological samples and factors influencing bioaccumulation and placental transfer. Additionally, we introduce the development of new technology readiness levels for methods used in the detection of MNPs in complex matrices. Importantly, this review integrates a broad scope of relevant information into one comprehensive document, providing a unified resource. We highlight specific requirements and areas for targeted research, including the development of dose-response relationships specific to early-life stages and novel strategies for assessing bioaccumulation and placental transfer of MNPs. By addressing these gaps, our roadmap aims to advance the development of a robust framework, ultimately enhancing the understanding and mitigation of risks associated with early-life exposure to MNPs.

Nanoenergetics are defined as a class of nanomaterials that possess the ability to release energy in certain situations. These properties have been studied and deepened in recent years, so much so that nanoenergetics have been introduced into the use of the weapons industry, among others. It is therefore an emerging reality that deserves attention with regard to potential harmful effects on human and environmental health. it has been suggested that nanoenergetics may have genotoxic and immunotoxic effects, among others. Problems related to exposure to nanonenergetics can therefore potentially affect both exposed workers (both in the production and use phase) and the civilian population, if used in war scenarios, for example,. Starting from these assumptions, the INNOTOX research project aims to contribute to the in-depth study of the toxicity of nanonenergetics, through an integrated approach involving experts in occupational and forensic medicine, nanotoxicology and bioengineering.

Abstract 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-1β 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.

Termite mushrooms are edible fungi that provide significant economic, nutritional, and medicinal value. However, identifying these mushroom species based on morphology and traditional knowledge is ineffective due to their short development time and seasonal nature. This study proposes a novel method for classifying termite mushroom species. The method utilizes Gradient Boosting machine learning techniques and sequence encoding on the Internal Transcribed Spacer (ITS) gene dataset to construct a machine learning model for identifying termite mushroom species. The model is trained using ITS sequences obtained from the National Center for Biotechnology Information (NCBI) and the Barcode of Life Data Systems (BOLD). Ensemble learning techniques are applied to classify termite mushroom species. The proposed model achieves good results on the test dataset, with an accuracy of 0.91 and an average AUCROC value of 0.99. To validate the model, eight ITS sequences collected from termite mushroom samples in An Linh commune, Phu Giao district, Binh Duong province, Vietnam were used as the test data. The results show consistent species identification with predictions from the NCBI BLAST software. The results of species identification were consistent with the NCBI BLAST prediction software. This machine-learning model shows promise as an automatic solution for classifying termite mushroom species. It can help researchers better understand the local growth of these termite mushrooms and develop conservation plans for this rare and valuable plant resource.

Abstract Chemicals containing Volatile Organic Compounds (VOCs) are commonly used in the machine carpet production. 1,3-butadiene and styrene are main components of the carpenter’s glue used in carpet factories. Exposition to these chemicals can lead to a number of adverse health effects. This is the first study of the human health risk assessment due to inhalational exposure to 1,3-butadiene (BD) and styrene (ST) performed among workers in the carpet factories in Kashan city, Iran. The importance of the study was related with the fact of high popularity of carpet production in the South Asia countries. Inhalation exposure to BD and ST were measured based on the National Institute for Occupational Safety and Health (NIOSH) 1024 and 1501 methods, respectively. The cancerogenic risk (CR) and non-cancerogenic risk described as Hazard Quotient (HQ) values were calculated based on the United States Environmental Protection Agency (USEPA) method. The sensitivity and uncertainty analysis were performed by the Monte Carlo simulation (MCS) technique. The average concentration measured of BD and ST during work shifts of employees were 0.039 mg m−3 (0.017 ppm) and 12.108 mg m−3 (2.84 ppm), respectively. The mean ± SD value of estimated cancerogenic risk in inhalation exposure to BD and ST were equal to 5.13 × 10–3 ± 3.85 × 10–4 and 1.44 × 10–3 ± 2.36 × 10–4, respectively exceeding the acceptable risk level of 10–6 defined by USEPA. The average non-carcinogenic risk (HQ) values of BD and ST were equal to 8.50 × 100 and 5.13 × 100, respectively exceeding the acceptable risk level of 1. As the results of our studies exceeded both cancerogenic and non-carcinogenic risk values it indicates that adverse health effects due to inhalational exposure to BD and ST for workers in the machine carpet industry are very likely. To avoid negative health effects protective measures for employees in the factories should be introduced immediately and furher detailed research are recommended.

Nanomaterials have a range of potential applications, however, toxicity remains a concern, limiting application and requiring extensive testing. Here, the authors report on a predictive framework made using a range of tests linking materials properties with toxicity, allowing the prediction of toxicity from physiochemical and biological properties.

Despite the increasing exploitation of nanomaterials (NMs) in an array of consumer products, there are uncertainties regarding their potential adverse impact on human health. Investigation of whether NMs activate a pro-inflammatory response is routinely used to assess their toxicity in in vitro and in vivo (rodent) studies. The use of zebrafish ( Danio rerio) to investigate inflammatory responses to chemicals, pathogens and injury has increased considerably over recent years. Zebrafish have also been used to investigate the role of inflammation in disease pathogenesis and for drug discovery. Availability of transgenic strains which express fluorescent proteins in immune cells (e.g. macrophages and neutrophils) enables the visualization and quantification of immune cell accumulation in the target site(s) of interest. We therefore propose that transgenic zebrafish have great utility for screening the toxicity of NMs via investigation of inflammatory responses. Indeed, we have successfully used non-protected life stages of transgenic zebrafish with fluorescent neutrophils (Tg(mpx:EGFP 114) to investigate inflammatory responses to NMs. The more widespread use of transgenic zebrafish in nanotoxicology could reduce the reliance placed on rodents and thereby enhance the implementation of the 3Rs principles. As zebrafish continue to grow in popularity it is timely to offer guidance to new users on their use. Here we will reflect on: exposure routes that can adopted to mimic human/rodent exposure, what transgenic strains and life stages are best suited to investigate inflammatory responses, selection criteria for zebrafish embryos/larvae, the inclusion of appropriate controls, the importance of dose selection and sample size, and how the (inflammatory) response can be quantified. It is hoped that our recommendations will support the development of standard protocols that can be used to assess whether NMs activate inflammatory responses. Importantly, the themes discussed are not restricted to NMs but relevant also to zebrafish application in ecotoxicology or human health focused studies.

Toxicity evaluation of engineered nanomaterials is challenging due to the ever increasing number of materials and because nanomaterials (NMs) frequently interfere with commonly used assays. Hence, there is a need for robust, high-throughput assays with which to assess their hazard potential. The present study aimed at evaluating the applicability of a genotoxicity assay based on the immunostaining and foci counting of the DNA repair protein 53BP1 (p53-binding protein 1), in a high-throughput format, for NM genotoxicity assessment. For benchmarking purposes, we first applied the assay to a set of eight known genotoxic agents, as well as X-ray irradiation (1 Gy). Then, a panel of NMs and nanobiomaterials (NBMs) was evaluated with respect to their impact on cell viability and genotoxicity, and to their potential to induce reactive oxygen species (ROS) production. The genotoxicity recorded using the 53BP1 assay was confirmed using the micronucleus assay, also scored via automated (high-throughput) microscopy. The 53BP1 assay successfully identified genotoxic compounds on the HCT116 human intestinal cell line. None of the tested NMs showed any genotoxicity using the 53BP1 assay, except the positive control consisting in (CoO)(NiO) NMs, while only TiO2 NMs showed positive outcome in the micronucleus assay. Only Fe3O4 NMs caused significant elevation of ROS, not correlated to DNA damage. Therefore, owing to its adequate predictivity of the genotoxicity of most of the tested benchmark substance and its ease of implementation in a high throughput format, the 53BP1 assay could be proposed as a complementary high-throughput screening genotoxicity assay, in the context of the development of New Approach Methodologies.

Poly (ethylene glycol) (PEG) is a widely used polymer in a variety of consumer products and in medicine. PEGylation refers to the conjugation of PEG to drugs or nanoparticles to increase circulation time and reduce unwanted host responses. PEG is viewed as being well-tolerated, but previous studies have identified anti-PEG antibodies and so-called pseudoallergic reactions in certain individuals. The increased use of nanoparticles as contrast agents or in drug delivery, along with the introduction of mRNA vaccines encapsulated in PEGylated lipid nanoparticles has brought this issue to the fore. Thus, while these vaccines have proven to be remarkably effective, rare cases of anaphylaxis have been reported, and this has been tentatively ascribed to the PEGylated carriers, which may trigger complement activation in susceptible individuals. Here, we provide a general overview of the use of PEGylated nanoparticles for pharmaceutical applications, and we discuss the activation of the complement cascade that might be caused by PEGylated nanomedicines for a better understanding of these immunological adverse reactions.

Engineered nanomaterials are potentially very useful for a variety of applications, but studies are needed to ascertain whether these materials pose a risk to human health. Here, we studied three benchmark nanomaterials (Ag nanoparticles, TiO2 nanoparticles, and multi-walled carbon nanotubes, MWCNTs) procured from the nanomaterial repository at the Joint Research Centre of the European Commission. Having established a sub-lethal concentration of these materials using two human cell lines representative of the immune system and the lungs, respectively, we performed RNA sequencing of the macrophage-like cell line after exposure for 6, 12, and 24 h. Downstream analysis of the transcriptomics data revealed significant effects on chemokine signaling pathways. CCR2 was identified as the most significantly upregulated gene in MWCNT-exposed cells. Using multiplex assays to evaluate cytokine and chemokine secretion, we could show significant effects of MWCNTs on several chemokines, including CCL2, a ligand of CCR2. The results demonstrate the importance of evaluating sub-lethal concentrations of nanomaterials in relevant target cells.

Comparative hazard identification of nanomaterials (NMs) can aid in the prioritisation for further toxicity testing. Here, we assessed the acute lung, systemic and liver responses in C57BL/6N mice for three NMs to provide a hazard ranking. A silver (Ag), non-functionalised zinc oxide (ZnO) and a triethoxycaprylylsilane functionalised ZnO NM suspended in water with 2% mouse serum were examined 24 hours following a single intratracheal instillation (I.T.). An acute pulmonary inflammation was noted (marked by a polymorphonuclear neutrophil influx) with cell damage (LDH and total protein) in broncho-alveolar lavage fluid (BALF) after administration of both non-functionalised and functionalised ZnO. The latter also induced systemic inflammation measured as an increase in blood neutrophils and a decrease in blood lymphocytes. Exposure to Ag NM was not accompanied by pulmonary inflammation or cytotoxicity, or by systemic inflammation. A decrease in glutathione levels was demonstrated in the liver following exposure to high doses of all three nanomaterials irrespective of any noticeable inflammatory or cytotoxic effects in the lung. By applying benchmark dose (BMD) modeling statistics to compare potencies of the NMs, we rank functionalised ZnO ranked the highest based on the largest number of affected endpoints, as well as the strongest responses observed after 24 hours. The non-functionalised ZnO NM gave an almost similar response, whereas Ag NM did not cause an acute response at similar doses.

Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry - hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO - uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques - precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially 'weak-embryotoxic' and ZnO and SiO2 NMs as 'non-embryotoxic'. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.