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Abstract: Colloidal quantum dots (QD) show great promise as fluorescent markers. The QD used in this study were obtained in aqueous medium rather than the widely used colloidal QD. Both methodologies used for the production of QD are associated with the presence of heavy metals such as cadmium (Cd). Here we investigate the short-term inhalation toxicity of water-soluble core-shell CdS/Cd(OH)2 QD. Male Wistar rats were head-nose exposed for 6h/day on 5 days at the technically maximum concentration (0.52mgCd/m3). Histological examination was performed directly after the last exposure. Additional rats were used for Cd organ burden determinations. Clinical parameters in blood, bronchoalveolar lavage fluid and lung tissue were determined 3 days after the last exposure. To analyze the reversibility or progression of effects, the examinations were performed again after a recovery period of 3 weeks. The results of the study indicate that CdS/Cd(OH)2 QD caused local neutrophil inflammation in the lungs that partially regressed after the 3-week recovery period. There was no evidence that QD were translocated to the central nervous system nor that a systemic acute phase response occurred. [Copyright &y& Elsevier]

Highlights: [•] We examined the inhalation toxicity and translocation ability of coated CdS/Cd(OH)2 quantum dots in rats. [•] Broncho-alveolar lavage fluid examinations and microscopy were performed. [•] Inflammatory process was observed by examination of broncho-alveolar lavage fluid. [•] Quantum dots caused less toxicity compared to compounds with larger particle sizes. [•] No translocation of the particles from lung to other organs were observed. [•] Small particle sizes are not the only factor triggering the toxic response or translocation. [Copyright &y& Elsevier]

Oxide-perovskites designed for automotive catalysts contain multiple metal elements whose presence is crucial to achieving the targeted performance. They are highly stable in exhaust operating conditions; however, little is known about their stability under physiological conditions. As some of the metallic components are hazardous to humans and the environment, perovskite benefits in cleaner air must be balanced with risks in a Safe and Sustainable Design (SSbD) approach. New approach methodologies (NAMs), including in chemico and in silico methods, were used for testing hazards and benefits, including catalytic activity and tolerance for temporary excess of oxygen under dynamic driving conditions. The composition and surface properties of six different lanthanum-based oxide-perovskites compromised their stability under lung physiological conditions, influencing the oxidative damage of the particles and the bioacessibility of leaching metals. We found consistent biotransformation of the oxide-perovskite materials at pH 4.5. The leached lanthanum ions, but not other metals, respeciated into lanthanum phosphate nanoparticles, which increased the overall oxidative damage in additive synergy. The NAM results in the presented SSbD approach were challenged by in vivo studies in rats and mice, which confirmed multicomponent clearance from lungs into urine and supported the comparative ranking of effects against well-characterized spinel materials. Among the perovskites, the version with reduced nickel content and doped with palladium offered the best SSbD balance, despite not improving the conventional benchmark catalytic performance and related sustainability benefits. Redesign by industry may be necessary to better fulfill all SSbD dimensions.

Glycopeptide antibiotics (GPAs) are peptide natural products used as last resort treatments for antibiotic resistant bacterial infections. They are produced by the sequential activities of a linear nonribosomal peptide synthetase (NRPS), which assembles the heptapeptide core of GPAs, and cytochrome P450 (Oxy) enzymes, which perform a cascade of cyclisation reactions. The GPAs contain proteinogenic and nonproteinogenic amino acids, including phenylglycine residues such as 4-hydroxyphenylglycine (Hpg). The ability to incorporate non-proteinogenic amino acids in such peptides is a distinctive feature of the modular architecture of NRPSs, with each module selecting and incorporating a desired amino acid. Here, we have exploited this ability to produce and characterise GPA derivatives containing fluorinated phenylglycine (F-Phg) residues through a combination of mutasynthesis, biochemical, structural and bioactivity assays. Our data indicate that the incorporation of F-Phg residues is limited by poor acceptance by the NRPS machinery, and that the phenol moiety normally present on Hpg residues is essential to ensure both acceptance by the NRPS and the sequential cyclisation activity of Oxy enzymes. The principles learnt here may prove useful for the future production of GPA derivatives with more favourable properties through mixed feeding mutasynthesis approaches., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Herein, a Safe-and-Sustainable-by-Design (SSbD) screening strategy on four different inorganic aerogel mats and two conventional mineral wools for ranking purposes is demonstrated. Given that they do not consist of particles, the release is first simulated, addressing three occupational exposure scenarios, realistic for their intended use as building insulators. No exposure to consumers nor to the environment is foreseen in the use phase, however, aerosols may be released during mat installation, posing an inhalation risk for workers. All four aerogel mats release more respirable dust than the benchmark materials and 60% thereof deposits in the alveolar region according to modelling tools. The collected aerogel dust allows for subsequent screening of hazard implications via two abiotic assays: 1) surface reactivity in human blood serum; 2) biodissolution kinetics in lung simulant fluids. Both aerogels and conventional insulators show similar surface reactivity. Differences in biodissolution are influenced by the specifically designed organic and inorganic structural modifications. Aerogel mats are better-performing insulators (2-fold lower thermal conductivity than the benchmark) However, this work demonstrates how investment decisions can be balanced with safety and sustainability aspects. Concepts of analogy and similarity thus support easily accessible methods to companies for safe and economically viable innovation with advanced materials., (© 2024 Wiley‐VCH GmbH.)

The natural soil environment of Streptomyces is characterized by variations in the availability of nitrogen, carbon, phosphate and sulfur, leading to complex primary and secondary metabolisms. Their remarkable ability to adapt to fluctuating nutrient conditions is possible through the utilization of a large amount of substrates by diverse intracellular and extracellular enzymes. Thus, Streptomyces fulfill an important ecological role in soil environments, metabolizing the remains of other organisms. In order to survive under changing conditions in their natural habitats, they have the possibility to fall back on specialized enzymes to utilize diverse nutrients and supply compounds from primary metabolism as precursors for secondary metabolite production. We aimed to summarize the knowledge on the C-, N-, P- and S-metabolisms in the genus Streptomyces as a source of building blocks for the production of antibiotics and other relevant compounds.

This manuscript discusses the challenges of applying New Approach Methodologies (NAMs) for safe by design and regulatory risk assessment of advanced nanomaterials (AdNMs). The authors propose a framework for Next Generation Risk Assessment of AdNMs involving NAMs that is aligned to the conventional risk assessment paradigm. This framework is exposure-driven, endpoint-specific, makes best use of pre-existing information, and can be implemented in tiers of increasing specificity and complexity of the adopted NAMs. The tiered structure of the approach, which effectively combines the use of existing data with targeted testing will allow safety to be assessed cost-effectively and as far as possible with even more limited use of vertebrates. The regulatory readiness of state-of-the-art emerging NAMs is assessed in terms of Transparency, Reliability, Accessibility, Applicability, Relevance and Completeness, and their appropriateness for AdNMs is discussed in relation to each step of the risk assessment paradigm along with providing perspectives for future developments in the respective scientific and regulatory areas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Nanomaterials (NMs) offer plenty of novel functionalities. Moreover, their physicochemical properties can be fine-tuned to meet the needs of specific applications, leading to virtually unlimited numbers of NM variants. Hence, efficient hazard and risk assessment strategies building on New Approach Methodologies (NAMs) become indispensable. Indeed, the design, the development and implementation of NAMs has been a major topic in a substantial number of research projects. One of the promising strategies that can help to deal with the high number of NMs variants is grouping and read-across. Based on demonstrated structural and physicochemical similarity, NMs can be grouped and assessed together. Within an established NM group, read-across may be performed to fill in data gaps for data-poor variants using existing data for NMs within the group. Establishing a group requires a sound justification, usually based on a grouping hypothesis that links specific physicochemical properties to well-defined hazard endpoints. However, for NMs these interrelationships are only beginning to be understood. The aim of this review is to demonstrate the power of bioinformatics with a specific focus on Machine Learning (ML) approaches to unravel the NM Modes-of-Action (MoA) and identify the properties that are relevant to specific hazards, in support of grouping strategies. This review emphasizes the following messages: 1) ML supports identification of the most relevant properties contributing to specific hazards; 2) ML supports analysis of large omics datasets and identification of MoA patterns in support of hypothesis formulation in grouping approaches; 3) omics approaches are useful for shifting away from consideration of single endpoints towards a more mechanistic understanding across multiple endpoints gained from one experiment; and 4) approaches from other fields of Artificial Intelligence (AI) like Natural Language Processing or image analysis may support automated extraction and interlinkage of information related to NM toxicity. Here, existing ML models for predicting NM toxicity and for analyzing omics data in support of NM grouping are reviewed. Various challenges related to building robust models in the field of nanotoxicology exist and are also discussed.

Microplastics have been detected in various food types, suggesting inevitable human exposure. A major fraction may originate from aerial deposition and could be contaminated by ubiquitous pollutants such as polycyclic aromatic hydrocarbons (PAHs). While data on the sorption of pollutants to microplastics are abundant, the subsequent desorption in the gastrointestinal tract (GIT) is less understood. This prompted us to systematically investigate the release of microplastics-sorbed PAHs at realistic loadings (44-95 ng/mg) utilizing a physiology-based in vitro model comprising digestion in simulated saliva, gastric, and small and large intestinal fluids. Using benzo[ a ]pyrene as a representative PAH, desorption from different microplastics based on low density polyethylene (LDPE), thermoplastic polyurethanes (TPUs), and polyamides (PAs) was investigated consecutively in all four GIT fluid simulants. The cumulative relative desorption (CRD) of benzo[ a ]pyrene was negligible in saliva simulant but increased from gastric (4 ± 1% - 15 ± 4%) to large intestinal fluid simulant (21 ± 1% - 29 ± 6%), depending on the polymer type. CRDs were comparable for ten different microplastics in the small intestinal fluid simulant, except for a polydisperse PA-6 variant (1-10 μm), which showed an exceptionally high release (51 ± 8%). Nevertheless, the estimated contribution of microplastics-sorbed PAHs to total human PAH dietary intake was very low (≤0.1%). Our study provides a systematic data set on the desorption of PAHs from microplastics in GIT fluid simulants., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: Significant variations exist in the forms of ZnO, making it impossible to test all forms in in vivo inhalation studies. Hence, grouping and read-across is a common approach under REACH to evaluate the toxicological profile of familiar substances. The objective of this paper is to investigate the potential role of dissolution, size, or coating in grouping ZnO (nano)forms for the purpose of hazard assessment. We performed a 90-day inhalation study (OECD test guideline no. (TG) 413) in rats combined with a reproduction/developmental (neuro)toxicity screening test (TG 421/424/426) with coated and uncoated ZnO nanoforms in comparison with microscale ZnO particles and soluble zinc sulfate. In addition, genotoxicity in the nasal cavity, lungs, liver, and bone marrow was examined via comet assay (TG 489) after 14-day inhalation exposure., Results: ZnO nanoparticles caused local toxicity in the respiratory tract. Systemic effects that were not related to the local irritation were not observed. There was no indication of impaired fertility, developmental toxicity, or developmental neurotoxicity. No indication for genotoxicity of any of the test substances was observed. Local effects were similar across the different ZnO test substances and were reversible after the end of the exposure., Conclusion: With exception of local toxicity, this study could not confirm the occasional findings in some of the previous studies regarding the above-mentioned toxicological endpoints. The two representative ZnO nanoforms and the microscale particles showed similar local effects. The ZnO nanoforms most likely exhibit their effects by zinc ions as no particles could be detected after the end of the exposure, and exposure to rapidly soluble zinc sulfate had similar effects. Obviously, material differences between the ZnO particles do not substantially alter their toxicokinetics and toxicodynamics. The grouping of ZnO nanoforms into a set of similar nanoforms is justified by these observations., (© 2024. The Author(s).)

To ensure the safe use of materials, one must assess the identity and quantity of exposure. Solid materials, such as plastics, metals, coatings and cements, degrade to some extent during their life cycle, and releases can occur during manufacturing, use and end-of-life. Releases (e.g., what is released, how does release happen, and how much material is released) depend on the composition and internal (nano)structures of the material as well as the applied stresses during the lifecycle. We consider, in some depth, releases from mechanical, weathering and thermal stresses and specifically address the use cases of fused-filament 3D printing, dermal contact, food contact and textile washing. Solid materials can release embedded nanomaterials, composite fragments, or micro- and nanoplastics, as well as volatile organics, ions and dissolved organics. The identity of the release is often a heterogenous mixture and requires adapted strategies for sampling and analysis, with suitable quality control measures. Control materials enhance robustness by enabling comparative testing, but reference materials are not always available as yet. The quantity of releases is typically described by time-dependent rates that are modulated by the nature and intensity of the applied stress, the chemical identity of the polymer or other solid matrix, and the chemical identity and compatibility of embedded engineered nanomaterials (ENMs) or other additives. Standardization of methods and the documentation of metadata, including all the above descriptors of the tested material, applied stresses, sampling and analytics, are identified as important needs to advance the field and to generate robust, comparable assessments. In this regard, there are strong methodological synergies between the study of all solid materials, including the study of micro- and nanoplastics. From an outlook perspective, we review the hazard of the released entities, and show how this informs risk assessment. We also address the transfer of methods to related issues such as tyre wear, advanced materials and advanced manufacturing, biodegradable polymers, and non-solid matrices. As the consideration of released entities will become more routine in industry via lifecycle assessment in Safe-and-Sustainable-by-Design practices, release assessments will require careful design of the study with quality controls, the use of agreed-on test materials and standardized methods where these exist and the adoption of clearly defined data reporting practices that enable data reuse, meta-analyses, and comparative studies., Competing Interests: Declaration of competing interest WW is employee of BASF SE, a company that markets several of the material classes that are in scope of this review., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Chemical degradation testing often involves monitoring the loss of a chemical or the evolution of a single diagnostic product through time. Here, we demonstrate a novel approach to tracing complex degradation networks using mass-spectrometry-based methods and open cheminformatics tools. Ester- and ether-based thermoplastic polyurethane (TPU_Ester and TPU_Ether) microplastics (350 μm) and microplastics-derived dissolved organic carbon (MP-DOC) were photoweathered in a simulated marine environment and subsequently analyzed by liquid chromatography coupled to high-resolution mass spectrometry. We formula-annotated 1342 and 2344 unique features in the MP-DOC of TPU_Ester and TPU_Ether, respectively. From these, we extracted 199 and 568 plausible parent-transformation product pairs via matching of features (a) with complementary increasing and decreasing trends (Spearman's correlation coefficient between normalized intensity and time), (b) spectral similarities of at least three accurate mass MS2 fragments, and (c) at least 3 ppm agreement between the theoretical and measured change in m / z between the parent-transformation product formula. Molecular network analysis revealed that both chain scission and cross-linking reactions occur dynamically rather than degradation proceeding in a monotonic progression to smaller or more oxygenated structures. Network nodes with the highest degree of centrality were tentatively identified using in silico fragmentation and can be prioritized for toxicity screening or other physicochemical properties of interest. This work has important implications for chemical transformation tracking in complex mixtures and may someday enable improved elucidation of environmental transformation rules (i.e., structure-reactivity relationships) and fate modeling.

Understanding the occurrence and transformation of microplastics when released into the environment is essential for risk assessment. The use of biodegradable polymers in agriculture can help to reduce microplastic accumulation in soil, since released fragments of such materials are not persistent and are further transformed into CO 2 and biomass (Wohlleben et al., 2023). To be able to monitor the fragmentation and biodegradation of these materials in soil, a validated extraction protocol is needed, which does not induce changes in the chemical and particle properties, additionally it should show high recoveries and matrix removal efficiency. A density-based extraction method in the centrifuge has the potential to remove a high amount of the soil matrix and is very selective for the polymer at the same time. Here we developed an efficient and non-destructive extraction protocol for biodegradable fragments from different soils using sequential centrifugation steps with varying densities and a freezing approach for sample collection. Although the focus of the present study was on biodegradable fragments, the technique can also be used for other types of microplastics with similar or lower density than the one tested for the method validation, but additional recovery tests for the target analyte are recommended.•A density-based extraction method for microplastics from soil, validated by recovery and stability tests using biodegradable polymers•Vessel changes and harsh chemical treatments are kept to a minimum., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All authors are employees of BASF, a company producing and marketing polymers, including biodegradable plastics., (© 2024 The Author(s).)

Extracting microplastics from complex matrices poses challenges due to the potential impact of harsh chemical treatments on microplastic properties. For fate and hazard assessment reliable techniques are needed to not only quantify the particle number but also to assess the physicochemical properties of environmental microplastics with minimum changes induced by extraction. Here we present the method development for an innovative and non-destructive extraction protocol based on a switchable calcium chloride density separation column. In contrast to commonly reported extraction protocols, the presented technique is suitable for targeted microplastic property analysis (e.g., surface chemistry and texture) by keeping chemical treatments (such as oxidation and enzymatic digestion) to a minimum. By adjusting the temperature we can control the aggregate state of the highly concentrated salt solution, allowing to separate the microplastics from matrix by cutting of purified, solidified samples. Harsh chemical treatments are avoided, as well as obstruction of microplastic extraction by adsorption to matrix components when passing the tap at the bottom of traditional density separation funnels. The use of microplastics that were prelabeled with a fluorescence dye helped to solve difficulties observed during method development by visual inspection before measurement of extraction efficiency: We spiked a blank compost with low-density polyethylene (LDPE) and polyamide (PA). Additionally, UV aged LDPE was used to demonstrate applicability to more hydrophilic, more environmentally relevant microplastics. The obtained initial results show high recovery of both unaged and aged LDPE over 97 wt.-% and an efficient compost removal but a lower and less robust recovery (between 68 and 18 wt.-%) for PA particles that are more challenging to extract due to an unfortunate synergistic combination of smaller particle size and higher density. Method adaptation to other microplastic types may still be necessary. In short:•A low-cost and simple approach without oxidation to extract (pre-aged) microplastics from compost•Method development by visual observation using fluorescent labelled microplastics and method validation by spike-recovery tests., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Some of the authors are employees of BASF, a company producing and marketing polymers, including plastics., (© 2024 The Authors. Published by Elsevier B.V.)

The dissolution of a nanomaterial (NM) in an in vitro simulant of the oro-gastrointestinal (OGI) tract is an important predictor of its biodurability in vivo . The cascade addition of simulated digestive juices (saliva, stomach and intestine), including inorganic/organic biomacromolecules and digestive enzymes (complete composition, referred to as "Type 1 formulation"), strives for realistic representation of chemical composition of the OGI tract. However, the data robustness requires consideration of analytical feasibility, such as the use of simplified media. Here we present a systematic analysis of the effects exerted by different digestive juice formulations on the dissolution% (or half-life values) of benchmark NMs ( e.g. , zinc oxide, titanium dioxide, barium sulfate, and silicon dioxide). The digestive juices were progressively simplified by removal of components such as organic molecules, enzymes, and inorganic molecules (Type 2, 3 and 4). The results indicate that the "Type 1 formulation" augments the dissolution via sequestration of ions by measurable factors compared to formulations without enzymes ( i.e. , Type 3 and 4). Type 1 formulation is thus regarded as a preferable option for predicting NM biodurability for hazard assessment. However, for grouping purposes, the relative similarity among diverse nanoforms (NFs) of a NM is decisive. Two similarity algorithms were applied, and additional case studies comprising NFs and non NFs of the same substance were included. The results support the grouping decision by simplified formulation (Type 3) as a robust method for screening and grouping purposes., Competing Interests: At the time of the study, JGK and WW were employees of BASF SE, a company producing nanomaterials. The other authors declare that they have no competing interests., (This journal is © The Royal Society of Chemistry.)

Polymers are a very large class of chemicals comprising often complex molecules with multiple functions used in everyday products. The EU Commission is seeking to develop environmental and human health standard information requirements (SIRs) for man-made polymers requiring registration (PRR) under a revised Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Regulation. Conventional risk assessment approaches currently used for small molecules may not apply to most polymers. Therefore, we propose a conceptual three-tiered regulatory approach for data generation to assess individual and groups of polymers requiring registration (PRR). A key element is the grouping of polymers according to chemistry, physico-chemical properties and hazard similarity. The limited bioavailability of many polymers is a prominent difference to many small molecules and is a key consideration of the proposed approach. Methods assessing potential for systemic bioavailability are integral to Tier 1. Decisions for further studies are based on considerations of properties and effects, combined with systemic bioavailability and use and exposure considerations. For many PRRs, Tier 1 data on hazard, use and exposure will likely be sufficient for achieving the protection goals of REACH. Vertebrate animal studies in Tiers 2 and 3 can be limited to targeted testing. The outlined approach aims to make use of current best scientific evidence and to reduce animal testing whilst providing data for an adequate level of protection., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This manuscript relates to work undertaken by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC; www.ecetoc.org). ECETOC is a scientific organisation which provides a collaborative space for scientists from industry, academia and governments. ECETOC is financed by its membership, which are the leading companies with interests in the manufacture and use of chemicals, biomaterials and pharmaceuticals (http://www.ecetoc.org/ecetoc-membership/member-companies/). Within the ECETOC Task Forces members do not receive compensation by ECETOC. The authors are employed by companies producing or otherwise working with polymers. The views expressed in this article are solely those of the co-authors and may not represent those of the sponsoring organizations., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

This is a reply to the Comment of Okhrimenko et al. in the same issue of RSC Advances . We discuss the arguments brought forward by said authors, oppose their objections and show the unchanged validity of our results., Competing Interests: All authors are employees of BASF SE, a competitor in the market of insulation materials., (This journal is © The Royal Society of Chemistry.)

Glycopeptide antibiotics (GPA) consist of a glycosylated heptapeptide backbone enriched in aromatic residues originating from the shikimate pathway. Since the enzymatic reactions within the shikimate pathway are highly feedback-regulated, this raises the question as to how GPA producers control the delivery of precursors for GPA assembly. We chose Amycolatopsis balhimycina, the producer of balhimycin, as a model strain for analyzing the key enzymes of the shikimate pathway. A. balhimycina contains two copies each of the key enzymes of the shikimate pathway, deoxy-d-arabino-heptulosonate-7-phosphate synthase (Dahp) and prephenate dehydrogenase (Pdh), with one pair (Dahp sec and Pdh sec ) encoded within the balhimycin biosynthetic gene cluster and one pair (Dahp prim and Pdh prim ) in the core genome. While overexpression of the dahp sec gene resulted in a significant (>4-fold) increase in balhimycin yield, no positive effects were observed after overexpression of the pdh prim or pdh sec genes. Investigation of allosteric enzyme inhibition revealed that cross-regulation between the tyrosine and phenylalanine pathways plays an important role. Tyrosine, a key precursor of GPAs, was found to be a putative activator of prephenate dehydratase (Pdt), which catalyzes the first step reaction from prephenate to phenylalanine in the shikimate pathway. Surprisingly, overexpression of pdt in A. balhimycina led to an increase in antibiotic production in this modified strain. In order to demonstrate that this metabolic engineering approach is generally applicable to GPA producers, we subsequently applied this strategy to Amycolatopsis japonicum and improved the production of ristomycin A, which is used in diagnosis of genetic disorders. Comparison of "cluster-specific" enzymes with the isoenzymes from the primary metabolism's pathway provided insights into the adaptive mechanisms used by producers to ensure adequate precursor supply and GPA yields. These insights further demonstrate the importance of a holistic approach in bioengineering efforts that takes into account not only peptide assembly but also adequate precursor supply., (Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

In analogy to the periodic system that groups elements by their similarity in structure and chemical properties, the hazard of chemicals can be assessed in groups having similar structures and similar toxicological properties. Here we review case studies of chemical grouping strategies that supported the assessment of hazard, exposure, and risk to human health. By the EU-REACH and the US-TSCA New Chemicals Program, structural similarity is commonly used as the basis for grouping, but that criterion is not always adequate and sufficient. Based on the lessons learned, we derive ten principles for grouping, including: transparency of the purpose, criteria, and boundaries of the group; adequacy of methods used to justify the group; and inclusion or exclusion of substances in the group by toxicological properties. These principles apply to initial grouping to prioritize further actions as well as to definitive grouping to generate data for risk assessment. Both can expedite effective risk management., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

FK-506 is a potent immunosuppressive macrocyclic polyketide with growing pharmaceutical interest, produced by Streptomyces tsukubaensis. However, due to low levels synthesized by the wild-type strain, biotechnological production of FK-506 is rather limited. Optimization strategies to enhance the productivity of S. tsukubaensis by means of genetic engineering have been established. In this work primarily global regulatory aspects with respect to the FK-506 biosynthesis have been investigated with the focus on the global Crp (cAMP receptor protein) regulator. In expression analyses and protein-DNA interaction studies, the role of Crp during FK-506 biosynthesis was elucidated. Overexpression of Crp resulted in two-fold enhancement of FK-506 production in S. tsukubaensis under laboratory conditions. Further optimizations using fermentors proved that the strategy described in this study can be transferred to industrial scale, presenting a new approach for biotechnological FK-506 production. KEY POINTS: • The role of the global Crp (cAMP receptor protein) regulator for FK-506 biosynthesis in S. tsukubaensis was demonstrated • Crp overexpression in S. tsukubaensis was applied as an optimization strategy to enhance FK-506 and FK-520 production resulting in two-fold yield increase., (© 2023. The Author(s).)

Sunlight exposure can naturally mitigate microplastics pollution in the surface ocean, however it results in emissions of dissolved organic carbon (DOC) whose characteristics and fate remain largely unknown. In this work, we investigated the effects of solar radiation on polyether (TPU_Ether) and polyester (TPU_Ester) thermoplastic polyurethane, and on a thermoset polyurethane (PU_Hardened). The microplastics were irradiated with simulated solar light with a UV dose of 350 MJ m -2 , which corresponds to roughly 15 months outdoor exposure at 31° N latitude. The particles were characterized using ATR-FTIR and elemental analysis. The DOC released to the aqueous phase was quantified by total organic carbon analysis and characterized by nontarget liquid chromatography coupled to high-resolution mass spectrometry. Polyurethane microplastics were degraded following mechanisms reconcilable with UV photo-oxidation. The carbon mass fraction released to the aqueous phase was 8.5 ± 0.5%, 3.7 ± 0.2%, and 2.8 ± 0.2% for TPU_Ether, TPU_Ester, and PU_Hardened, respectively. The corresponding DOC release rates, expressed as mg carbon per UV dose were 0.023, 0.013, and 0.010 mg MJ -1 for TPU_Ether, TPU_Ester and PU_Hardened, respectively. Roughly three thousand unique by-products were released from photo-weathered TPUs, whereas 540 were detected in the DOC of PU_Hardened. This carbon pool was highly complex and dynamic in terms of physicochemical properties and susceptibility to further photodegradation after dissolution from the particles. Our results show that plastics photodegradation in the ocean requires chemical assessment of the DOC emissions in addition to the analysis of aged microplastics and that polymer chemistry influences the chain scission products.

The new recommended definition of a nanomaterial, 2022/C 229/01, adopted by the European Commission in 2022, will have a considerable impact on European Union legislation addressing chemicals, and therefore tools to implement this new definition are urgently needed. The updated NanoDefiner framework and its e-tool implementation presented here are such instruments, which help stakeholders to find out in a straightforward way whether a material is a nanomaterial or not. They are two major outcomes of the NanoDefine project, which is explicitly referred to in the new definition. This work revisits the framework and e-tool, and elaborates necessary adjustments to make these outcomes applicable for the updated recommendation. A broad set of case studies on representative materials confirms the validity of these adjustments. To further foster the sustainability and applicability of the framework and e-tool, measures for the FAIRification of expert knowledge within the e-tool's knowledge base are elaborated as well. The updated framework and e-tool are now ready to be used in line with the updated recommendation. The presented approach may serve as an example for reviewing existing guidance and tools developed for the previous definition 2011/696/EU, particularly those adopting NanoDefine project outcomes.

In this work, we present the first inhibitor of GlnA2Sc, a gamma-glutamylpolyamine synthetase, which allows Streptomyces coelicolor to detoxify high concentrations of polyamines and to utilize them as a carbon or nitrogen source. GlnA2 belongs to the class of glutamine synthetase-like (GS-like) enzymes that catalyze the glutamylation of different nitrogen-containing compounds. Whereas a number of inhibitors for GS are known, none of them are known to inhibit GlnA2. In this work, PPU268, an inhibitor for GlnA2 is presented that is structurally derived from the prototypic GS inhibitor-methionine sulfoximine (MSO). It combines two features: the binding mechanism of MSO and the amine substrate specificity of GlnA2Sc. This inhibitor is a novel compound to block the polyamine utilization in bacteria resulting in the inability to detoxify polyamines. This may offer a possibility to develop novel therapeutic strategies to combat actinobacterial human pathogens that encounter polyamines in the course of the infection processes., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Whole-cell antibacterial assays are particularly suitable for fast detection and semi-quantification of bioactivities in extracts or other solutions such as microbial culture supernatants. As Actinomycetales, including the members of the genus Streptomyces, are one of the most potent "suppliers" of antibiotics and other bioactive compounds, there is a strong interest in the development of useful assays enabling early identification of such valuable producers. Furthermore, such assays facilitate the screening of a large collection of clones for the detection of engineered "super-producers" that are essential for industrial manufacturing of the respective product.In this protocol, we describe a whole-cell assay for a fast detection of antimicrobial agents in culture supernatants. As model, Streptomyces fradiae DSM 41546, the producer of the antibiotic tylosin, and the test strain Bacillus subtilis ATCC 6051 are used. Culture supernatants of S. fradiae DSM 41546 and controls are diluted, and their antibiotic activity is tested against B. subtilis ATCC 6051. For the dilutions of tylosin-containing culture supernatant, a clear concentration-dependent growth inhibition effect on B. subtilis ATCC 6051 is demonstrated. In contrast, dilutions of the culture supernatant lacking tylosin do not inhibit the growth of B. subtilis. Moreover, including defined concentrations of tylosin facilitates the semi-quantification of tylosin in the culture supernatants.Our results confirm the applicability of the assay in fast screenings for antimicrobial products in culture supernatants. The protocol can be used as positive control within screening campaigns in the drug discovery field., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Manufacturing and functionalizing materials at the nanoscale has led to the generation of a whole array of nanoforms (NFs) of substances varying in size, morphology, and surface characteristics. Due to financial, time, and ethical considerations, testing every unique NF for adverse effects is virtually impossible. Use of hypothesis-driven grouping and read-across approaches, as supported by the GRACIOUS Framework, represents a promising alternative to case-by-case testing that will make the risk assessment process more efficient. Through application of appropriate grouping hypotheses, the Framework facilitates the assessment of similarity between NFs, thereby supporting grouping and read-across of information, minimizing the need for new testing, and aligning with the 3R principles of replacement, reduction, and refinement of animals in toxicology studies. For each grouping hypothesis an integrated approach to testing and assessment (IATA) guides the user in data gathering and acquisition to test the hypothesis, following a structured format to facilitate efficient decision-making. Here we present the template used to generate the GRACIOUS grouping hypotheses encompassing information relevant to “Lifecycle, environmental release, and human exposure”, “What they are: physicochemical characteristics”, “Where they go: environmental fate, uptake, and toxicokinetics”, and “What they do: human and environmental toxicity”. A summary of the template-derived hypotheses focusing on human health is provided, along with an overview of the IATAs generated by the GRACIOUS project. We discuss the application and flexibility of the template, providing the opportunity to expand the application of grouping and read-across in a logical, evidence-based manner to a wider range of NFs and substances.

The release of fragments from plastic products, that is, secondary microplastics, is a major concern in the context of the global plastic pollution. Currently available (thermoplastic) polyurethanes [(T)PU] are not biodegradable and therefore should be recycled. However, the ester bond in (T)PUs might be sufficiently hydrolysable to enable at least partial biodegradation of polyurethane particles. Here, we investigated biodegradation in compost of different types of (T)PU to gain insights into their fragmentation and biodegradation mechanisms. The studied (T)PUs varied regarding the chemistry of their polymer backbone (aromatic/aliphatic), hard phase content, cross-linking degree, and presence of a hydrolysis-stabilizing additive. We developed and validated an efficient and non-destructive polymer particle extraction process for partially biodegraded (T)PUs based on ultrasonication and density separation. Our results showed that biodegradation rates and extents decreased with increasing cross-linking density and hard-segment content. We found that the presence of a hydrolysis stabilizer reduced (T)PU fragmentation while not affecting the conversion of (T)PU carbon into CO 2 . We propose a biodegradation mechanism for (T)PUs that includes both mother particle shrinkage by surface erosion and fragmentation. The presented results help to understand structure-degradation relationships of (T)PUs and support recycling strategies.

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, SiO 2 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., (© 2022. The Author(s).)

Tacrolimus (FK506) is a macrolide widely used as immunosuppressant to prevent transplant rejection. Synthetic production of FK506 is not efficient and costly, whereas the biosynthesis of FK506 is complex and the level produced by the wild type strain, Streptomyces tsukubaensis , is very low. We therefore engineered FK506 biosynthesis and the supply of the precursor L-lysine to generate strains with improved FK506 yield. To increase FK506 production, first the intracellular supply of the essential precursor lysine was improved in the native host S. tsukubaensis NRRL 18488 by engineering the lysine biosynthetic pathway. Therefore, a feedback deregulated aspartate kinase AskSt* of S. tsukubaensis was generated by site directed mutagenesis. Whereas overexpression of AskSt* resulted only in a 17% increase in FK506 yield, heterologous overexpression of a feedback deregulated AskCg* from Corynebacterium glutamicum was proven to be more efficient. Combined overexpression of AskCg* and DapASt, showed a strong enhancement of the intracellular lysine pool following increase in the yield by approximately 73% compared to the wild type. Lysine is coverted into the FK506 building block pipecolate by the lysine cyclodeaminase FkbL. Construction of a ∆fkbL mutant led to a complete abolishment of the FK506 production, confirming the indispensability of this enzyme for FK506 production. Chemical complementation of the ∆fkbL mutant by feeding pipecolic acid and genetic complementation with fkbL as well as with other lysine cyclodeaminase genes ( pipAf, pipASt , originating from Actinoplanes friuliensis and Streptomyces pristinaespiralis , respectively) completely restored FK506 production. Subsequently, FK506 production was enchanced by heterologous overexpression of PipAf and PipASp in S. tsukubaensis . This resulted in a yield increase by 65% compared to the WT in the presence of PipAf from A. friuliensis . For further rational yield improvement, the crystal structure of PipAf from A. friuliensis was determined at 1.3 Å resolution with the cofactor NADH bound and at 1.4 Å with its substrate lysine. Based on the structure the Ile91 residue was replaced by Val91 in PipAf, which resulted in an overall increase of FK506 production by approx. 100% compared to the WT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Schulz, Schall, Stehle, Breitmeyer, Krysenko, Mitulski and Wohlleben.)