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The xenobiotic metabolism in the lung, an organ of first entry of xenobiotics into the organism, is crucial for inhaled compounds entering this organ intentionally (e.g. drugs) and unintentionally (e.g. work place and environmental compounds). Additionally, local metabolism by enzymes preferentially or exclusively occurring in the lung is important for favorable or toxic effects of xenobiotics entering the organism also by routes other than by inhalation. The data collected in this review show that generally activities of cytochromes P450 are low in the lung of all investigated species and in vitro models. Other oxidoreductases may turn out to be more important, but are largely not investigated. Phase II enzymes are generally much higher with the exception of UGT glucuronosyltransferases which are generally very low. Insofar as data are available the xenobiotic metabolism in the lung of monkeys comes closed to that in the human lung; however, very few data are available for this comparison. Second best rate the mouse and rat lung, followed by the rabbit. Of the human in vitro model primary cells in culture, such as alveolar macrophages and alveolar type II cells as well as the A549 cell line appear quite acceptable. However, (1) this generalization represents a temporary oversimplification born from the lack of more comparable data; (2) the relative suitability of individual species/models is different for different enzymes; (3) when more data become available, the conclusions derived from these comparisons quite possibly may change.

Studies on the metabolic fate of medical drugs, skin care products, cosmetics and other chemicals intentionally or accidently applied to the human skin have become increasingly important in order to ascertain pharmacological effectiveness and to avoid toxicities. The use of freshly excised human skin for experimental investigations meets with ethical and practical limitations. Hence information on xenobiotic-metabolizing enzymes (XME) in the experimental systems available for pertinent studies compared with native human skin has become crucial. This review collects available information of which—taken with great caution because of the still very limited data—the most salient points are: in the skin of all animal species and skin-derived in vitro systems considered in this review cytochrome P450 (CYP)-dependent monooxygenase activities (largely responsible for initiating xenobiotica metabolism in the organ which provides most of the xenobiotica metabolism of the mammalian organism, the liver) are very low to undetectable. Quite likely other oxidative enzymes [e.g. flavin monooxygenase, COX (cooxidation by prostaglandin synthase)] will turn out to be much more important for the oxidative xenobiotic metabolism in the skin. Moreover, conjugating enzyme activities such as glutathione transferases and glucuronosyltransferases are much higher than the oxidative CYP activities. Since these conjugating enzymes are predominantly detoxifying, the skin appears to be predominantly protected against CYP-generated reactive metabolites. The following recommendations for the use of experimental animal species or human skin in vitro models may tentatively be derived from the information available to date: for dermal absorption and for skin irritation esterase activity is of special importance which in pig skin, some human cell lines and reconstructed skin models appears reasonably close to native human skin. With respect to genotoxicity and sensitization reactive-metabolite-reducing XME in primary human keratinocytes and several reconstructed human skin models appear reasonably close to human skin. For a more detailed delineation and discussion of the severe limitations see the Conclusions section in the end of this review.

While in vitro testing is used to identify hazards of chemicals, nominal in vitro assay concentrations may misrepresent potential in vivo effects and do not provide dose–response data which can be used for a risk assessment. We used reverse dosimetry to compare in vitro effect concentrations-to-in vivo doses causing toxic effects related to endocrine disruption. Ten compounds (acetaminophen, bisphenol A, caffeine, 17α-ethinylestradiol, fenarimol, flutamide, genistein, ketoconazole, methyltestosterone, and trenbolone) have been tested in the yeast estrogen screening (YES) or yeast androgen-screening (YAS) assays for estrogen and androgen receptor binding, as well as the H295R assay (OECD test guideline no. 456) for potential interaction with steroidogenesis. With the assumption of comparable concentration–response ratios of these effects in the applied in vitro systems and the in vivo environment, the lowest observed effect concentrations from these assays were extrapolated to oral doses (LOELs) by reverse dosimetry. For extrapolation, an eight-compartment Physiologically Based Toxicokinetic (PBTK) rat model based on in vitro and in silico input data was used. The predicted LOEL was then compared to the LOEL actually observed in corresponding in vivo studies (YES/YAS assay versus uterotrophic or Hershberger assay and steroidogenesis assay versus pubertal assay or generation studies). This evaluation resulted in 6 out of 10 compounds for which the predicted LOELs were in the same order of magnitude as the actual in vivo LOELs. For four compounds, the predicted LOELs differed by more than tenfold from the actual in vivo LOELs. In conclusion, these data demonstrate the applicability of reverse dosimetry using a simple PBTK model to serve in vitro–in silico-based risk assessment, but also identified cases and test substance were the applied methods are insufficient.

Background Microplastics have been repeatedly detected in the human body, yet uncertainties surround their bioavailability and fate due to experimental challenges and limitations, especially regarding their nano-sized counterparts. Knowing that toxicokinetics information is essential for accurate risk assessment and management, this research aimed to (1) evaluate different sample preparation and quantification methods for nanoplastics particles in mammalian tissue, and (2) investigate the lung retention, bioavailability and fate of these particles. Methods In this study, rats inhaled aerosols with up to 50 mg/m 3 of Nile Red-labeled polystyrene (PS-NR) or unlabeled polyamide particles (PA-6) particles for 28 days. The tissues were analyzed for the presence of polymer particles. PS-NR were quantified in formalin-fixed tissue by confocal fluorescence laser microscopy with semi-automatic imaging analysis, and PA-6 particles were quantified in dried tissues by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS). Results PA-6 deposition was detected and quantified in lung and lymph nodes. Deposition of PS-NR was quantified in lungs and lung-draining lymph nodes, but no particles were detected in the liver, spleen, and kidneys. The lung burdens and translocation to the draining lymph nodes were similar for both particles, and particles were still detectable after the end of the exposure periods (five weeks for PS-NR and 13 weeks for PA-6). Conclusions This work highlights limitations and applicability of the various methods for sample preparation, detecting and quantifying polymer particles in mammalian tissues. In addition, it provides reliable data on the internal dose of inhaled polymer particles. Graphical Abstract

This review is based on the lecture presented at the April 2010 nanomaterials safety assessment Postsatellite to the 2009 EUROTOX Meeting and summarizes genotoxicity investigations on nanomaterials published in the open scientific literature (up to 2008). Special attention is paid to the relationship between particle size and positive versus negative outcome, as well as the dependence of the outcome on the test used. Salient conclusions and outstanding recommendations emerging from the information summarized in this review are as follows: recognize that nanomaterials are not all the same; therefore know and document what nanomaterial has been tested and in what form; take nanomaterials specific properties into account; in order to make your results comparable with those of others and on other nanomaterials: use or at least include in your studies standardized methods; use in vivo studies to put in vitro results into perspective; take uptake and distribution of the nanomaterial into account; and in order to become able to make extrapolations to risk for human: learn about the mechanism of nanomaterials genotoxic effects. Past experience with standard non-nanosubstances already had shown that mechanisms of genotoxic effects can be complex and their elucidation can be demanding, while there often is an immediate need to assess the genotoxic hazard. Thus, a practical and pragmatic approach to genotoxicity investigations of novel nanomaterials is the use of a battery of standard genotoxicity testing methods covering a wide range of mechanisms. Application of these standard methods to nanomaterials demands, however, adaptations, and the interpretation of results from the genotoxicity testing of nanomaterials needs additional considerations exceeding those used for standard size materials.

Background Carbon nanotubes, graphene, graphite nanoplatelets and carbon black are seemingly chemically identical carbon-based nano-materials with broad technological applications. Carbon nanotubes and carbon black possess different inhalation toxicities, whereas little is known about graphene and graphite nanoplatelets. Methods In order to compare the inhalation toxicity of the mentioned carbon-based nanomaterials, male Wistar rats were exposed head-nose to atmospheres of the respective materials for 6 hours per day on 5 consecutive days. Target concentrations were 0.1, 0.5, or 2.5 mg/m 3 for multi-wall carbon nanotubes and 0.5, 2.5, or 10 mg/m 3 for graphene, graphite nanoplatelets and low-surface carbon black. Toxicity was determined after end of exposure and after three-week recovery using broncho-alveolar lavage fluid and microscopic examinations of the entire respiratory tract. Results No adverse effects were observed after inhalation exposure to 10 mg/m 3 graphite nanoplatelets or relatively low specific surface area carbon black. Increases of lavage markers indicative for inflammatory processes started at exposure concentration of 0.5 mg/m 3 for multi-wall carbon nanotubes and 10 mg/m 3 for graphene. Consistent with the changes in lavage fluid, microgranulomas were observed at 2.5 mg/m 3 multi-wall carbon nanotubes and 10 mg/m 3 graphene. In order to evaluate volumetric loading of the lung as the key parameter driving the toxicity, deposited particle volume was calculated, taking into account different methods to determine the agglomerate density. However, the calculated volumetric load did not correlate to the toxicity, nor did the particle surface burden of the lung. Conclusions The inhalation toxicity of the investigated carbon-based materials is likely to be a complex interaction of several parameters. Until the properties which govern the toxicity are identified, testing by short-term inhalation is the best option to identify hazardous properties in order to avoid unsafe applications or select safer alternatives for a given application.

Background A standard short-term inhalation study (STIS) was applied for hazard assessment of 13 metal oxide nanomaterials and micron-scale zinc oxide. Methods Rats were exposed to test material aerosols (ranging from 0.5 to 50 mg/m 3 ) for five consecutive days with 14- or 21-day post-exposure observation. Bronchoalveolar lavage fluid (BALF) and histopathological sections of the entire respiratory tract were examined. Pulmonary deposition and clearance and test material translocation into extra-pulmonary organs were assessed. Results Inhaled nanomaterials were found in the lung, in alveolar macrophages, and in the draining lymph nodes. Polyacrylate-coated silica was also found in the spleen, and both zinc oxides elicited olfactory epithelium necrosis. None of the other nanomaterials was recorded in extra-pulmonary organs. Eight nanomaterials did not elicit pulmonary effects, and their no observed adverse effect concentrations (NOAECs) were at least 10 mg/m 3 . Five materials (coated nano-TiO 2 , both ZnO, both CeO 2 ) evoked concentration-dependent transient pulmonary inflammation. Most effects were at least partially reversible during the post-exposure period. Based on the NOAECs that were derived from quantitative parameters, with BALF polymorphonuclear (PMN) neutrophil counts and total protein concentration being most sensitive, or from the severity of histopathological findings, the materials were ranked by increasing toxic potency into 3 grades: lower toxic potency: BaSO 4 ; SiO 2 .acrylate (by local NOAEC); SiO 2 .PEG; SiO 2 .phosphate; SiO 2 .amino; nano-ZrO 2 ; ZrO 2 .TODA; ZrO 2 .acrylate; medium toxic potency: SiO 2 .naked; higher toxic potency: coated nano-TiO 2 ; nano-CeO 2 ; Al-doped nano-CeO 2 ; micron-scale ZnO; coated nano-ZnO (and SiO 2 .acrylate by systemic no observed effect concentration (NOEC)). Conclusion The STIS revealed the type of effects of 13 nanomaterials, and micron-scale ZnO, information on their toxic potency, and the location and reversibility of effects. Assessment of lung burden and material translocation provided preliminary biokinetic information. Based upon the study results, the STIS protocol was re-assessed and preliminary suggestions regarding the grouping of nanomaterials for safety assessment were spelled out.

The tissue distribution and toxicity of intravenously administered nanoparticles of titanium dioxide (TiO 2 ) (>10 wt.% at <100 nm size) were investigated because of the fundamental importance to obtain information on the kinetics of this widely used nanoparticle in a situation of 100% bioavailability. Male Wistar rats were treated with single intravenous injections of a suspension of TiO 2 in serum (5 mg/kg body weight), and the tissue content of TiO 2 was determined 1, 14, and 28 days later. Biochemical parameters and antigens in serum were also assessed to determine potential pathological changes. The health and behavior of the animals were normal throughout the study. There were no detectable levels of TiO 2 in blood cells, plasma, brain, or lymph nodes. The TiO 2 levels were highest in the liver, followed in decreasing order by the levels in the spleen, lung, and kidney, and highest on day 1 in all organs. TiO 2 levels were retained in the liver for 28 days, there was a slight decrease in TiO 2 levels from day 1 to days 14 and 28 in the spleen, and a return to control levels by day 14 in the lung and kidney. There were no changes in the cytokines and enzymes measured in blood samples, indicating that there was no detectable inflammatory response or organ toxicity. Overall, rats exposed to TiO 2 nanoparticles by a route that allows immediate systemic availability showed expected tissue distribution, no obvious toxic health effects, no immune response, and no change in organ function. Therefore, even with 100% bioavailability of the 5 mg/kg TiO 2 dose afforded by the intravenous route of administration, there were no remarkable toxic effects evident in the experimental animals. These results indicate that TiO 2 nanoparticles could be used safely in low doses.

Background The oral uptake of nanoparticles is an important route of human exposure and requires solid models for hazard assessment. While the systemic availability is generally low, ingestion may not only affect gastrointestinal tissues but also intestinal microbes. The gut microbiota contributes essentially to human health, whereas gut microbial dysbiosis is known to promote several intestinal and extra-intestinal diseases. Gut microbiota-derived metabolites, which are found in the blood stream, serve as key molecular mediators of host metabolism and immunity. Results Gut microbiota and the plasma metabolome were analyzed in male Wistar rats receiving either SiO 2 (1000 mg/kg body weight/day) or Ag nanoparticles (100 mg/kg body weight/day) during a 28-day oral gavage study. Comprehensive clinical, histopathological and hematological examinations showed no signs of nanoparticle-induced toxicity. In contrast, the gut microbiota was affected by both nanoparticles, with significant alterations at all analyzed taxonomical levels. Treatments with each of the nanoparticles led to an increased abundance of Prevotellaceae, a family with gut species known to be correlated with intestinal inflammation. Only in Ag nanoparticle-exposed animals, Akkermansia , a genus known for its protective impact on the intestinal barrier was depleted to hardly detectable levels. In SiO 2 nanoparticles-treated animals, several genera were significantly reduced, including probiotics such as Enterococcus . From the analysis of 231 plasma metabolites, we found 18 metabolites to be significantly altered in Ag-or SiO 2 nanoparticles-treated rats. For most of these metabolites, an association with gut microbiota has been reported previously. Strikingly, both nanoparticle-treatments led to a significant reduction of gut microbiota-derived indole-3-acetic acid in plasma. This ligand of the arylhydrocarbon receptor is critical for regulating immunity, stem cell maintenance, cellular differentiation and xenobiotic-metabolizing enzymes. Conclusions The combined profiling of intestinal microbiome and plasma metabolome may serve as an early and sensitive indicator of gut microbiome changes induced by orally administered nanoparticles; this will help to recognize potential adverse effects of these changes to the host.

Poorly soluble low toxicity particles (PSLT) have long been a concept in particle toxicology and regulatory frameworks addressing inhalation hazards. The term PSLT refers to particles that exhibit low toxicity and minimal solubility in biological fluids, leading to prolonged retention in the lungs and potential overload effects. Historically, PSLT has been used to distinguish materials whose adverse effects are primarily driven by particle burden rather than intrinsic chemical toxicity. While the “low toxicity” (LT) component of the definition has been examined to a certain degree (Driscoll and Borm in Inhal Toxicol 32(2):53-62, 2020), the poorly soluble (PS)-criterion does not yet have a precise definition although it is critically influencing the interpretation of toxicological inhalation repeated dose studies and hazard classifications. An ECETOC Task Force (TF) was formed to define criteria for “poorly soluble” particles (PSPs). This paper presents a quantitative non-animal approach for defining PSP using a model particle, with a focus on its potential to cause volumetric lung overload and affect macrophage clearance mechanisms. The analysis allows to calculate a dissolution rate that would lead to a lung burden of about 1 µL/g of lung tissue (lung overload threshold according to Morrow (Morrow in Fundam Appl Toxicol 10(3):369, 1988)). Below this threshold dissolution rate, this specific model particle would qualify as PSP. In addition, a formula was presented to translate abiotic dissolution rates into biotic (rat) dissolution rates to allow a PS-assessment in an animal-free system. As proof of concept, the TF collected existing in vivo data from member companies, publicly available literature of presumed PS-substances, and reference materials. The collected data revealed that most substances exhibited dissolution rates below the critical threshold and that the lung burden at no observed adverse effect concentrations (NOAECs) remained below the lung overload limit. Importantly, this threshold dissolution rate can differ from particle to particle, depending on factors such as agglomerate density, particle size distribution, and expected concentration. Thus, it should be evaluated on a case-by-case basis. Graphical Abstract Highlights Dissolution rate threshold: For a model particle, a dissolution rate threshold was calculated for PSP. This dissolution threshold for PSP varies on the basis of several factors and should be assessed on a case-by-case basis. Animal-free PS-assessment: a formula was designed to translate abiotic dissolution rates into biotic (rat) dissolution rates to allow a PS-assessment in an animal free system. Lung burden assessment: Comparing the determined and modeled lung burdens of the collected dataset with the respective NOAEC/LOAEC mostly revealed relevant effects above a lung burden threshold of 1 µL/g lung. Common Toxicological Pattern: 28- and 90-day inhalation toxicity data of particles with different chemistries revealed pulmonary foreign body inflammatory reactions as a prevalent toxicological response.