Considerations for the Identification and Conveyance of Clinical Pathology Findings in Preclinical Toxicity Studies: Results From the 9th ESTP International Expert Workshop.

The European Society of Toxicologic Pathology (ESTP) organized a panel of 24 international experts from many fields of toxicologic clinical pathology (e.g., industry, academia, and regulatory) that came together in 2021 to align the use of terminology to convey the importance of clinical pathology findings in preclinical toxicity studies. An additional goal consisted of how to identify important findings in standard and nonstandard clinical pathology associated endpoints. This manuscript summarizes the information and opinions discussed and shared at the ninth ESTP International Expert Workshop, April 5 to 6, 2022. In addition to terminology usage, the workshop considered topics related to the identification and conveyance of the importance of test item-related findings. These topics included sources of variability, comparators, statistics, reporting, correlations to other study data, nonstandard biomarkers, indirect/secondary findings, and an overall weight-of-evidence approach.

Toxicological inhalation studies in rats to substantiate grouping of zinc oxide nanoforms.

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.

Eutectic Media Open a Synthetic Route to Oligocitrazinic Acid Fluorophores of Purple Hue.

Under isochoric and solvent-free conditions, the reaction between ammonium formate and citric acid results in a deeply purple reaction product with fluorescent properties. This brings this reaction in the realm of bio-based fluorophores and bottom-up carbon nanodots from citric acid. The reaction conditions are optimized in terms of UV-vis spectroscopic properties and, subsequently, the main reaction product is separated. While the structural analysis does not give any indication for carbon nanodots in a general sense, it points towards the formation of molecular fluorophores that consist of oligomerized citrazinic acid derivatives. Furthermore, EPR spectroscopy reveals the presence of stable free radicals in the product. We hypothesize that such open-shell structures may play a general role in molecular fluorophores from citric acid and are not yet sufficiently explored. Therefore, we believe that analysis of these newly discovered fluorophores may contribute to a better understanding of the properties of fluorophores and CND from citric acid in general.

On-Chip Direct Laser Writing of PAN-Based Carbon Supercapacitor Electrodes.

The carbonization of polyacrylonitrile (PAN) by direct laser writing to produce microsupercapacitors directly on-chip is reported. The process is demonstrated by producing interdigitated carbon finger electrodes directly on a printed circuit board (PCB), which is then employed to characterize the supercapacitor electrodes. By varying the laser power, the process can be tuned from carbonization to material ablation. This allows to not only convert pristine PAN films into carbon electrodes, but also to pattern and cut away non-carbonized material to produce completely freestanding carbon electrodes. While the carbon electrodes adhere well to the printed circuit board, non-carbonized PAN is peeled off the substrate. Specific capacities as high as 260 µF cm⁻2 are achieved in a supercapacitor with 16 fingers.

Gut microbiome and plasma metabolome changes in rats after oral gavage of nanoparticles: sensitive indicators of possible adverse health effects.

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 SiO2 (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 SiO2 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 SiO2 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.

Carbon Nanodots for All-in-One Photocatalytic Hydrogen Generation.

Carbon nanodots (CNDs) were photochemically altered to produce dihydrogen under light irradiation. Within the complex structure of CNDs, photo-oxidation takes place at citrazinic acid molecular fluorophore sites. Important is the fact that the resulting CND materials have a dual function. On one hand, they absorb light, and on the other hand, they photo- and electrocatalytically produce dihydrogen from water and seawater, without any external photosensitizer or cocatalyst. Record HER activities of 15.15 and 19.70 mmol(H2) g(catalyst)-1 h-1 were obtained after 1 h of 75 mW/cm2 Xe lamp illumination, from water and seawater, respectively. This impressive performance outweighs the remaining structural uncertainties. A full-fledged physicochemical investigation based on an arsenal of steady-state and time-resolved spectroscopic characterizations together with microscopy enabled a comprehensive look into the reaction mechanism. For an efficient dihydrogen formation, a precatalytic activation by means of reduction with a sacrificial electron donor is imperative.

Graphene's Role in Emerging Trends of Capacitive Energy Storage.

Technological breakthroughs in energy storage are being driven by the development of next-generation supercapacitors with favorable features besides high-power density and cycling stability. In this innovation, graphene and its derived materials play an active role. Here, the research status of graphene supercapacitors is analyzed. Recent progress is outlined in graphene assembly, exfoliation, and processing techniques. In addition, electrochemical and electrical attributes that are increasingly valued in next-generation supercapacitors are highlighted along with a summary of the latest research addressing chemical modification of graphene and its derivatives for future supercapacitors. The challenges and solutions discussed in the review hopefully will shed light on the commercialization of graphene and a broader genre of 2D materials in energy storage applications.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - part II: how can key events of relevant adverse outcome pathways be addressed in toxicological assessments?

The current understanding of thyroid-related adverse outcome pathways (AOPs) with adverse neurodevelopmental outcomes in mammals has been reviewed. This served to establish if standard rodent toxicity test methods and in vitro assays allow identifying thyroid-related modes-of-action potentially leading to adverse neurodevelopmental outcomes, and the human relevance of effects - in line with the European Commission's Endocrine Disruptor Criteria. The underlying hypothesis is that an understanding of the key events of relevant AOPs provides insight into differences in incidence, magnitude, or species sensitivity of adverse outcomes. The rodent studies include measurements of serum thyroid hormones, thyroid gland pathology and neurodevelopmental assessments, but do not directly inform on specific modes-of-action. Opportunities to address additional non-routine parameters reflecting critical events of AOPs in toxicological assessments are presented. These parameters appear relevant to support the identification of specific thyroid-related modes-of-action, provided that prevailing technical limitations are overcome. Current understanding of quantitative key event relationships is often weak, but would be needed to determine if the triggering of a molecular initiating event will ultimately result in an adverse outcome. Also, significant species differences in all processes related to thyroid hormone signalling are evident, but the biological implications thereof (including human relevance) are often unknown. In conclusion, careful consideration of the measurement (e.g. timing, method) and interpretation of additional non-routine parameters is warranted. These findings will be used in a subsequent paper to propose a testing strategy to identify if a substance may elicit maternal thyroid hormone imbalance and potentially also neurodevelopmental effects in the progeny.

Progress towards an OECD reporting framework for transcriptomics and metabolomics in regulatory toxicology.

Omics methodologies are widely used in toxicological research to understand modes and mechanisms of toxicity. Increasingly, these methodologies are being applied to questions of regulatory interest such as molecular point-of-departure derivation and chemical grouping/read-across. Despite its value, widespread regulatory acceptance of omics data has not yet occurred. Barriers to the routine application of omics data in regulatory decision making have been: 1) lack of transparency for data processing methods used to convert raw data into an interpretable list of observations; and 2) lack of standardization in reporting to ensure that omics data, associated metadata and the methodologies used to generate results are available for review by stakeholders, including regulators. Thus, in 2017, the Organisation for Economic Co-operation and Development (OECD) Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) launched a project to develop guidance for the reporting of omics data aimed at fostering further regulatory use. Here, we report on the ongoing development of the first formal reporting framework describing the processing and analysis of both transcriptomic and metabolomic data for regulatory toxicology. We introduce the modular structure, content, harmonization and strategy for trialling this reporting framework prior to its publication by the OECD.

Carbon Nanodots for Charge-Transfer Processes.

In recent years, carbon nanodots (CNDs) have emerged as an environmentally friendly, biocompatible, and inexpensive class of material, whose features sparked interest for a wide range of applications. Most notable is their photoactivity, as exemplified by their strong luminescence. Consequently, CNDs are currently being investigated as active components in photocatalysis, sensing, and optoelectronics. Charge-transfer interactions are common to all these areas. It is therefore essential to be able to fine-tune both the electronic structure of CNDs and the electronic communication in CND-based functional materials. The complex, but not completely deciphered, structure of CNDs necessitates, however, a multifaceted strategy to investigate their fundamental electronic structure and to establish structure-property relationships. Such investigations require a combination of spectroscopic methods, such as ultrafast transient absorption and fluorescence up-conversion techniques, electrochemistry, and modeling of CNDs, both in the absence and presence of other photoactive materials. Only a sound understanding of the dynamics of charge transfer, charge shift, charge transport, etc., with and without light makes much-needed improvements in, for example, photocatalytic processes, in which CNDs are used as either photosensitizers or catalytic centers, possible. This Account addresses the structural, photophysical, and electrochemical properties of CNDs, in general, and the charge-transfer chemistry of CNDs, in particular. Pressure-synthesized CNDs (pCNDs), for which citric acid and urea are used as inexpensive and biobased precursor materials, lie at the center of attention. A simple microwave-assisted thermolytic reaction, performed in sealed vessels, yields pCNDs with a fairly homogeneous size distribution of ∼1-2 nm. The narrow and excitation-independent photoluminescence of pCNDs contrasts with that seen in CNDs synthesized by other techniques, making pCNDs optimal for in-depth physicochemical analyses. The atomistic and electronic structures of CNDs were also analyzed by quantum chemical modeling approaches that led to a range of possible structures, ranging from heavily functionalized, graphene-like structures to disordered amorphous particles containing small sp2 domains. Both the electron-accepting and -donating performances of CNDs make the charge-transfer chemistry of CNDs rather versatile. Both covalent and noncovalent synthetic approaches have been explored, resulting in architectures of various sizes. CNDs, for example, have been combined with molecular materials ranging from electron-donating porphyrins and extended tetrathiafulvalenes to electron-accepting perylendiimides, or nanocarbon materials such as polymer-wrapped single-walled carbon nanotubes. In every case, charge-separated states formed as part of the reaction cascades initiated by photoexcitation. Charge-transfer assemblies including CNDs have also played a role in technological applications: for example, a proof-of-concept dye-sensitized solar cell was designed and tested, in which CNDs were adsorbed on the surface of mesoporous anatase TiO2. The wide range of reported electron-donor-acceptor systems documents the versatility of CNDs as molecular building blocks, whose electronic properties are tunable for the needs of emerging technologies.

Toward a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - part I: which parameters from human studies are most relevant for toxicological assessments?

The 2018 European Food Safety Authority/European Chemicals Agency Guidance on the Identification of Endocrine Disruptors lacks clarity on how the presence or absence of substance-induced maternal thyroid hormone imbalance, or the potential for subsequent deleterious consequences in child neurodevelopment, should be established by toxicological assessments. To address these uncertainties, this narrative review evaluates human evidence on how altered maternal thyroid function may be associated with child neurodevelopmental outcomes; and seeks to identify parameters in human studies that appear most relevant for toxicological assessments. Serum levels of free thyroxine (fT4) and thyroid stimulating hormone (TSH) are most frequently measured when assessing thyroid function in pregnant women, whereas a broad spectrum of neurodevelopmental parameters is used to evaluate child neurodevelopment. The human data confirms an association between altered maternal serum fT4 and/or TSH and increased risk for child neurodevelopmental impairment. Quantitative boundaries of effects indicative of increased risks need to be established. Moreover, it is unknown if altered serum levels of total T4, free or total triiodothyronine, or parameters unrelated to serum thyroid hormones might be more relevant indicators of such effects. None of the human studies established a link between substance-mediated liver enzyme induction and increased serum thyroid hormone clearance, let alone further to child neurodevelopmental impairment. This review identifies research needs to contribute to the development of toxicity testing strategies, to reliably predict whether substances have the potential to impair child neurodevelopment via maternal thyroid hormone imbalance.

Adversity Considerations for Thyroid Follicular Cell Hypertrophy and Hyperplasia in Nonclinical Toxicity Studies: Results From the 6th ESTP International Expert Workshop.

The European Society of Toxicologic Pathology organized an expert workshop in May 2018 to address adversity considerations related to thyroid follicular cell hypertrophy and/or hyperplasia (FCHH), which is a common finding in nonclinical toxicity studies that can have important implications for risk assessment of pharmaceuticals, food additives, and environmental chemicals. The broad goal of the workshop was to facilitate better alignment in toxicologic pathology and regulatory sciences on how to determine adversity of FCHH. Key objectives were to describe common mechanisms leading to thyroid FCHH and potential functional consequences; provide working criteria to assess adversity of FCHH in context of associated findings; and describe additional methods and experimental data that may influence adversity determinations. The workshop panel was comprised of representatives from the European Union, Japan, and the United States. Participants shared case examples illustrating issues related to adversity assessments of thyroid changes. Provided here are summary discussions, key case presentations, and panel recommendations. This information should increase consistency in the interpretation of adverse changes in the thyroid based on pathology findings in nonclinical toxicity studies, help integrate new types of biomarker data into the review process, and facilitate a more systematic approach to communicating adversity determinations in toxicology reports.

ERGO: Breaking Down the Wall between Human Health and Environmental Testing of Endocrine Disrupters.

ERGO (EndocRine Guideline Optimization) is the acronym of a European Union-funded research and innovation action, that aims to break down the wall between mammalian and non-mammalian vertebrate regulatory testing of endocrine disruptors (EDs), by identifying, developing and aligning thyroid-related biomarkers and endpoints (B/E) for the linkage of effects between vertebrate classes. To achieve this, an adverse outcome pathway (AOP) network covering various modes of thyroid hormone disruption (THD) in multiple vertebrate classes will be developed. The AOP development will be based on existing and new data from in vitro and in vivo experiments with fish, amphibians and mammals, using a battery of different THDs. This will provide the scientifically plausible and evidence-based foundation for the selection of B/E and assays in lower vertebrates, predictive of human health outcomes. These assays will be prioritized for validation at OECD (Organization for Economic Cooperation and Development) level. ERGO will re-think ED testing strategies from in silico methods to in vivo testing and develop, optimize and validate existing in vivo and early life-stage OECD guidelines, as well as new in vitro protocols for THD. This strategy will reduce requirements for animal testing by preventing duplication of testing in mammals and non-mammalian vertebrates and increase the screening capacity to enable more chemicals to be tested for ED properties.

Potassium Poly(Heptazine Imide): Transition Metal-Free Solid-State Triplet Sensitizer in Cascade Energy Transfer and [3+2]-cycloadditions.

Polymeric carbon nitride materials have been used in numerous light-to-energy conversion applications ranging from photocatalysis to optoelectronics. For a new application and modelling, we first refined the crystal structure of potassium poly(heptazine imide) (K-PHI)-a benchmark carbon nitride material in photocatalysis-by means of X-ray powder diffraction and transmission electron microscopy. Using the crystal structure of K-PHI, periodic DFT calculations were performed to calculate the density-of-states (DOS) and localize intra band states (IBS). IBS were found to be responsible for the enhanced K-PHI absorption in the near IR region, to serve as electron traps, and to be useful in energy transfer reactions. Once excited with visible light, carbon nitrides, in addition to the direct recombination, can also undergo singlet-triplet intersystem crossing. We utilized the K-PHI centered triplet excited states to trigger a cascade of energy transfer reactions and, in turn, to sensitize, for example, singlet oxygen (1 O2 ) as a starting point to synthesis up to 25 different N-rich heterocycles.

Laser-Assisted Lattice Recovery of Graphene by Carbon Nanodot Incorporation.

Producing highly oriented graphene is a major challenge that constrains graphene from fulfilling its full potential in technological applications. The exciting properties of graphene are impeded in practical bulk materials due to lattice imperfections that hinder charge mobility. A simple method to improve the structural integrity of graphene by utilizing laser irradiation on a composite of carbon nanodots (CNDs) and 3D graphene is presented. The CNDs attach themselves to defect sites in the graphene sheets and, upon laser-assisted reduction, patch defects in the carbon lattice. Spectroscopic experiments reveal graphitic structural recovery of up to 43% and electrical conductivity four times larger than the original graphene. The composites are tested as electrodes in electrochemical capacitors and demonstrate extremely fast RC time constant as low as 0.57 ms. Due to their low defect concentrations, the reduced graphene oxide-carbon nanodot (rGO-CND) composites frequency response is sufficiently fast to operate as AC line filters, potentially replacing today's electrolytic capacitors. Using this methodology, demonstrated is a novel line filter with one of the fastest capacitive responses ever reported, and an aerial capacitance of 68.8 mF cm-2 . This result emphasizes the decisive role of structural integrity for optimizing graphene in electronic applications.

Report from the BfR expert hearing on practicability of hormonal measurements: recommendations for experimental design of toxicological studies with integrated hormonal end points.

This publication summarizes discussions that were held during an international expert hearing organized by the German Federal Institute for Risk Assessment (BfR) in Berlin, Germany, in October 2017. The expert hearing was dedicated to providing practical guidance for the measurement of circulating hormones in regulatory toxicology studies. Adequate measurements of circulating hormones have become more important given the regulatory requirement to assess the potential for endocrine disrupting properties for all substances covered by the plant protection products and biocidal products regulations in the European Union (EU). The main focus was the hypothalamus-pituitary-thyroid axis (HPT) and the hypothalamus-pituitary-gonadal axis (HPG). Insulin, insulin-like growth factor 1 (IGF-1), parathyroid hormone (PTH) and vitamins A and D were also discussed. During the hearing, the experts agreed on specific recommendations for design, conduct and evaluation of acceptability of studies measuring thyroid hormones, thyroid stimulating hormone and reproductive hormones as well as provided some recommendations for insulin and IGF-1. Experts concluded that hormonal measurements as part of the test guidelines (TGs) of the Organisation for Economic Co-operation and Development (OECD) were necessary on the condition that quality criteria to guarantee reliability and reproducibility of measurements are adhered to. Inclusion of the female reproductive hormones in OECD TGs was not recommended unless the design of the study was modified to appropriately measure hormone concentrations. The current report aims at promoting standardization of the experimental designs of hormonal assays to allow their integration in OECD TGs and highlights research needs for better identification of endocrine disruptors using hormone measurements.

Practical considerations for developmental thyroid toxicity assessments: What's working, what's not, and how can we do better?

Thyroid hormones (THs; T3 and T4) play a role in development of cardiovascular, reproductive, immune and nervous systems. Thus, interpretation of TH changes from rodent studies (during pregnancy, in fetuses, neonates, and adults) is critical in hazard characterization and risk assessment. A roundtable session at the 2017 Society of Toxicology (SOT) meeting brought together academic, industry and government scientists to share knowledge and different perspectives on technical and data interpretation issues. Data from a limited group of laboratories were compiled for technical discussions on TH measurements, including good practices for reliable serum TH data. Inter-laboratory historical control data, derived from immunoassays or mass spectrometry methods, revealed: 1) assay sensitivities vary within and across methodologies; 2) TH variability is similar across animal ages; 3) laboratories generally achieve sufficiently sensitive TH quantitation levels, although issues remain for lower levels of serum TH and TSH in fetuses and postnatal day 4 pups; thus, assay sensitivity is critical at these life stages. Best practices require detailed validation of rat serum TH measurements across ages to establish assay sensitivity and precision, and identify potential matrix effects. Finally, issues related to data interpretation for biological understanding and risk assessment were discussed, but their resolution remains elusive.

Screening Supramolecular Interactions between Carbon Nanodots and Porphyrins.

We present an in-depth investigation regarding the electron-accepting nature of pressure-synthesized carbon nanodots (pCNDs) in combination with porphyrins as excited-state electron donors. To this end, electrostatic attractions involving negative charges, which are present on the pCND surface, are essential to govern the hybrid assembly, on one hand, and charge separation, on the other hand.

Carbon Nanodots as Feedstock for a Uniform Hematite-Graphene Nanocomposite.

High degrees of dispersion are a prerequisite for functional composite materials for applications in electronics such as sensors, charge and data storage, and catalysis. The use of small precursor materials can be a decisive factor in achieving a high degree of dispersion. In this study, carbon nanodots are used to fabricate a homogeneous, finely dispersed Fe2 O3 -graphene composite aerogel in a one-step conversion process from a precursor mixture. The laser-assisted conversion of small size carbon nanodots enables a uniform distribution of 6.5 nm Fe2 O3 nanoparticles during the formation of a highly conductive carbon matrix. Structural and electrochemical characterization shows that the features of both material entities are maintained and successfully integrated. The presence of Fe2 O3 nanoparticles has a positive effect on the active surface area of the carbon aerogel and thus on the capacitance of the material. This is demonstrated by testing the performance of the composite in supercapacitors. Faradaic reactions are exploited in an aqueous electrolyte through the high accessible surface of the incorporated small Fe2 O3 nanoparticles boosting the specific capacitance of the 3D turbostratic graphene network significantly.

Differences in the toxicity of cerium dioxide nanomaterials after inhalation can be explained by lung deposition, animal species and nanoforms.

Considerable differences in pulmonary responses have been observed in animals exposed to cerium dioxide nanoparticles via inhalation. These differences in pulmonary toxicity might be explained by differences in lung deposition, species susceptibility or physicochemical characteristics of the tested cerium dioxide nanoforms (i.e. same chemical substance, different size, shape, surface area or surface chemistry). In order to distinguish the relative importance of these different influencing factors, we performed a detailed analysis of the data from several inhalation studies with different exposure durations, species and nanoforms, namely published data on NM211 and NM212 (JRC repository), NanoAmor (commercially available) and our published and unpublished data on PROM (industry provided). Data were analyzed by comparing the observed pulmonary responses at similar external and internal dose levels. Our analyses confirm that rats are more sensitive to developing pulmonary inflammation compared to mice. The observed differences in responses do not result purely from differences in the delivered and retained doses (expressed in particle mass as well as surface area). In addition, the different nanoforms assessed showed differences in toxic potency likely due to differences in their physicochemical parameters. Primary particle and aggregate/agglomerate size distributions have a substantial impact on the deposited dose and consequently on the pulmonary response. However, in our evaluation size could not fully explain the difference observed in the analyzed studies indicating that the pulmonary response also depends on other physicochemical characteristics of the particles. It remains to be determined to what extent these findings can be generalized to other poorly soluble nanomaterials.