The safety of titanium dioxide nanoparticles (TiO2 NPs) has been a subject of debate for over two decades, primarily due to the lack of consensus on their toxicity. A comprehensive understanding of the molecular-level toxicity of TiO2 NPs is essential for accurate safety evaluations and effective risk mitigation strategies. Thus, this study aims to elucidate the relationship between the physicochemical properties of TiO2 NPs and their pulmonary toxicity at the molecular level. Additionally, it seeks to determine whether these properties and the corresponding transcriptomic responses can facilitate the categorization of TiO2 nanoforms into groups with similar pulmonary hazards. Through the integration of bioinformatics and machine learning algorithms to analyze genome-wide transcriptomic profiles, we identified size, specific surface area, reactive oxygen species (ROS) production, crystalline structure, and surface modification as key determinants of TiO2 NP toxicity at the transcriptomic level. Furthermore, we observed that different nanoforms of TiO2 NPs, characterized by varying properties, can elicit distinct molecular-level responses, indicating that transcriptomic pathways are subject to different modes of perturbation. Our findings offer valuable insights into the safety considerations of TiO2 NPs and lay the groundwork for future strategies to group nanoforms with similar patterns of hazards.

Toxicity pathways of engineered carbon nanomaterials, which have many and diverse industrial applications, include oxidative stress primarily due to the generation of reactive oxygen species (ROS). This study on 13 carbon black, 6 Graphene nanoplatelets (GNP), and 3 nanodiamond materials identified physicochemical properties driving their ROS production. ROS production was cross-validated using different assays: electron paramagnetic resonance (EPR) with and without (bulk) CPH spin probe, and cellular and acellular fluorescence measurements using the DCFH2-DA probe. ROS production was highly correlated between assays, however, the EPR spin-count without CPH (i.e., intrinsic ROS of bulk materials) was not correlated with the surface-related ROS production captured by the other assays. ROS production was also closely linked to cell membrane damage through hemolytic potential (r=0.96). Among physicochemical properties, ROS production was primarily related to BET specific surface area (SSA). The variation in ROS production not explained by SSA was 4-fold for sp2-hybridized carbon black and GNP materials and 100-fold when also considering sp3-hybridized carbon nanodiamond materials. This variation in ROS production was related to surface properties and could be accurately modelled (R2=0.99) based on surface-sensitive analysis using x-ray photoelectron spectroscopy (XPS). The XPS-based model showed that ROS production increased with carbon sp2-hybridization and sulfur oxide surface groups while it was inhibited by the total non-oxidized sulfur and oxygen surface content. Surface functionalization thus appears to regulate ROS production of common carbon nanomaterials by at least a factor of 4, and simple surface-specific analyses may be useful in the design of safer carbon nanomaterials.

Background:Inhalation exposure is the gold standard when assessing pulmonary tox-icity.However,it typically requires substantial amounts of test material.Intratracheal instillation is an alternative administration technique,where the test substance is suspended in a liquid vehicle and deposited into the lung via the trachea.Instillation requires minimal test material,delivers an exact dose deep into the lung,and is less labor-intensive than inhalation exposures.However,one shortcoming is that the pro-cedure may induce short-term inflammation.To minimize this,we tested different modifications of the technique to identify the potential for refinement. Methods:First,we tested whether previous findings of increased inflammation could be confirmed.Next,we tested whether instillation with a disposable 1 mL syringe with ball-tipped steel-needle(Disposable-syringe/steel-needle)induced less inflammation than the use of our standard set-up,a 250 μ L reusable glass syringe with a disposable plastic catheter(Glass-syringe/plastic-catheter).Finally,we tested if access to pelleted and liquid feed prior to instillation affected inflammation.We evaluated inflammation by neutrophil numbers in bronchoalveolar fluid 24 h post-exposure. Results:Vehicle-instilled mice showed a small increase in neutrophil numbers com-pared to untreated mice.Neutrophil numbers were slightly elevated in the groups in-stilled with Disposable-syringe/steel-needle;an interaction with feed type indicated that the increase in neutrophils was more pronounced in combination with feed pel-lets compared to liquid feed.We found no difference between the feed types when using the Glass-syringe/plastic-catheter combination. Conclusion:The Glass-syringe/plastic-catheter combination induced the least exposure-related inflammation,confirming this as a preferred instillation procedure.

Understanding the biokinetics of nanoparticles will support the identification of target organs for toxicological endpoints. We investigated the biokinetics of poorly soluble nanomaterials carbon black, multi-walled carbon nanotubes (MWCNT), cerium oxide (CeO2), titanium dioxide (TiO2), crystalline silica (SiO2) in inhalation studies in rodents (the soluble amorphous silica was also included). By reviewing research papers on the inhalation of these substances, we collected physico-chemical data and elemental distribution to organs, urine, and feces. Carbon black, MWCNT, cerium, and titanium accumulated during exposure and persisted in the lung post-exposure (still present at >3000 h). For silica, the amorphous form resulted in silicon accumulation in the lungs. Silicon was increased in the blood. Lymph node accumulation was observed for MWCNT, cerium, and titanium. Liver accumulation was observed for cerium and titanium. Cerium and silicon were increased in the spleen. Titanium accumulated and remained in the spleen (>4000 h). MWCNT were increased in several organs, some of which had a persistent presence of this material. In conclusion, we collected data on the biodistribution of five nanomaterials that, except for amorphous silica, are poorly soluble. The poorly soluble materials or their elements were persistent in the lungs but also showed persistence in other organs. In addition, the data on lung content supports Haber's rule, with titanium being deposited to a greater extent at exposure end than the other materials. Lung deposition seems relatively linear for the collected MMAD values, indicating size may be less important than previously suggested regarding alveolar deposition of the sub-2-micrometer size.

Studies on in vitro-in vivo correlations of inflammatory and genotoxic responses are needed to advance new approach methodologies. Here, we assessed pro-inflammatory and genotoxic responses by 13 nanosized metal oxides (nMeOx) and quartz (DQ12) in alveolar epithelial cells (A549) and macrophages (THP-1a) exposed in submerged conditions, and in A549:THP-1a co-cultures in air-liquid interface (ALI) system. Soluble nMeOx produced the highest IL-8 expression in A549 and THP-1a cells in submerged conditions (>= 2-fold, p < 0.05), whereas only CuO caused a strong response in co-cultures exposed in the ALI system (13-fold, p < 0.05). IL-8 expression in A549 cells with concentrations as nMeOx specific surface area (SSA) correlated with neutrophil influx in mice (r = 0.89-0.98, p < 0.05). Similarly, IL-8 expression in THP-1a cell with concentrations as mass and SSA (when excluding soluble nMeOx) correlated with neutrophil influx in mice (r = 0.81-0.84, p < 0.05). DNA strand breaks (SB) was measured by the comet assay. We used a scoring system that categorizes effects in standard deviation units for comparison of genotoxicity in different models. Concordant genotoxicity was observed between SB levels in vitro (A549 and co-culture) and in vivo (broncho-alveolar lavage fluid cells and lung tissue). In conclusion, this study shows in vitro-in vivo correlations of nMeOx-induced inflammatory and genotoxic responses.

At the turn of the twentieth century, nanotechnology was regarded as the new innovation potential. However, increased industrial use of nanomaterials raised concerns whether nanomaterial exposure could pose occupational health risks similar to asbestos. In 2012, the National Research Centre for the Working Environment (NFA) established the ‘Danish Centre for Nanosafety’ with the aim to provide evidence-based knowledge for policy advice on occupational handling of nanomaterials. Relevant stakeholders including the Danish Working Environment Authority (WEA), the Danish Environment Protection Agency, and the social partners were engaged. The project achieved societal impact in various ways: NFA produced documentation for health-based occupational exposure limits for three engineered nanomaterials and diesel exhaust nanoparticles at the request of WEA. Denmark adopted a national occupational exposure limit for diesel particles in 2021. WEA made a guidance document on safe handling of nanomaterials. The Working Environment Council published fact sheets based on knowledge from NFA aimed at both nanomaterial providers and enterprises using nanomaterials. A revised law made it compulsory to register the presence of nanomaterials in products in the Danish Product Registry. Finally, the Danish Centre for Nanosafety supported development of a free, web-based control-banding tool for occupational risk assessment and -management of nanomaterials (NanoSafer.org).

commenting on the suggested health-based occupational exposure limit for zinc oxide made by the National Research Centre for the Working Environment (NFA) in Denmark (Hadrup et al. 2021b).In 2018, Christian Monsé and co-workers made a controlled human exposure study (Monse et al. 2018), which reproduced the dose-dependent ZnO-induced acute phase response in human volunteers that we have previously observed in mice (Hadrup et al. 2019;Jacobsen et al. 2015;Saber et al. 2022), and enabled NFA to derive a health-based occupational exposure limit based on human data.Our suggestion was 0.05 mg/m 3 ZnO.In the recent short communication, Monsé and et al. argues that a higher OEL for ZnO could be justified.We would like to address some of their arguments.We agree with Monsé et al. that the No Observed Effect Concentration (NOEC) for systemic acute phase response is 0.5 mg/m 3 ZnO for 4 h.We furthermore consider it a No Observed Adverse Effect Concentration (NOAEC), because we consider the induced effects as adverse.As also mentioned by Monsé et al., Brand et al. showed, in their controlled human exposure studies, that the biologically relevant dose is the total daily dose calculated as time x concentration (Brand et al. 2019).We argue that the same dose-response relationship would be expected in the range of the NOAEC, and therefore, the derived NOAEC for an 8-h working day should be 0.25 mg/m 3 (ECHA 2012).ZnO exposure induces dose-dependent acute phase response in humans and mice (Hadrup et al.

This study collects toxicity data from animal inhalation studies of some nanomaterials and their bulk and ionic counterparts. To allow potential grouping and interpretations, we retrieved the primary physicochemical and exposure data to the extent possible for each of the materials. Reviewed materials are compounds (mainly elements, oxides and salts) of carbon (carbon black, carbon nanotubes, and graphene), silver, cerium, cobalt, copper, iron, nickel, silicium (amorphous silica and quartz), titanium (titanium dioxide), and zinc (chemical symbols: Ag, C, Ce, Co, Cu, Fe, Ni, Si, Ti, TiO2, and Zn). Collected endpoints are: a) pulmonary inflammation, measured as neutrophils in bronchoalveolar lavage (BAL) fluid at 0-24 hours after last exposure; and b) genotoxicity/carcinogenicity. We present the dose descriptors no-observed-adverse-effect concentrations (NOAECs) and lowest-observed-adverse-effect concentrations (LOAECs) for 88 nanomaterial investigations in data-library and graph formats. We also calculate 'the value where 25% of exposed animals develop tumors' (T25) for carcinogenicity studies. We describe how the data may be used for hazard assessment of the materials using carbon black as an example. The collected data also enable hazard comparison between different materials. An important observation for poorly soluble particles is that the NOAEC for neutrophil numbers in general lies around 1 to 2 mg/m(3). We further discuss why some materials' dose descriptors deviate from this level, likely reflecting the effects of the ionic form and effects of the fiber-shape. Finally, we discuss that long-term studies, in general, provide the lowest dose descriptors, and dose descriptors are positively correlated with particle size for near-spherical materials.

Tungsten is used in several applications and human exposure may occur. To assess its pulmonary toxicity, we exposed male mice to nose-only inhalation of tungsten particles at 9, 23 or 132 mg/m3 (Low, Mid and High exposure) (45 min/day, 5 days/week for 2 weeks). Increased genotoxicity (assessed by comet assay) was seen in bronchoalveolar (BAL) fluid cells at Low and High exposure. We measured acellular ROS production, and cannot exclude that ROS contributed to the observed genotoxicity. We saw no effects on body weight gain, pulmonary inflammation, lactate dehydrogenase or protein in BAL fluid, pathology of liver or kidney, or on sperm counts. In conclusion, tungsten showed non-dose dependent genotoxicity in the absence of inflammation and therefore interpreted to be primary genotoxicity. Based on genotoxicity, a Lowest Observed Adverse Effect Concentration (LOAEC) could be set at 9 mg/m3. It was not possible to establish a No Adverse Effect Concentration (NOAEC).

Many in vitro and in vivo studies have shown that exposure to carbon nanotubes (CNTs) is associated with inflammation, oxidative stress and genotoxicity, although there is a paucity of studies on these effects in the pleural cavity. In the present study, we investigated adverse outcomes of pleural exposure to multi-walled CNTs (MWCNT-7, NM-401 and NM-403) and single-walled CNTs (NM-411). Female C57BL/6 mice were exposed to 0.2 or 5 µg of CNTs by intra-pleural injection and sacrificed one-year post-exposure. Exposure to long and straight types of MWCNTs (i.e. MWCNT-7 and NM-401) was associated with decreased number of macrophages and increased number of neutrophils and eosinophils in pleural lavage fluid. Increased protein content in the pleural lavage fluid was also observed in mice exposed to MWCNT-7 and NM-401. The concentration of mesothelin was increased in mice exposed to MWCNT-7 and NM-411. Levels of DNA strand breaks and DNA oxidation damage, measured by the comet assay, were unaltered in cells from pleural scrape. Extra-pleural effects were seen in CNT exposed mice, including enlarged and pigmented mediastinal lymph nodes (all four types of CNTs), pericardial plaques (MWCNT-7 and NM-401), macroscopic abnormalities on the liver (MWCNT-7) and ovaries/uterus (NM-411). In conclusion, the results demonstrate that intra-pleural exposure to long and straight MWCNTs is associated with adverse outcomes. Certain observations such as increased content of mesothelin in pleural lavage fluid and ovarian/uterine abnormalities in mice exposed to NM-411 suggests that exposure to SWCNTs may also be associated with some adverse outcomes.

Animal experiments are highly relevant models for the assessment of toxicological effects of engineered nanomaterials (ENMs), due to lack of biomonitoring and epidemiological studies. However, the expanding number of ENMs with different physico-chemical properties strains this approach, as there are ethical concerns and economical challenges with the use of animals in toxicology. There is an urgent need for cell culture models that predict the level of toxicological responses in vivo, consequently reducing or replacing the use of animals in nanotoxicology. However, there is still a limited number of studies on in vitro-in vivo correlation of toxicological responses following ENMs exposure. In this review, we collected studies that have compared in vitro and in vivo toxic effects caused by ENMs. We discuss the influence of cell culture models and exposure systems on the predictability of in vitro models to equivalent toxic effects in animal lungs after pulmonary exposure to ENMs. In addition, we discuss approaches to qualitatively or quantitatively compare the effects in vitro and in vivo. The magnitude of toxicological responses in cells that are exposed in submerged condition is not systematically different from the response in cells exposed in air-liquid interface systems, and there appears to be similar ENMs hazard ranking between the two exposure systems. Overall, we show that simple in vitro models with cells exposed to ENMs in submerged condition can be used to predict toxic effects in vivo, and identify future strategies to improve the associations between in vitro and in vivo ENMs-induced pulmonary toxicity. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.

Background The EU-project GRACIOUS developed an Integrated Approach to Testing and Assessment (IATA) to support grouping high aspect ratio nanomaterials (HARNs) presenting a similar inhalation hazard. Application of grouping reduces the need to assess toxicity on a case-by-case basis and supports read-across of hazard data from substances that have the data required for risk assessment (source) to those that lack such data (target). The HARN IATA, based on the fibre paradigm for pathogenic fibres, facilitates structured data gathering to propose groups of similar HARN and to support read-across by prompting users to address relevant questions regarding HARN morphology, biopersistence and inflammatory potential. The IATA is structured in tiers, allowing grouping decisions to be made using simple in vitro or in silico methods in Tier1 progressing to in vivo approaches at the highest Tier3. Here we present a case-study testing the applicability of GRACIOUS IATA to form an evidence-based group of multiwalled carbon nanotubes (MWCNT) posing a similar predicted fibre-hazard, to support read-across and reduce the burden of toxicity testing. Results The case-study uses data on 15 different MWCNT, obtained from the published literature. By following the IATA, a group of 2 MWCNT was identified (NRCWE006 and NM-401) based on a high degree of similarity. A pairwise similarity assessment was subsequently conducted between the grouped MWCNT to evaluate the potential to conduct read-across and fill data gaps required for regulatory hazard assessment. The similarity assessment, based on expert judgement of Tier 1 assay results, predicts both MWCNT are likely to cause a similar acute in vivo hazard. This result supports the possibility for read-across of sub-chronic and chronic hazard endpoint data for lung fibrosis and carcinogenicity between the 2 grouped MWCNT. The implications of accepting the similarity assessment based on expert judgement of the MWCNT group are considered to stimulate future discussion on the level of similarity between group members considered sufficient to allow regulatory acceptance of a read-across argument. Conclusion This proof-of-concept case-study demonstrates how a grouping hypothesis and IATA may be used to support a nuanced and evidence-based grouping of 'similar' MWCNT and the subsequent interpolation of data between group members to streamline the hazard assessment process.

To reduce, replace, and refine in vivo testing, there is increasing emphasis on the development of more physiologically relevant in vitro test systems to improve the reliability of non-animal-based methods for hazard assessment. When developing new approach methodologies, it is important to standardize the protocols and demonstrate the methods can be reproduced by multiple laboratories. The aim of this study was to assess the transferability and reproducibility of two advanced in vitro liver models, the Primary Human multicellular microtissue liver model (PHH) and the 3D HepG2 Spheroid Model, for nanomaterial (NM) and chemical hazard assessment purposes. The PHH model inter-laboratory trial showed strong consistency across the testing sites. All laboratories evaluated cytokine release and cytotoxicity following exposure to titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles. No significant difference was observed in cytotoxicity or IL-8 release for the test materials. The data were reproducible with all three laboratories with control readouts within a similar range. The PHH model ZnO induced the greatest cytotoxicity response at 50.0 μg/mL and a dose-dependent increase in IL-8 release. For the 3D HepG2 spheroid model, all test sites were able to construct the model and demonstrated good concordance in IL-8 cytokine release and genotoxicity data. This trial demonstrates the successful transfer of new approach methodologies across multiple laboratories, with good reproducibility for several hazard endpoints.

Carbon black exposure causes oxidative stress, inflammation and genotoxicity. The objective of this systematic review was to assess the contributions of primary (i.e. direct formation of DNA damage) and secondary genotoxicity (i.e., DNA lesions produced indirectly by inflammation) to the overall level of DNA damage by carbon black. The database is dominated by studies that have measured DNA damage by the comet assay. Cell culture studies indicate a genotoxic action of carbon black, which might be mediated by oxidative stress. Many in vivo studies originate from one laboratory that has investigated the genotoxic effects of Printex 90 in mice by intra-tracheal instillation. Meta-analysis and pooled analysis of these results demonstrate that Printex 90 exposure is associated with a slightly increased level of DNA strand breaks in bronchoalveolar lavage cells and lung tissue. Other types of genotoxic damage have not been investigated as thoroughly as DNA strand breaks, although there is evidence to suggest that carbon black exposure might increase the mutation frequency and cytogenetic endpoints. Stratification of studies according to concurrent inflammation and DNA damage does not indicate that carbon black exposure gives rise to secondary genotoxicity. Even substantial pulmonary inflammation is at best only associated with a weak genotoxic response in lung tissue. In conclusion, the review indicates that nanosized carbon black is a weak genotoxic agent and this effect is more likely to originate from a primary genotoxic mechanism of action, mediated by e.g., oxidative stress, than inflammation-driven (secondary) genotoxicity.

sient ROS play an important role in the defense mechanism of immune cells and redox signaling, whereas prolonged ROS elevation is involved in disease development and progression.With global progress in the AOP development field, multiple, slightly nuanced KEs related to ROS have been created in the AOP-Wiki 2 .Many of these KEs are highly similar and largely redundant, which has catalyzed a need to create harmonized consensus KEs on ROS that can be shared in a modular fashion between closely related pathways and networks.To address this need, a consortium of ROS and AOP experts has been formed to discuss the "Mystery of ROS" and develop consensus KEs for this field.This meeting report summarizes initial efforts of the "Mystery of ROS" consortium to harmonize the ROS-related KEs currently available in the AOP-Wiki.The two new modular KEs reflect discussion from the group relating to the effects of ROS presenting a "double-edged sword" by describing the concepts of "up-regulation of ROS" and "diminished protective response." Summary of the discussions in the consortiumThe international online conferences on the Mystery of ROS took place on May 31, 2021 and October 8, 2021.At the first conference on Mystery of ROS (I), a brief introduction of the ROS collaboration was followed by eight presentations, which are listed in Table 1.

Improved strategies are required for testing nanomaterials (NMs) to make hazard and risk assessment more efficient and sustainable. Including reduced reliance on animal models, without decreasing the level of human health protection. Acellular detection of reactive oxygen species (ROS) may be useful as a screening assay to prioritize NMs of high concern. To improve reliability and reproducibility, and minimize uncertainty, a standard operating procedure (SOP) has been developed for the detection of ROS using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH2-DA) assay. The SOP has undergone an inter- and intra-laboratory comparison, to evaluate robustness, reliability, and reproducibility, using representative materials (ZnO, CuO, Mn2O3, and BaSO4 NMs), and a number of calibration tools to normalize data. The SOP includes an NM positive control (nanoparticle carbon black (NPCB)), a chemical positive control (SIN-1), and a standard curve of fluorescein fluorescence. The interlaboratory comparison demonstrated that arbitrary fluorescence units show high levels of partner variability; however, data normalization improved variability. With statistical analysis, it was shown that the SIN-1 positive control provided an extremely high level of reliability and reproducibility as a positive control and as a normalization tool. The NPCB positive control can be used with a relatively high level of reproducibility, and in terms of the representative materials, the reproducibility CuO induced-effects was better than for Mn2O3. Using this DCFH2-DA acellular assay SOP resulted in a robust intra-laboratory reproduction of ROS measurements from all NMs tested, while effective reproduction across different laboratories was also demonstrated; the effectiveness of attaining reproducibility within the interlaboratory assessment was particle-type-specific.

Some implantable medical devices contain silver. We aimed to assess at what amount implanted silver becomes toxic. Silver was elevated in bodily fluids and tissues surrounding silver-containing implants. Silver released from implants also distributes to blood and other tissues; there is evidence to suggest silver can pass the blood-brain-barrier. Silver can be deposited as nano-sized particles in various tissues. Such particles, in addition to silver, often contain other elements too, e.g., selenium and sulfur. Silver released from implants seems to stay in the body for long periods (years). Reported excretion pathways following implantation are urinary and fecal ones. Reported toxicological effects were virtually all local reactions surrounding the implants. Argyria is a blue-gray discoloration of the skin due to deposited silver granules. Localized argyria has been described after the implantation of acupuncture needles and silver-coated prostheses, although the presence of silver was tested only for and shown in the former. Other toxicological effects include local tissue reactivity and examples of neurotoxic and vascular effects. We did not include genotoxicity studies in the present publication as we recently evaluated silver to be genotoxic. Carcinogenicity studies were absent. We conclude that local toxicity of implanted silver can be foreseen in some situations.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are formed as a result of natural cellular processes, intracellular signaling, or as adverse responses associated with diseases or exposure to oxidizing chemical and non-chemical stressors. The action of ROS and RNS, collectively referred to as reactive oxygen and nitrogen species (RONS), has recently become highly relevant in a number of adverse outcome pathways (AOPs) that capture, organize, evaluate and portray causal relationships pertinent to adversity or disease progression. RONS can potentially act as a key event (KE) in the cascade of responses leading to an adverse outcome (AO) within such AOPs, but are also known to modulate responses of events along the AOP continuum without being an AOP event itself. A substantial discussion has therefore been undertaken in a series of workshops named "Mystery or ROS" to elucidate the role of RONS in disease and adverse effects associated with exposure to stressors such as nanoparticles, chemical, and ionizing and non-ionizing radiation. This review introduces the background for RONS production, reflects on the direct and indirect effects of RONS, addresses the diversity of terminology used in different fields of research, and provides guidance for developing a harmonized approach for defining a common event terminology within the AOP developer community.

We spend most of our time indoors; however, little is known about the effects of exposure to aerosol particles indoors. We aimed to determine differences in relative toxicity and physicochemical properties of PM2.5 collected simultaneously indoors (PM2.5 INDOOR ) and outdoors (PM2.5 OUTDOOR ) in 15 occupied homes in southern Sweden. Collected particles were extracted from filters, pooled (indoor and outdoor separately), and characterized for chemical composition and endotoxins before being tested for toxicity in mice via intratracheal instillation. Various endpoints including lung inflammation, genotoxicity, and acute-phase response in lung and liver were assessed 1, 3, and 28 days post-exposure. Chemical composition of particles used in toxicological assessment was compared to particles analyzed without extraction. Time-resolved particle mass and number concentrations were monitored. PM2.5 INDOOR showed higher relative concentrations (μg mg-1 ) of metals, PAHs, and endotoxins compared to PM2.5 OUTDOOR . These differences may be linked to PM2.5 INDOOR causing significantly higher lung inflammation and lung acute-phase response 1 day post-exposure compared to PM2.5 OUTDOOR and vehicle controls, respectively. None of the tested materials caused genotoxicity. PM2.5 INDOOR displayed higher relative toxicity than PM2.5 OUTDOOR under the studied conditions, that is, wintertime with reduced air exchange rates, high influence of indoor sources, and relatively low outdoor concentrations of PM. Reducing PM2.5 INDOOR exposure requires reduction of both infiltration from outdoors and indoor-generated particles.