Lack of pulmonary fibrogenicity and carcinogenicity of titanium dioxide nanoparticles in 26-week inhalation study in rasH2 mouse model

Background: With the rapid development of alternative methods based on the spirit of animal welfare, the publications of animal studies evaluating endpoints such as cancer have been extremely reduced. There have been no systemic inhalation exposure studies of titanium dioxide nanoparticles (TiO2 NPs) using CByB6F1-Tg(HRAS)2Jic (rasH2) 26-week study mice model for detecting carcinogenicity. Methods: Male and female rasH2 mice were exposed to 2, 8 or 32 mg/m3 of TiO2 NPs for 6 hours/day, 5 days/week for 26 weeks using a whole-body inhalation exposure system, with reference to the Organization for Economic Co-operation and Development principles of Good Laboratory Practice. All tissues including lungs, and blood were collected and subjected to biological and histopathological analyses. Additionally, Ki67 positive index were evaluated in mice lung alveolar epithelial type 2 cell (AEC2). Results: This study established a stable method for generating and exposing TiO2 NPs aerosol, and clarified the dose-response relationship by TiO2 NPs inhalation to rasH2 mice. TiO2 NPs exposure induced deposition of particles in lungs and mediastinal lymph nodes in a dose-dependent manner in each exposure group. Additionally, alveolar inflammation was only observed in 32 mg/m3 exposure group in both the sexes. Exposure to TiO2 NPs, as well as other organs, did not increase the incidence of lung tumors in any group, and pulmonary fibrosis and pre-neoplastic lesions were not observed in all groups. Finally, the cell proliferative activity of AEC2 was examined, and it was not increased by exposure to TiO2 NPs. Conclusions: This is the first report showing the lack of pulmonary fibrogenicity and carcinogenicity (no evidence of carcinogenic activity) of TiO2 NPs in 26-week inhalation study in rasH2 mice exposed up to 32 mg/m3, which is considered to be a high concentration. Macrophages undergoing phagocytosis due to TiO2 NPs exposure formed inflammatory foci in the alveolar regions of exposed mice but did not develop fibrosis or hyperplasia or tumors. Moreover, the cell proliferative ability of AEC2 in lesions was not increased. In addition, no carcinogenicity was observed for any organs other than the lungs in this study.

Genotoxicity and Gene Expression in the Rat Lung Tissue following Instillation and Inhalation of Different Variants of Amorphous Silica Nanomaterials (aSiO2 NM)

Several reports on amorphous silica nanomaterial (aSiO2 NM) toxicity have been questioning their safety. Herein, we investigated the in vivo pulmonary toxicity of four variants of aSiO2 NM: SiO2_15_Unmod, SiO2_15_Amino, SiO2_7 and SiO2_40. We focused on alterations in lung DNA and protein integrity, and gene expression following single intratracheal instillation in rats. Additionally, a short-term inhalation study (STIS) was carried out for SiO2_7, using TiO2_NM105 as a benchmark NM. In the instillation study, a significant but slight increase in oxidative DNA damage in rats exposed to the highest instilled dose (0.36 mg/rat) of SiO2_15_Amino was observed in the recovery (R) group. Exposure to SiO2_7 or SiO2_40 markedly increased oxidative DNA lesions in rat lung cells of the exposure (E) group at every tested dose. This damage seems to be repaired, since no changes compared to controls were observed in the R groups. In STIS, a significant increase in DNA strand breaks of the lung cells exposed to 0.5 mg/m3 of SiO2_7 or 50 mg/m3 of TiO2_NM105 was observed in both groups. The detected gene expression changes suggest that oxidative stress and/or inflammation pathways are likely implicated in the induction of (oxidative) DNA damage. Overall, all tested aSiO2 NM were not associated with marked in vivo toxicity following instillation or STIS. The genotoxicity findings for SiO2_7 from instillation and STIS are concordant; however, changes in STIS animals were more permanent/difficult to revert.

Experimental justification of the maximum possible concentration (MPC) of dichlorohexafluorobutene in a working area

To date, there have been no exposure standards for air concentrations of 1,4-dichlorohexafluorobutene (DCHF) in the work areas. The study was aimed to assess the toxicity of DCHF and to evaluate health hazard in acute, subacute, and chronic experiments. It was found that the substance was highly hazardous, DL50 in mice after intragastric injection was 79.0 mg/kg, СL50 was 229.0 mg/m3, and in rats these values were 86,0 mg/kg and 670,0 mg/m3. In animals, DCHF had a moderate local irritative effect on animal skin and ocular mucous membranes, as well as the skin resorptive effect. The 18.2 mg/m3 threshold limit concentration for a single inhalation exposure to DCHF was defined based on the changes in behavior responses and blood parameters. The 30-day subacute inhalation experiment revealed the pronounced cumulative effect of the substance. The 4-months chronic inhalation study showed that the exposure of experimental rats to 16.8 mg/m3 concentration of DCHF resulted in impaired function of central nervous system and cardiac activity, altered hematologic, biochemical, acid-base, and blood gas values, as well as in morphological alterations in lungs, which persisted after the 30-day recovery period. The chronic exposure threshold defined for DCHF was 2.2 mg/m3, and the defined no observable effect level was 0.24 mg/m3. Based on the study results, the maximum permissible concentration of DCHF in the air of the working area of 0.2 mg/m3 was confirmed and approved, the substance was assigned hazard class 2, vapor + aerosol + (specific protection of skin and eyes required). Gas chromatographic method using electron-capture detection for determination of DCHF mass air concentration in the work areas has been developed and approved.

Discriminating Spontaneous From Cigarette Smoke and THS 2.2 Aerosol Exposure-Related Proliferative Lung Lesions in A/J Mice by Using Gene Expression and Mutation Spectrum Data

Mice, especially A/J mice, have been widely employed to elucidate the underlying mechanisms of lung tumor formation and progression and to derive human-relevant modes of action. Cigarette smoke (CS) exposure induces tumors in the lungs; but, non-exposed A/J mice will also develop lung tumors spontaneously with age, which raises the question of discriminating CS-related lung tumors from spontaneous ones. However, the challenge is that spontaneous tumors are histologically indistinguishable from the tumors occurring in CS-exposed mice. We conducted an 18-month inhalation study in A/J mice to assess the impact of lifetime exposure to Tobacco Heating System (THS) 2.2 aerosol relative to exposure to 3R4F cigarette smoke (CS) on toxicity and carcinogenicity endpoints. To tackle the above challenge, a 13-gene gene signature was developed based on an independent A/J mouse CS exposure study, following by a one-class classifier development based on the current study. Identifying gene signature in one data set and building classifier in another data set addresses the feature/gene selection bias which is a well-known problem in literature. Applied to data from this study, this gene signature classifier distinguished tumors in CS-exposed animals from spontaneous tumors. Lung tumors from THS 2.2 aerosol-exposed mice were significantly different from those of CS-exposed mice but not from spontaneous tumors. The signature was also applied to human lung adenocarcinoma gene expression data (from The Cancer Genome Atlas) and discriminated cancers in never-smokers from those in ever-smokers, suggesting translatability of our signature genes from mice to humans. A possible application of this gene signature is to discriminate lung cancer patients who may benefit from specific treatments (i.e., EGFR tyrosine kinase inhibitors). Mutational spectra from a subset of samples were also utilized for tumor classification, yielding similar results. “Landscaping” the molecular features of A/J mouse lung tumors highlighted, for the first time, a number of events that are also known to play a role in human lung tumorigenesis, such as Lrp1b mutation and Ros1 overexpression. This study shows that omics and computational tools provide useful means of tumor classification where histopathological evaluation alone may be unsatisfactory to distinguish between age- and exposure-related lung tumors.

A 7-month inhalation toxicology study in C57BL/6 mice demonstrates reduced pulmonary inflammation and emphysematous changes following smoking cessation or switching to e-vapor products

Cigarette smoking causes serious diseases, including lung cancer, atherosclerotic coronary artery disease, peripheral vascular disease, chronic bronchitis, and emphysema. While cessation remains the most effective approach to minimize smoking-related disease, alternative non-combustible tobacco-derived nicotine-containing products may reduce disease risks among those unable or unwilling to quit. E-vapor aerosols typically contain significantly lower levels of smoke-related harmful and potentially harmful constituents; however, health risks of long-term inhalation exposures are unknown. We designed a 7-month inhalation study in C57BL/6 mice to evaluate long-term respiratory toxicity of e-vapor aerosols compared to cigarette smoke and to assess the impact of smoking cessation (Cessation group) or switching to an e-vapor product (Switching group) after 3 months of exposure to 3R4F cigarette smoke (CS). There were no significant changes in in-life observations (body weights, clinical signs) in e-vapor groups compared to the Sham Control. The 3R4F CS group showed reduced respiratory function during exposure and had lower body weight and showed transient signs of distress post-exposure. Following 7 months of exposure, e-vapor aerosols resulted in no or minimal increase in pulmonary inflammation, while exposure to 3R4F CS led to impairment of lung function and caused marked lung inflammation and emphysematous changes. Biological changes observed in the Switching group were similar to the Cessation group. 3R4F CS exposure dysregulated the lung and nasal tissue transcriptome, while these molecular effects were substantially lower in the e-vapor group. Results from this study demonstrate that in comparison with 3R4F CS, e-vapor aerosols induce substantially lower biological responses including pulmonary inflammation and emphysematous changes, and that complete switching from CS to e-vapor products significantly reduces biological changes associated with CS in C57BL/6 mice.

Preclinical Safety Assessment of 2 Inhaled Single-Domain Antibodies in the Cynomolgus Monkey

The safety of 2 single domain antibodies (dAbs) was evaluated by inhalation toxicology studies in the cynomolgus monkey. In the first case study, a 14-day repeat-dose study evaluating an anti-thymic stromal lymphopoietin (anti-TSLP) dAb resulted in minimal mononuclear inflammatory cell infiltrates in the lungs, increases in lymphocytes in bronchoalveolar lavage fluid, and development of antidrug antibodies (ADAs). In a 6-week inhalation study, there was an increase in incidence and/or severity of mononuclear cell infiltrates in the lung, increased cellularity in the tracheobronchial lymph node (TBLN), and development of ADA. The second case study evaluated a change in duration of inhalation dosing, a different route of exposure (intravenous or IV), and recovery following an off-dose period with an anti-tumor necrosis factor receptor 1 dAb. A 7-day repeat-dose inhalation study and a 14-day IV study produced no microscopic effects in the lung, whereas a 14-day inhalation study resulted in moderate increases in pulmonary perivascular/peribronchiolar/alveolar lymphocytic infiltrates and increased cellularity in the TBLN, with partial and full recovery, respectively, after 14 days. The lung and lymph node findings seen after inhalation of either dAb were considered secondary to the immunogenic response to a human protein and were considered nonadverse.

Parametric Optimization of an Air–Liquid Interface System for Flow-Through Inhalation Exposure to Nanoparticles: Assessing Dosimetry and Intracellular Uptake of CeO2 Nanoparticles

Air–liquid interface (ALI) systems have been widely used in recent years to investigate the inhalation toxicity of many gaseous compounds, chemicals, and nanomaterials and represent an emerging and promising in vitro method to supplement in vivo studies. ALI exposure reflects the physiological conditions of the deep lung more closely to subacute in vivo inhalation scenarios compared to submerged exposure. The comparability of the toxicological results obtained from in vivo and in vitro inhalation data is still challenging. The robustness of ALI exposure scenarios is not yet well understood, but critical for the potential standardization of these methods. We report a cause-and-effect (C&E) analysis of a flow through ALI exposure system. The influence of five different instrumental and physiological parameters affecting cell viability and exposure parameters of a human lung cell line in vitro (exposure duration, relative humidity, temperature, CO2 concentration and flow rate) was investigated. After exposing lung epithelia cells to a CeO2 nanoparticle (NP) aerosol, intracellular CeO2 concentrations reached values similar to those found in a recent subacute rat inhalation study in vivo. This is the first study showing that the NP concentration reached in vitro using a flow through ALI system were the same as those in an in vivo study.

In Vitro and In Vivo Evaluation of the Toxic Effects of Dodecylguanidine Hydrochloride

The toxicity profiles of the widely used guanidine-based chemicals have not been fully elucidated. Herein, we evaluated the in vitro and in vivo toxicity of eight guanidine-based chemicals, focusing on inhalation toxicity. Among the eight chemicals, dodecylguanidine hydrochloride (DGH) was found to be the most cytotoxic (IC50: 0.39 μg/mL), as determined by the water soluble tetrazolium salts (WST) assay. An acute inhalation study for DGH was conducted using Sprague-Dawley rats at 8.6 ± 0.41, 21.3 ± 0.83, 68.0 ± 3.46 mg/m3 for low, middle, and high exposure groups, respectively. The levels of lactate dehydrogenase, polymorphonuclear leukocytes, and cytokines (MIP-2, TGF-β1, IL-1β, TNF-α, and IL-6) in the bronchoalveolar lavage fluid increased in a concentration-dependent manner. Histopathological examination revealed acute inflammation with necrosis in the nasal cavity and inflammation around terminal bronchioles and alveolar ducts in the lungs after DGH inhalation. The LC50 of DGH in rats after exposure for 4 h was estimated to be >68 mg/m3. Results from the inhalation studies showed that DGH was more toxic in male rats than in female rats. Overall, DGH was found to be the most cytotoxic chemical among guanidine-based chemicals. Exposure to aerosols of DGH could induce harmful pulmonary effects on human health.

Novel Toxicology Program to Support the Development of Inhaled VentaProst

Currently, off-label continuous administration of inhaled epoprostenol is used to manage hemodynamics during mitral valve surgery. A toxicology program was developed to support the use of inhaled epoprostenol during mechanical ventilation as well as pre- and postsurgery via nasal prongs. To support use in patients using nasal prongs, a Good Laboratory Practice (GLP), 14-day rat, nose-only inhalation study was performed. No adverse findings were observed at ∼50× the dose rate received by patient during off-label use. To simulate up to 48 hours continuous aerosol exposure during mechanical ventilation, a GLP toxicology study was performed using anesthetized, intubated, mechanically ventilated dogs. Dogs inhaled epoprostenol at approximately 6× and 13× the dose rate reported in off-label human studies. This novel animal model required establishment of a dog intensive care unit providing sedation, multisystem support, partial parenteral nutrition, and management of the intubated mechanically ventilated dogs for the 48-hour duration of study. Aerosol was generated by a vibrating mesh nebulizer with novel methods required to determine dose and particle size in-vitro. Continuous pH 10.5 epoprostenol was anticipated to be associated with lung injury; however, no adverse findings were observed. As no toxicity at pH 10.5 was observed with a formulation that required refrigeration, a room temperature stable formulation at pH 12 was evaluated in the same ventilated dog model. Again, there were no adverse findings. In conclusion, current toxicology findings support the evaluation of inhaled epoprostenol at pH 12 in surgical patients with pulmonary hypertension for up to 48 hours continuous exposure.