Nanobodies to multiple spike variants and inhalation of nanobody-containing aerosols neutralize SARS-CoV-2 in cell culture and hamsters.

The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single-domain (VHH) antibodies, also known as nanobodies, that target the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein and neutralize the Wuhan strain of the virus. In this study, we present a new generation of anti-RBD nanobodies with superior properties. The primary representative of this group, Re32D03, neutralizes Alpha to Delta as well as Omicron BA.2.75; other members neutralize, in addition, Omicron BA.1, BA.2, BA.4/5, and XBB.1. Crystal structures of RBD-nanobody complexes reveal how ACE2-binding is blocked and also explain the nanobodies' tolerance to immune escape mutations. Through the cryo-EM structure of the Ma16B06-BA.1 Spike complex, we demonstrated how a single nanobody molecule can neutralize a trimeric spike. We also describe a method for large-scale production of these nanobodies in Pichia pastoris, and for formulating them into aerosols. Exposing hamsters to these aerosols, before or even 24 h after infection with SARS-CoV-2, significantly reduced virus load, weight loss and pathogenicity. These results show the potential of aerosolized nanobodies for prophylaxis and therapy of coronavirus infections.

Toxicokinetics of Nanoparticles Deposited in Lungs Using Occupational Exposure Scenarios.

Various synthetic powders with primary particle sizes at the nanoscale and a high commercial impact have been studied using Wistar rats. The test materials were metal oxides, i.e., TiO2, ZnO and amorphous silica, and carbon black (technical soot). Dosing schemes were in the regular ranges typically used in subacute rat studies to simulate occupational exposure scenarios (mg range). Nanoscaled particle agglomerates have the potential to disintegrate and translocate as individual nanoparticles to remote locations following deposition in the lungs. The toxicokinetic fate of metal oxides post-inhalation in lungs/organs was investigated (i) by chemical analysis of the retained particulate/dissolved matter and (ii) by visualization of particles in various remote organs using transmission electron microscopy (TEM). The three titanium dioxides (NM-103, NM-104, NM-105; JRC coding) showed a very slow dissolution in lung fluids. In contrast, the coated ZnO (NM-111) dissolved quickly and was eliminated from the body within approximately 1 day. The precipitated amorphous silica (NM-200) showed a partial dissolution. Chemical analysis in lungs (particulate and soluble TiO2) and in remote organs (liver and brain) showed a small solubility effect under physiological conditions. The translocation to remote organs was negligible. This confirms that for poorly soluble TiO2 particles there was no considerable translocation to the liver and brain. The chemical analysis of zinc demonstrated a very rapid dissolution of ZnO particles after deposition in the lungs. Statistically significant increases in Zn levels in the lungs were detectable only on day 1 post-exposure (NM-111). Overall, no relevant amounts of increased NM-111 in the ionic or particulate matter were detected in any body compartment. Amorphous silica (NM-200) particles were found in the cytoplasm of intraalveolar macrophages in the lung and the cytoplasm of macrophages in the lung associated lymph node. Interestingly, these particles were found in a few animals of all treatment groups (1, 2.5, and 5 mg/m3 NM-200) even after 91 days post-exposure. In all other organs of the NM-200 treated animals such as the nasal epithelium, trachea, larynx, liver, spleen, kidney, and mesenteric lymph node no particles were found at any time point investigated. Carbon black was tagged internally ("intrinsically") with a γ tracer (7beryllium; half-time: 53.3 days). Due to limited amounts, the test item (0.3 mg per rat lung) was intratracheally instilled into the lungs. This dose avoided a particle overload effect, meaning that the toxicokinetic fate of carbon black could be followed under the approximated physiological conditions of lung clearance. Analysis of the γ labeled carbon black confirmed conclusively that there was no evidence for the translocation of carbon black beyond the lung into the blood or other body compartments. Very small amounts were only detected in lung-associated lymph nodes (LALN). On day 20 post-treatment, upon necropsy, both carbon black samples were practically exclusively found in lungs (75.1% and 91.0%, respectively) and in very small amounts in the lung-associated lymph nodes (LALN), i.e., ~0.5%. In the other organs/tissues, the test item was not significantly detectable. Separation of leukocytes and cell-free supernatant of a bronchoalveolar lavagate by centrifugation revealed that carbon black was completely located in the cell sediment, indicating total engulfment by alveolar macrophages. In conclusion, in occupational settings the nanomaterials titanium dioxide, zinc oxide, amorphous silica, and carbon black acted as microscaled agglomerates, not as individual nanoparticles. They displayed no potential to translocate beyond the lung into the blood compartment. Besides lungs, very small particulate amounts were detected only in LALN. This finding is consistent with the behavior of microscaled poorly soluble particles. Overall, there was no evidence of translocation of the nanomaterials following pulmonary exposures.

Assessing the impact of exposome on the course of chronic obstructive pulmonary disease and cystc fibrosis: The REMEDIA European Project Approach.

Because of the direct interaction of lungs with the environment, respiratory diseases are among the leading causes of environment-related deaths in the world. Chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) are two highly debilitating diseases that are of particular interest in the context of environmental studies; they both are characterized by a similar progressive loss of lung function with small bronchi alterations, and a high phenotypic variability of unknown origin, which prevents a good therapeutic efficacy. In the last years, there has been an evolution in the apprehension of the study of diseases going from a restricted "one exposure, one disease" approach to a broader concept with other associating factors, the exposome. The overall objective of the REMEDIA project is to extend the understanding of the contribution of the exposome to COPD and CF diseases. To achieve our aim, we will (1) exploit data from existing cohorts and population registries to create a unified global database gathering phenotype and exposome information; (2) develop a flexible individual sensor device combining environmental and biomarker toolkits; (3) use a versatile atmospheric simulation chamber to simulate the health effects of complex exposomes; (4) use machine learning supervised analyses and causal inference models to identify relevant risk factors; and (5) develop econometric and cost-effectiveness models to assess the costs, performance, and cost-effectiveness of a selection of prevention strategies. The results will be used to develop guidelines to better predict disease risks and constitute the elements of the REMEDIA toolbox. The multidisciplinary approach carried out by the REMEDIA European project should represent a major breakthrough in reducing the morbidity and mortality associated with COPD and CF diseases.

Breath-Triggered Drug Release System for Preterm Neonates.

A major disadvantage of inhalation therapy with continuous drug delivery is the loss of medication during expiration. Developing a breath-triggered drug release system can highly decrease this loss. However, there is currently no breath-triggered drug release directly inside the patient interface (nasal prong) for preterm neonates available due to their high breathing frequency, short inspiration time and low tidal volume. Therefore, a nasal prong with an integrated valve releasing aerosol directly inside the patient interface increasing inhaled aerosol efficiency is desirable. We integrated a miniaturized aerosol valve into a nasal prong, controlled by a double-stroke cylinder. Breathing was simulated using a test lung for preterm neonates on CPAP respiratory support. The inhalation flow served as a trigger signal for the valve, releasing humidified surfactant. Particle detection was performed gravimetrically (filter) and optically (light extinction). The integrated miniaturized aerosol valve enabled breath-triggered drug release inside the patient interface with an aerosol valve response time of <25 ms. By breath-triggered release of the pharmaceutical aerosol as a bolus during inhalation, the inhaled aerosol efficiency was increased by a factor of >4 compared to non-triggered release. This novel nasal prong with integrated valve allows breath-triggered drug release directly inside the nasal prong with short response time.

Detection of Breathing Movements of Preterm Neonates by Recording Their Abdominal Movements with a Time-of-Flight Camera.

In order to deliver an aerosolized drug in a breath-triggered manner, the initiation of the patient's inspiration needs to be detected. The best-known systems monitoring breathing patterns are based on flow sensors. However, due to their large dead space volume, flow sensors are not advisable for monitoring the breathing of (preterm) neonates. Newly-developed respiratory sensors, especially when contact-based (invasive), can be tested on (preterm) neonates only with great effort due to clinical and ethical hurdles. Therefore, a physiological model is highly desirable to validate these sensors. For developing such a system, abdominal movement data of (preterm) neonates are required. We recorded time sequences of five preterm neonates' abdominal movements with a time-of-flight camera and successfully extracted various breathing patterns and respiratory parameters. Several characteristic breathing patterns, such as forced breathing, sighing, apnea and crying, were identified from the movement data. Respiratory parameters, such as duration of inspiration and expiration, as well as respiratory rate and breathing movement over time, were also extracted. This work demonstrated that respiratory parameters of preterm neonates can be determined without contact. Therefore, such a system can be used for breathing detection to provide a trigger signal for breath-triggered drug release systems. Furthermore, based on the recorded data, a physiological abdominal movement model of preterm neonates can now be developed.

Novel Test Bench for Inhaler Characterization Including Real-Time Determination of Output, Output Rate, and Liquid Water Content of Delivered Aerosols.

Background: Developing new (triggered) or improving existing inhaler systems for (preterm) neonates and adults requires test benches for the determination of aerosol output and aerosol output rate. Furthermore, real-time measurement of aerosol output and output rate is advantageous with respect to both development costs and development time, especially when using liquid or humidified dry aerosols. The current standard test procedures following ISO 27427, however, are time-consuming. Moreover, these procedures are not applicable to inhalers for preterm neonates, due to their high breathing frequency, low tidal volume, and the dead space in commercially available test benches. We are describing a novel test bench approach combining gravimetric and optical detection to facilitate real-time measurement of aerosol output, aerosol output rate, and aerosol liquid water content in inhalation systems for (preterm) neonates and adults. Methods: We integrated a laser-based optical measurement unit into test benches for inhalers for adults and preterm neonates, based on ISO 27427. Breathing was simulated by a sine pump for adults and by a test lung for preterm neonates on continuous positive airway pressure respiratory support. Dry or humidified aerosol was released by a continuous powder aerosolizer system. Simultaneous particle measurement by gravimetry (filter) and light extinction (laser system) was performed using the novel test benches. Results: We developed test benches for inhalers for (preterm) neonates and adults in accordance with ISO 27427, combining optical and gravimetric particle detection. Optical and gravimetric measurements conducted with these test benches were highly correlated, thus enabling real-time measurement of aerosol output and output rate. In addition, our test benches are suitable to determine the aerosol water content in situ directly at the patient interface. Conclusion: This novel test bench allows characterization of inhalation devices in real time and therefore will accelerate optimization and development cycles. Conformity with ISO 27427 allows its use in various applications.

Assessment of the Power Required for Optimal Use of Current Inhalation Devices.

Background: Inhalation of medications is the cornerstone in the treatment of patients with lung diseases. A variety of inhalation devices exists and each device has specific requirements to achieve optimum inhalation of the drug. The goal of this study was to establish a clear overview on performance requirements of standard inhalation devices that should be met by the patient's breathing power and to develop a new method to measure the individual performance data. Materials and Methods: An optimum and still acceptable required breathing power (P in watts) was calculated for each device with the aid of individual device flow rates (determined by a literature search) and the flow resistances (by measuring the pressure drop over the different inhalation devices). For the in vivo part of the study, peak inspiratory flow and peak inspiratory pressure drop were measured in 21 adult patients with asthma or chronic obstructive pulmonary disease and healthy volunteers and the peak inspiratory power (PIPO in watts) was calculated. Results: Nearly no power is needed to achieve optimum results when using pressurized metered dose inhalers. For dry powder inhalers, the required power depends on the specific inhalation device. Conclusions: Inhalation devices impose differing demands on the inspiratory breathing power of patients. To ensure adequate use of the different devices, a cheap and simple assessment of patients' PIPO may be one option.

Supraglottic Atomization of Surfactant in Spontaneously Breathing Lambs Receiving Continuous Positive Airway Pressure.

OBJECTIVES: To determine the short-term tolerance, efficacy, and lung deposition of supraglottic atomized surfactant in spontaneously breathing lambs receiving continuous positive airway pressure. DESIGN: Prospective, randomized animal study. SETTING: Animal research laboratory. SUBJECTS: Twenty-two preterm lambs on continuous positive airway pressure (132 ± 1 d gestational age). INTERVENTIONS: Animals receiving continuous positive airway pressure via binasal prongs at 8 cm H2O were randomized to receive atomized surfactant at approximately 60-minute of life (atom; n = 15) or not (control; n = 7). The atom group received 200 mg/kg of poractant alfa (Curosurf; Chiesi Farmaceutici SpA, Parma, Italy) over 45 minutes via a novel atomizer located in the upper pharynx that synchronized surfactant delivery with the inspiratory phase. MEASUREMENTS AND MAIN RESULTS: Arterial blood gas, regional distribution of tidal ventilation (electrical impedance tomography), and carotid blood flow were recorded every 15 minutes until 90 minutes after stabilizing on continuous positive airway pressure. Gas exchange, respiratory rate, and hemodynamic variables, including carotid blood flow, remained stable during surfactant treatment. There was a significant improvement in arterial alveolar ratio after surfactant delivery in the atom group (p < 0.05; Sidak posttests), while there was no difference in PaCO2. Electrical impedance tomography data showed a more uniform pattern of ventilation in the atom group. In the atom group, the median (interquartile range) deposition of surfactant in the lung was 32% (22-43%) of the delivered dose, with an even distribution between the right and the left lungs. CONCLUSIONS: In our model of spontaneously breathing lambs receiving CPAP, supraglottic atomization of Curosurf via a novel device was safe, improved oxygenation and ventilation homogeneity compared with CPAP only, and provided a relatively large lung deposition suggesting clinical utility.

Effects from a 90-day inhalation toxicity study with cerium oxide and barium sulfate nanoparticles in rats.

BACKGROUND: Nanomaterials like cerium oxide and barium sulfate are frequently processed in industrial and consumer products and exposure of humans and other organisms is likely. Generally less information is given on health effects and toxicity, especially regarding long-term exposure to low nanoparticle doses. Since inhalation is still the major route of uptake the present study focused on pulmonary effects of CeO2NM-212 (0.1, 0.3, 1.0, 3.0 mg/m3) and BaSO4NM-220 nanoparticles (50.0 mg/m3) in a 90-day exposure setup. To define particle-related effects and potential mechanisms of action, observations in histopathology, bronchoalveolar lavage and immunohistochemistry were linked to pulmonary deposition and clearance rates. This further allows evaluation of potential overload related effects. RESULTS: Lung burden values increased with increasing nanoparticle dose levels and ongoing exposure. At higher doses, cerium clearance was impaired, suggesting lung overload. Barium elimination was extremely rapid and without any signs of overload. Bronchoalveolar lavage fluid analysis and histopathology revealed lung tissue inflammation with increasing severity and post-exposure persistency for CeO2. Also, marker levels for genotoxicity and cell proliferation were significantly increased. BaSO4 showed less inflammation or persistency of effects and particularly affected the nasal cavity. CONCLUSION: CeO2 nanoparticles penetrate the alveolar space and affect the respiratory tract after inhalation mainly in terms of inflammation. Effects at low dose levels and post-exposure persistency suggest potential long-term effects and a notable relevance for human health. The generated data might be useful to improve nanoparticle risk assessment and threshold value generation. Mechanistic investigations at conditions of non-overload and absent inflammation should be further investigated in future studies.

Biological effects of carbon black nanoparticles are changed by surface coating with polycyclic aromatic hydrocarbons.

BACKGROUND: Carbon black nanoparticles (CBNP) are mainly composed of carbon, with a small amount of other elements (including hydrogen and oxygen). The toxicity of CBNP has been attributed to their large surface area, and through adsorbing intrinsically toxic substances, such as polycyclic aromatic hydrocarbons (PAH). It is not clear whether a PAH surface coating changes the toxicological properties of CBNP by influencing their physicochemical properties, through the specific toxicity of the surface-bound PAH, or by a combination of both. METHODS: Printex®90 (P90) was used as CBNP; the comparators were P90 coated with either benzo[a]pyrene (BaP) or 9-nitroanthracene (9NA), and soot from acetylene combustion that bears various PAHs on the surface (AS-PAH). Oxidative stress and IL-8/KC mRNA expression were determined in A549 and bronchial epithelial cells (16HBE14o-, Calu-3), mouse intrapulmonary airways and tracheal epithelial cells. Overall toxicity was tested in a rat inhalation study according to Organization for Economic Co-operation and Development (OECD) criteria. Effects on cytochrome monooxygenase (Cyp) mRNA expression, cell viability and mucociliary clearance were determined in acute exposure models using explanted murine trachea. RESULTS: All particles had similar primary particle size, shape, hydrodynamic diameter and ζ-potential. All PAH-containing particles had a comparable specific surface area that was approximately one third that of P90. AS-PAH contained a mixture of PAH with expected higher toxicity than BaP or 9NA. PAH-coating reduced some effects of P90 such as IL-8 mRNA expression and oxidative stress in A549 cells, granulocyte influx in the in vivo OECD experiment, and agglomeration of P90 and mucus release in the murine trachea ex vivo. Furthermore, P90-BaP decreased particle transport speed compared to P90 at 10 µg/ml. In contrast, PAH-coating induced IL-8 mRNA expression in bronchial epithelial cell lines, and Cyp mRNA expression and apoptosis in tracheal epithelial cells. In line with the higher toxicity compared to P90-BaP and P90-9NA, AS-PAH had the strongest biological effects both ex vivo and in vivo. CONCLUSIONS: Our results demonstrate that the biological effect of CBNP is determined by a combination of specific surface area and surface-bound PAH, and varies in different target cells.

Pulmonary toxicity of printer toner following inhalation and intratracheal instillation.

The pulmonary effects of a finished toner were evaluated in intratracheal instillation and inhalation studies, using toners with external additives (titanium dioxide nanoparticles and amorphous silica nanoparticles). Rats received an intratracheal dose of 1 mg or 2 mg of toner and were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months. The toner induced pulmonary inflammation, as evidenced by a transient neutrophil response in the low-dose groups and persistent neutrophil infiltration in the high-dose groups. There were increased concentrations of heme oxygenase-1 (HO-1) as a marker of oxidative stress in the bronchoalveolar lavage fluid (BALF) and the lung. In a 90-day inhalation study, rats were exposed to well-dispersed toner (mean of MMAD: 3.76 µm). The three mass concentrations of toner were 1, 4 and 16 mg/m(3) for 13 weeks, and the rats were sacrificed at 6 days and 91 days after the end of the exposure period. The low and medium concentrations did not induce neutrophil infiltration in the lung of statistical significance, but the high concentration did, and, in addition, upon histopathological examination not only showed findings of inflammation but also of fibrosis in the lung. Taken together, the results of our studies suggest that toners with external additives lead to pulmonary inflammation and fibrosis at lung burdens suggest beyond the overload. The changes observed in the pulmonary responses in this inhalation study indicate that the high concentration (16 mg/m(3)) is an LOAEL and that the medium concentration (4 mg/m(3)) is an NOAEL.

Toxicity profile of the GATA-3-specific DNAzyme hgd40 after inhalation exposure.

DNAzymes are single-stranded catalytic DNA molecules that bind and cleave specific sequences in a target mRNA molecule. Their potential as novel therapeutic agents has been demonstrated in a variety of disease models. However, no studies have yet addressed their toxicology and safety pharmacology profiles in detail. Here we describe a detailed toxicological analysis of inhaled hgd40, a GATA-3-specific DNAzyme designed for the treatment of allergic bronchial asthma. Subacute toxicity, immunotoxicity, and respiratory, cardiovascular, and CNS safety pharmacology were analyzed in rodents and non-rodents, and genotoxicity was assessed in human peripheral blood. Overall, hgd40 was very well tolerated when delivered by aerosol inhalation or slow intravenous infusion. Only marginal reversible histopathological changes were observed in the lungs of rats receiving the highest dose of inhaled hgd40. The changes consisted of slight mononuclear cell infiltration and alveolar histiocytosis, and moderate hyperplasia of bronchus-associated lymphoid tissue. No local or systemic adverse effects were observed in dogs. No compound-related respiratory, cardiovascular, or CNS adverse events were observed. The only relevant immunological findings were very slight dose-dependent changes in interleukin-10 and interferon-γ levels in bronchoalveolar lavage fluid. Taken together, these results support direct delivery of a DNAzyme via inhalation for the treatment of respiratory disease.

Assessment of pulmonary function and serum substance levels in newborn and juvenile rats.

In the context of pharmaceutical development today, studies for pediatric drug approval are requested more and more often by the regulatory authorities. The developing lung represents a potential target in juvenile toxicity studies. Due to physiological differences in prenatal and postnatal development between humans and standard animal models, experimental methods have to be modified to assess pulmonary function, and basic data on respiratory parameters need to be provided. Daily nose-only inhalation exposure from postnatal days 4 to 21 using a model substance (verapamil HCl) and plethysmographic measurements between postnatal days 2 and 50 were performed noninvasively in conscious juvenile Wistar (WU) rats. The methods proved to be feasible and did not interfere with normal growth and development of the animals. Both techniques therefore permit new insights to support human neonatal risk assessment and therefore these animal models are suitable for regulatory studies.

Genotoxicity testing of sulfur dioxide (SO2) in a mouse bone marrow micronucleus test complemented with hematological endpoints.

Sulfur dioxide (SO2) is a non-flammable, non-explosive, colorless gas. It is a ubiquitous environmental pollutant and an important chemical intermediate in several industrial processes. The toxicological properties of SO2, including its genotoxic potential, have been studied extensively. The majority of the available in vitro data indicate a lack of genotoxicity of SO2, while for sulfite salts some positive results have been reported. However, recent in vivo studies, using Kunming albino mice, have pointed to in vivo clastogenicity of SO2. To re-evaluate these positive findings, a bone-marrow micronucleus test according to OECD Guideline No. 474 was performed. NMRI mice (m/f) were exposed by inhalation via whole-body exposure to 0 (clean air), 2.7, 8, 27, or 80mg/m3 (0, 1, 3, 10, or 30ppm) SO2 for 4h/day on 7 consecutive days. Animals were sacrificed 24h after start of the last exposure, and blood samples (for complementing hematology) and bone marrow smears (for analysis of micronuclei) were prepared. Under the conditions used, exposure to SO2 caused no acute toxicity, mortality, or reduction in body weight. Compared with the clean-air controls, hematological parameters such as hematocrit, hemoglobin, erythrocyte/platelet/total leukocyte counts, differential white blood cell counts, and indicators of blood formation (reticulocyte counts, ratio of polychromatic to normochromatic erythrocytes in the bone marrow) remained unchanged by SO2 treatment. Unlike the previously reported studies on micronucleus formation, SO2 did not induce micronuclei in polychromatic erythrocytes of the bone marrow, whereas the positive control cyclophosphamide (60mg/kg body weight) was quite effective in this respect. Interestingly, SO2 treatment significantly enhanced malondialdehyde levels in erythrocyte lysates (TBARS method), indicating SO2-mediated oxidative stress, but also demonstrating systemic availability of the inhaled SO2. In conclusion, the present study could not reproduce the genotoxicity findings of the previously reported studies. SO2 is thus considered non-genotoxic in polychromatic erythrocytes in the bone marrow of NMRI mice under the conditions and in the concentrations used.

Low cytotoxicity of solid lipid nanoparticles in in vitro and ex vivo lung models.

The aim of this study was to investigate the potential cytotoxicity of solid lipid nanoparticles (SLN) for human lung as a suitable drug delivery system (DDS). Therefore we used a human alveolar epithelial cell line (A549) and murine precision-cut lung slices (PCLS) to estimate the tolerable doses of these particles for lung cells. A549 cells (in vitro) and precision-cut lung slices (ex vivo) were incubated with SLN20 (20% phospholipids in the lipid matrix of the particles) and SLN50 (50% phospholipids in the lipid matrix of the particles) in increasing concentrations. The cytotoxic effects of SLN were evaluated in vitro by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Vitality of lung slices was controlled by staining with calcein AM/ethidium homodimer 1 using confocal laser scanning microscopy and followed by quantitative image analysis with IMARIS software. A549 cell line revealed a middle effective concentration (EC(50)) for MTT assay for SLN20 of 4080 microg/ml and for SLN50 of 1520 microg/ml. The cytotoxicity in terms of LDH release showed comparable EC(50) values of 3431 microg/ml and 1253 microg/ml for SLN20 and SLN50, respectively. However, in PCLS we determined only SLN50 cytotoxic values with a concentration of 1500 microg/ml. The lung slices seem to be a more sensitive test system. SLN20 showed lower toxic values in all test systems. Therefore we conclude that SLN20 could be used as a suitable DDS for the lung, from a toxicological point of view.

Effects of di-n-butylamine on the respiratory system of Wistar (WU) rats after subchronic inhalation.

Aim of the study was to investigate the potential toxic effects of di-n-butylamine (DBA), a known skin and eye irritating compound, on the respiratory tract after inhalation exposure for up to 91 days in male and female rats [Crl:(WI)WU BR]. To check whether and to what degree the no-observed-(adverse)-effect level (NO(A)EL) decreases with increasing study duration, serial sacrifices were performed after 3 and 28 days, respectively. Based on two dose range-finding studies, the concentrations for this study were determined with 0 (clean air), 50, 150, and 450 mg/m(3). Animals were exposed for 3 days (6 h/day) 28, and 91 days (5 days/wk, 6 h/day), respectively, and immediately sacrificed thereafter. The results show clear irritating effects only in the upper part of the respiratory tract, that is, the nasal cavities. While after 3 and 28 days effects were found only in the high-dose group, slight adaptive effects, expressed as mucous (goblet) cell hyperplasia, could be diagnosed in the medium- and low-dose groups after 91 days of exposure. Pathological changes were most prominent after 3 days of exposure. In the lung, only marginal effects could be observed (increased relative lung weight only in females of the high concentration after 28 days, slight, not statistically significant histopathological effects in the high concentration after 3 days, no effects on parameters of bronchoalveolar lavage fluid), while no effects were found in the remaining groups.