90-day nose-only inhalation toxicity study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Sprague-Dawley rats.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. Repeated dose inhalation toxicity data on NNK, particularly relevant to cigarette smoking, however, is surprisingly limited. Hence, there is a lack of direct information available on the carcinogenic and potential non-carcinogenic effects of NNK via inhalational route exposure. In the present study, the subchronic inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 23 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.2, 0.8, 3.2, or 7.8 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.0066, 0.026, 0.11, or 0.26 mg/L air) for 1 h/day for 90 consecutive days. Toxicity was evaluated by assessing body weights; food consumption; clinical pathology; histopathology; organ weights; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); tissue levels of the DNA adduct O6-methylguanine; blood and bone marrow micronucleus (MN) frequency; and bone marrow DNA strand breaks (comet assay). The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic lesions in the nose. Although the genotoxic biomarker O6-methylguanine was detected, genotoxicity from NNK exposure was negative in the MN and comet assays. The Lowest-Observed-Adverse-Effect-Level (LOAEL) was 0.8 mg/kg BW/day or 0.026 mg/L air of NNK for 1 h/day for both sexes. The No-Observed-Adverse-Effect-Level (NOAEL) was 0.2 mg/kg BW/day or 0.0066 mg/L air of NNK for 1 h/day for both sexes. The results of this study provide new information relevant to assessing the human exposure hazard of NNK.

Advancement of the Infant Air-Jet Dry Powder Inhaler (DPI): Evaluation of Different Positive-Pressure Air Sources and Flow Rates.

PURPOSE: In order to improve the delivery of dry powder aerosol formulations to the lungs of infants, this study implemented an infant air-jet platform and explored the effects of different air sources, flow rates, and pulmonary mechanics on aerosolization performance and aerosol delivery through a preterm nose-throat (NT) in vitro model. METHODS: The infant air-jet platform was actuated with a positive-pressure air source that delivered the aerosol and provided a full inhalation breath. Three different air sources were developed to provide highly controllable positive-pressure air actuations (using actuation volumes of ~10 mL for the preterm model). While providing different flow waveform shapes, the three air sources were calibrated to produce the same flow rate magnitude (Q90: 90th percentile of flow rate). Multiple air-jet DPI designs were coupled with the air sources and evaluated with a model spray-dried excipient enhanced growth formulation. RESULTS: Compared to other designs, the D1-Single air-jet DPI provided improved performance with low variability across all three air sources. With the tested D1-Single air-jet and Timer air source, reducing the flow rate from 4 to 1.7 L/min marginally decreased the aerosol size and significantly increased the lung delivery efficiency above 50% of the loaded dose. These results were not impacted by the presence of downstream pulmonary mechanics (resistance and compliance model). CONCLUSIONS: The selected design was capable of providing an estimated >50% lung delivery efficiency of a model spray-dried formulation and was not influenced by the air source, thereby enabling greater flexibility for platform deployment in different environments.

Formaldehyde-induced hematopoietic stem and progenitor cell toxicity in mouse lung and nose.

Formaldehyde (FA), an economically important and ubiquitous chemical, has been classified as a human carcinogen and myeloid leukemogen. However, the underlying mechanisms of leukemogenesis remain unclear. Unlike many classical leukemogens that damage hematopoietic stem/progenitor cells (HSC/HPC) directly in the bone marrow, FA-as the smallest, most reactive aldehyde-is thought to be incapable of reaching the bone marrow through inhalation exposure. A recent breakthrough study discovered that mouse lung contains functional HSC/HPC that can produce blood cells and travel bi-directionally between the lung and bone marrow, while another early study reported the presence of HSC/HPC in rat nose. Based on these findings, we hypothesized that FA inhalation could induce toxicity in HSC/HPC present in mouse lung and/or nose rather than in the bone marrow. To test this hypothesis, we adapted a commercially available protocol for culturing burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte, macrophage (CFU-GM) colonies from bone marrow and spleen to also enable culture of these colonies from mouse lung and nose, a novel application of this assay. We reported that in vivo exposure to FA at 3 mg/m3 or ex vivo exposure up to 400 µM FA decreased the formation of both colony types from mouse lung and nose as well as from bone marrow and spleen. These findings, to the best of our knowledge, are the first empirically to show that FA exposure can damage mouse pulmonary and olfactory HSC/HPC and provide potential biological plausibility for the induction of leukemia at the sites of entry rather than the bone marrow.

Nonsurgical Rhinoplasty Using Injectable Fillers: A Safety Review of 2488 Procedures.

Background: Injectable fillers are used worldwide to improve the appearance of the nose by nonsurgical methods. The procedure is not without risks, as blindness and skin necrosis have been reported as a consequence of filler injections in the nose. Objective: To determine an overall adverse event (AE) rate for the nonsurgical rhinoplasty (NSR) procedure and to assess whether previous surgical rhinoplasty increases the odds of an AE. Methods: A retrospective chart review of 2275 patients and 2488 NSR procedures for a 10-year period from a single physician injector was conducted. Results: The overall procedural AE rate was 7.6%, with five cases (0.20%) considered serious (ischemia and necrosis). Previous surgical rhinoplasty patients had a greater AE rate (10.8%) than those patients without previous surgery (7.4%), with a significant odds ratio of 1.51 (95% confidence interval: 1.03-2.18); p = 0.032. Injecting the tip and sidewall of the nose had the highest AE rates for both categories of patients. Conclusions: NSR is a relatively safe procedure with the majority of AEs common injection site reactions. Patients with previous surgical rhinoplasty demonstrated significantly increased odds of an AE potentially due to surgical changes in anatomy.

Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia.

Background: Cilia are actin based cellular protrusions conserved from algae to complex multicellular organisms like Homo sapiens. Respiratory motile cilia line epithelial cells of the tracheobronchial tree, beat in a synchronous, metachronal wave, moving inhaled pollutants and pathogens cephalad. Their role in both congenital disorders like primary ciliary dyskinesia (PCD) to acquired disorders like chronic obstructive pulmonary disease (COPD) continues to evolve. In this current body of work we outline a protocol optimized to reciliate human nasal epithelial cells and mouse tracheal cells in vitro. Using this protocol, we knocked down known cilia genes, as well as use a small molecule inhibitor of Notch, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl Ester (DAPT), to assess the effect of these on ciliogenesis in order to show the validity of our protocol. Methods: Tracheas were harvested from wild-type, adult C57B6 mice, pronase digested and sloughed off epithelial cells grown to confluence in stationary culture on rat-tail collagen coated wells. Upon reaching confluence, collagen was digested and cells placed suspension culture protocol to reciliate the cells. Using this suspension culture protocol, we employed siRNA gene knockdown to assay gene functions required for airway ciliogenesis. Knock down of Dynein axonemal heavy chain 5 (Dnah5), a ciliary structural protein, was confirmed using immunostaining. Mouse tracheal cells were treated in suspension with varying doses of DAPT, an inhibitor of Notch, with the purpose of evaluating its effect and dose response on ciliogenesis. The optimum dose was then used on reciliating human nasal epithelial cells. Results: siRNA knockdown of Foxj1 prevented ciliation, consistent with its role as a master regulator of motile cilia. Knockdown of Dnai1 and Dnah5 resulted in immotile cilia, and Cand1 knockdown, a centrosome protein known to regulate centrosome amplification, inhibited airway ciliogenesis. Dnah5 knockdown was confirmed with significantly decreased immunostaining of cilia for this protein. Inhibiting Notch signaling by inhibiting gamma secretase with DAPT enhanced the percentage of ciliation, and resulted in longer cilia that beat with higher frequency in both mouse and human airway epithelia. Conclusions: Modifying existing reciliation protocols to suit both human nasal epithelial and mouse tracheal tissue, we have shown that knockdown of known cilia-related genes have the expected effects. Additionally, we have demonstrated the optimal dosage for significantly improving reciliation of airway epithelia using DAPT. Given that cilia length and function are significantly compromised in COPD, these findings open up interesting avenues for further exploration.

Characterization and Treatment of SARS-CoV-2 in Nasal and Bronchial Human Airway Epithelia.

In the current COVID-19 pandemic context, proposing and validating effective treatments represents a major challenge. However, the scarcity of biologically relevant pre-clinical models of SARS-CoV-2 infection imposes a significant barrier for scientific and medical progress, including the rapid transition of potentially effective treatments to the clinical setting. We use reconstituted human airway epithelia to isolate and then characterize the viral infection kinetics, tissue-level remodeling of the cellular ultrastructure, and transcriptional early immune signatures induced by SARS-CoV-2 in a physiologically relevant model. Our results emphasize distinctive transcriptional immune signatures between nasal and bronchial HAE, both in terms of kinetics and intensity, hence suggesting putative intrinsic differences in the early response to SARS-CoV-2 infection. Most important, we provide evidence in human-derived tissues on the antiviral efficacy of remdesivir monotherapy and explore the potential of the remdesivir-diltiazem combination as an option worthy of further investigation to respond to the still-unmet COVID-19 medical need.

Nose-to-brain drug delivery system with ligand/cell-penetrating peptide-modified polymeric nano-micelles for intracerebral gliomas.

We previously developed a nose-to-brain delivery system using poly(ethylene glycol)-polycaprolactone block polymeric micelles modified by a cell-penetrating peptide, Tat (PEG-PCL-Tat). This system showed excellent delivery of the anti-cancer drug camptothecin to the brain and improved therapeutic efficacy in a brain tumor model. However, improvements are necessary to selectively deliver drugs to tumor sites once they enter the brain, and avoid toxic side effects to normal brain tissue. In this study, to develop tumor-selective novel polymeric micelles, mixed micelles consisting of Tat-conjugated polymer micelles and stearoyl-modified bombesin (Bom/PEG-PCL-Tat) were designed. The GRPR selectivity, cellular uptake, and cytotoxicity in C6 glioma cells as well as the intracerebral drug distribution and therapeutic efficacy of Bom/PEG-PCL-Tat mixed micelles after intranasal administration in C6 glioma orthotropic grafted rats were evaluated. Selective cellular uptake and marked cytotoxic effects against GRPR-expressing C6 glioma cells were observed, as well as C6 tumor tissue-specific accumulation in vivo. Rats treated with camptothecin subsequent to a brain tumor graft survived longer when the drug was delivered by Bom/PEG-PCL-Tat mixed micelles than by PEG-PCL-Tat micelles.

Nasal timosaponin BII dually sensitive in situ hydrogels for the prevention of Alzheimer's disease induced by lipopolysaccharides.

Alzheimer's disease (AD) is a common and severe brain disease with a high mortality among the elders, but no highly efficient medications are currently available. For example, timosaponin BII, an efficient anti-AD agent, has low oral bioavailability. Here, timosaponin BII was formulated in a temperature/ion-sensitive in situ hydrogel (ISG) that was well transformed into gels in the nasal environment. Timosaponin BII protected the PC12 cells injured by lipopolysaccharides (LPS) by decreasing TNF-α and IL-1ß and stabilizing F-actin. Timosaponin BII ISGs were intranasally administered to the mice every day for 38 days. On Day 36, LPS was injected to the mice to establish an AD model. Morris water maze experiments showed that the number of the animals that were able to cross the platform returned to normal and the total distance over which the animals moved in the open field also increased, which demonstrated that the spatial memory and spontaneous behavior were improved after treatment compared to the model. Moreover, an AD improver, inducible nitric oxide synthase (iNOS) in the brain, was reduced after treatment. High brain targeting effect of timosaponin BII ISGs was confirmed by in vivo fluorescence imaging. The nasal timosaponin BII dually sensitive ISGs can serve as a promising medication for local prevention of AD.

Nitinol actuated soft structures towards transnasal drug delivery: a pilot cadaver study.

Sudden hearing loss can be treated noninvasively by administering drugs to the middle ear (≈1 ml) via the eustachian tube. The nasopharyngeal cavity requires high dexterity manipulation as it is restricted by the nasal vestibule, and precise drug delivery through the small cavity can allow previously unreachable areas to be reconsidered for localized delivery. Nitinol has shape memory capabilities and can be used for distal actuation accessed from small lumen and a tortuous path. The drug delivery device (DDD) is a soft and needle-sized (2 mm) and comprises of Nitinol, ribbon spring, and a drug delivery tube. By controlling the input voltage to the Nitinol, bending of the device at different angles could be achieved, and the ribbon spring works antagonistically to the Nitinol to revert to the initial position once deactivated. The actuation of the device and its corresponding bending are calculated in vitro and found to have a bending angle ranging between 36.2 and 66.8° for applied voltages of 1.2-2.0 V, with surface temperature of 45.6-154 °C. The DDD is able to actuate 200 cycles with ≈91-76% retention of bending performance, with a temperature increase of ≈8.5-9% when 1.2-2.0 V is applied. Addition of soft insulating material shows ≈34-62% reduction in the surface temperature in the first cycle and ≈37-59% over 200 cycles when actuated at 1.2-2.0 V. The active steering and navigation capabilities of the DDD are demonstrated in simulated environments (based on the eustachian tube dimensions of adult and infant). Preclinical testing in human cadavers is demonstrated and suggests the developed DDD controlled by varying the input voltages for bending, and mechanically varied drug delivery may be a feasible option for localized drug delivery in eustachian tube. Graphical abstract.

ORKAMBI-Mediated Rescue of Mucociliary Clearance in Cystic Fibrosis Primary Respiratory Cultures Is Enhanced by Arginine Uptake, Arginase Inhibition, and Promotion of Nitric Oxide Signaling to the Cystic Fibrosis Transmembrane Conductance Regulator Channel.

ORKAMBI, a combination of the corrector, lumacaftor, and the potentiator, ivacaftor, partially rescues the defective processing and anion channel activity conferred by the major cystic fibrosis-causing mutation, F508del, in in vitro studies. Clinically, the improvement in lung function after ORKAMBI treatment is modest and variable, prompting the search for complementary interventions. As our previous work identified a positive effect of arginine-dependent nitric oxide signaling on residual F508del-Cftr function in murine intestinal epithelium, we were prompted to determine whether strategies aimed at increasing arginine would enhance F508del-cystic fibrosis transmembrane conductance regulator (CFTR) channel activity in patient-derived airway epithelia. Now, we show that the addition of arginine together with inhibition of intracellular arginase activity increased cytosolic nitric oxide and enhanced the rescue effect of ORKAMBI on F508del-CFTR-mediated chloride conductance at the cell surface of patient-derived bronchial and nasal epithelial cultures. Interestingly, arginine addition plus arginase inhibition also enhanced ORKAMBI-mediated increases in ciliary beat frequency and mucociliary movement, two in vitro CF phenotypes that are downstream of the channel defect. This work suggests that strategies to manipulate the arginine-nitric oxide pathway in combination with CFTR modulators may lead to improved clinical outcomes. SIGNIFICANCE STATEMENT: These proof-of-concept studies highlight the potential to boost the response to cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators, lumacaftor and ivacaftor, in patient-derived airway tissues expressing the major CF-causing mutant, F508del-CFTR, by enhancing other regulatory pathways. In this case, we observed enhancement of pharmacologically rescued F508del-CFTR by arginine-dependent, nitric oxide signaling through inhibition of endogenous arginase activity.

Reversal of Post-filler Vision Loss and Skin Ischaemia with High-Dose Pulsed Hyaluronidase Injections.

Nose filler injections are very popular in many Asian countries to improve nose shape and projection. However, due to the vascular supply of nose from the ophthalmic artery and its communication with branches of the facial artery in this region, there could be a possibility of ophthalmic complications in case of an accidental intra-arterial injection of filler material. This may cause devastating complications of partial or complete vision loss with or without associated cutaneous ischaemic changes. We present a case report of a patient who developed features of vascular involvement after two ml of HA filler injection in the nasal dorsum, tip and columella. The patient initially developed tell-tale signs of impending skin necrosis in the nasal and forehead skin followed by ptosis, severe pain and progressive vision loss in the right eye until a point where the patient could only perceive light. The patient was managed with multiple doses of hyaluronidase in the involved skin and two doses of retrobulbar injection for vision loss. Significant recovery in the skin and ophthalmic components occurred within 20 days of filler injection. This case demonstrates that recovery of the ischaemic ophthalmic and cutaneous changes secondary to probable intra-arterial injection could be accomplished using combined retrobulbar and periorbital intracutaneous injections of high-dose pulsed hyaluronidase. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The Effect of Sedating Agents on Drug-Induced Sleep Endoscopy Findings.

OBJECTIVE: Drug-induced sleep endoscopy (DISE) has gained interest for upper airway evaluation in patients with snoring and obstructive sleep apnea (OSA), and different drugs have been used to induce sedation. Nevertheless, all drugs have presented specific advantages and disadvantages with differential effects on respiratory physiology. This study evaluated and compared the effects of midazolam, propofol and dexmedetomidine on DISE findings, O2 nadir, and bispectral index (BIS) in the same sample of patients. STUDY DESIGN: Case series prospective study. METHODS: Consecutive patients who elected to undergo surgery for OSA treatment and were intolerant to conservative therapies underwent DISE with propofol, dexmedetomidine, and midazolam between July 2015 and July 2016. RESULTS: Fifty-two patients were analyzed, and 43 (82.7%) were men. Agreement among drugs for both degree and patterns of obstruction was excellent at all sites (velum, oropharynx, and epiglottis) except for the tongue base. Dexmedetomidine had the least complete collapse sites and highest O2 nadir and was the only drug for which apnea severity and obstruction levels (upper, lower, or combined) were correlated. The variability among drug treatments for the BIS index was considerable, and propofol had the lowest variability and average value. CONCLUSION: Drug selection had a relevant influence in DISE findings. Compared with dexmedetomidine, midazolam and propofol presented higher incidence of tongue base collapse, lower O2 levels, and lower BIS index values. Propofol resulted in an O2 nadir that most resembled that observed during polysomnography. The BIS index variability differed among drugs, and its use was considered relevant for sedation orientation. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:506-513, 2019.

Evaluation of Staphylococcus aureus eradication therapy in orthopaedic surgery.

PURPOSE: Despite WHO recommendations, there is currently no national screening and eradication policy for the detection of methicillin-sensitive Staphylococcus aureus (MSSA) in the UK prior to elective orthopaedic surgery. This study aimed to evaluate the effectiveness of current standard methicillin-resistant S. aureus (MRSA) eradication therapies in the context of S. aureus (both MRSA and MSSA) decolonization in an elective orthopaedic population. METHODOLOGY: A total of 100 patients awaiting joint replacement surgery who were positive for S. aureus on PCR nasal screening underwent the current standard MRSA pre-operative decolonization regimen for 5 days. Prior to commencement of the eradication therapy, swabs of the anterior nares, throat and perineum were taken for culture. Further culture swabs were taken at 48-96 h following treatment, at hospital admission for surgery and at hospital discharge. Following the completion of treatment, patients were asked to provide feedback on their experience using Likert rating scales. The primary outcome of this study was S. aureus clearance 48-96 h following eradication treatment.Results/Key Findings. Clearance of S. aureus 48-96 h following treatment was 94 % anterior nares, 66 % throat and 88 % groin. Mean completion with nasal mupirocin was 98 %. There was no statistically significant recolonization effect between the end of the eradication treatment period and the day of surgery (P>0.05) at a median time of 10 days. CONCLUSION: Current MRSA decolonisation regimens are well tolerated and effective for MSSA decolonization for the anterior nares and groin. The decolonization effect is preserved for at least 10 days following treatment.

Control of canine idiopathic nasal hyperkeratosis with a natural skin restorative balm: a randomized double-blind placebo-controlled study.

BACKGROUND: Nasal hyperkeratosis may cause discomfort in dogs by predisposing them to fissures and secondary bacterial infection. Approaches to treatment have been described anecdotally; the effectiveness of such therapies remains unproven. HYPOTHESIS/OBJECTIVES: To investigate the efficacy of a balm containing essential oils and essential fatty acids in dogs with idiopathic nasal hyperkeratosis. ANIMALS: Client-owned dogs with noncomplicated nasal hyperkeratosis. METHODS: The study was conducted as a randomized, double-blind, placebo-controlled clinical trial with parallel group design and two month follow-up period. Dogs received daily topical application of a commercial balm product (group DBB) or placebo (aqueous gelling agent with preservatives, group PB). The main outcome variables were lichenification, dryness, suppleness and extent of lesions. Subjective owner satisfaction index score was a secondary variable. Evaluation was performed on days (D)0, 30 and 60. Response to treatment was assessed as the change from baseline to each examination day for each criterion. RESULTS: Forty eight dogs, principally French (26 of 48) and English (seven of 48) bulldogs, were included and 39 completed the study. No major adverse events were reported. On D60, changes from baseline for lichenification, lesion extent, suppleness and total score were -31.2%, -18.3%, -72.8% and -36.8% in group DBB (23 dogs) and -11.9%, 2.3%, -42.1% and -14% in group PB (16 dogs), respectively. The total score was significantly improved on D60 in group DBB compared to PB (Wilcoxon-Mann-Whitney U-test, P = 0.0016). CONCLUSIONS AND CLINICAL IMPORTANCE: The balm proved safe and helpful in managing canine idiopathic noncomplicated nasal hyperkeratosis.

Lipopolysaccharide induces autophagy by targeting the AMPK-mTOR pathway in Human Nasal Epithelial Cells.

Chronic rhinosinusitis (CRS) is a well-known disease encountered in the department of otorhinolaryngology, yet little is known about its pathogenesis. Autophagy, a lysosome-dependent degradation process, has been reported to be involved in the process of many chronic inflammatory diseases. Here we tried to evaluate the function of autophagy in CRS as well as explore the related mechanisms. We first stained light chain 3B (LC3B) with immunohistochemistry in uncinate tissues (UT) from patients with and without CRS and found that its expression was up-regulated in CRS patients. Then, Human Nasal Epithelial Cells (HNEpC) were treated with lipopolysaccharide (LPS), one of the most common pathogenic elements in CRS, and we found that autophagy was induced in a dose- and time-dependent manner. This is supported by a rise in the expression of light chain 3B-II (LC3B-II), accumulation of GFP-LC3 vesicles, as well as decreased p62 expression. Furthermore, we found that LPS promoted AMPK phosphorylation and inactived mTOR, while AMPK inhibition by compound C significantly attenuated LPS-induced autophagy. Besides, treatment of HNEpC with LPS increased the amount of Toll-like receptor 4 (TLR4) while inhibiting TLR4 by Polymyxin B (PMB) declined autophagy caused by LPS. Taken together, our study first demonstrated that LPS caused autophagy in HNEpC, and this process was AMPK-mTOR dependent. These data suggested the relationship between LPS and autophagy in the pathogenesis of CRS.

Comparative pulmonary toxicity of inhaled metalworking fluids in rats and mice.

Metalworking fluids (MWFs) are complex formulations designed for effective lubricating, cooling, and cleaning tools and parts during machining operations. Adverse health effects such as respiratory symptoms, dermatitis, and cancer have been reported in workers exposed to MWFs. Several constituents of MWFs have been implicated in toxicity and have been removed from the formulations over the years. However, animal studies with newer MWFs demonstrate that they continue to pose a health risk. This investigation examines the hypothesis that unrecognized health hazards exist in currently marketed MWF formulations that are presumed to be safe based on hazard assessments of individual ingredients. In vivo 13-week inhalation studies were designed to characterize and compare the potential toxicity of four MWFs: Trim VX, Cimstar 3800, Trim SC210, and Syntilo 1023. Male and female Wistar Han rats or Fischer 344N/Tac rats and B6C3F1/N mice were exposed to MWFs via whole-body inhalation at concentrations of 0, 25, 50, 100, 200, or 400 mg/m3 for 13 weeks, after which, survival, body and organ weights, hematology and clinical chemistry, histopathology, and genotoxicity were assessed following exposure. Although high concentrations were used, survival was not affected and toxicity was primarily within the respiratory tract of male and female rats and mice. Minor variances in toxicity were attributed to differences among species as well as in the chemical components of each MWF. Pulmonary fibrosis was present only in rats and mice exposed to Trim VX. These data confirm that newer MWFs have the potential to cause respiratory toxicity in workers who are repeatedly exposed via inhalation.