Analysis of the interrelationship of the pulmonary irritation and elicitation thresholds in rats sensitized with 1,6-hexamethylene diisocyanate (HDI).

This paper summarizes a range of experimental data central for developing a science-based approach for hazard identification of monomeric and polymeric aliphatic 1,6-hexamethylene diisocyanate (HDI). The dose-response curve of HDI-induced pulmonary responses in naïve or dermally sensitized rats after one or several inhalation priming exposures was examined in the Brown Norway (BN) rat asthma model. Emphasis was directed to demonstrate the need and the difficulty in selecting an appropriate pulmonary dose when much of the inhaled chemically reactive vapor may concentration dependently be retained in the upper airways of obligate nose-breathing rats. The course taken acknowledges the experimental challenges in identifying an elicitation threshold for HDI-monomer near or above the saturated vapor concentration or in the presence of a HDI-polymer aerosol. The inhalation threshold dose on elicitation was determined based on a fixed concentration (C) × variable exposure duration (t) protocol for improving inhalation dosimetry of the lower airways. Neutrophilic granulocytes (PMN) in bronchoalveolar lavage (BAL) fluid in equally inhalation primed naïve and dermally sensitized rats were used to define the inhalation elicitation threshold C × t. Sensitized rats elaborated markedly increased PMN challenged sensitized rats relative to equally challenged naïve rats at 5625 mg HDI/m(3) × min (75 mg/m(3) for 75 min). PMN were essentially indistinguishable at 900 mg HDI/m(3) × min. By applying adjustment factors accounting for both inter-species differences in inhalation dosimetry and intra-species susceptibility, the workplace human-equivalent threshold C × t was estimated to be in the range of the current ACGIH TLV® of HDI. Thus, this rat "asthma" model was suitable to demonstrate elicitation thresholds for HDI-vapor after one or several inhalation priming exposures and seems to be suitable to derive occupational exposure values (OELs) for diisocyanates in general.

Effect of PEEP on phosgene-induced lung edema: pilot study on dogs using protective ventilation strategies.

Various therapeutic regimes have been proposed for treatment of phosgene-induced acute lung injury (P-ALI). Most of these treatments rely on late-stage supportive measures to maintain the oxygenation of the lung. This exploratory proof-of-concept study on Beagle dogs focused on protective positive end-expiratory pressure (PEEP) ventilation, initiated early at the yet asymptomatic stage after phosgene exposure. Conscious, spontaneously breathing dogs were head-only exposed to a potentially lethal inhalation dose of phosgene (870 ppm × min). Shortly after exposure, the dogs were anesthetized, intubated and then subjected to mechanical ventilation (PEEP; tidal volume (VT)=10-12 mL/kg body weight, 40 breaths/min) at 0, 4, or 12 cm H2O over a post-exposure period of 8h (one dog per setting). For reference, one additional dog received the same dose of phosgene without anesthesia and mechanical ventilation. Time-course changes of hematocrit, leukocytes, and thrombocytes were determined in peripheral blood. At necropsy, changes lung weights, bronchoalveolar lavage, and histology were used to assess the efficacy of treatment. The most salient outcome in the non-ventilated dog was a time-related hemoconcentration and leukocytosis and autopsy findings suggestive of pulmonary congestion and edema. The pulmonary epithelium of the major airways was generally intact; however, in their lumen inflammatory cells, cellular debris and mucus were present. Relative to the dog receiving no intervention, the lung edema was markedly alleviated by PEEP at both 4 and 12 cm H2O but not at 0 cm H2O PEEP. In summary, the time-dependent progression into a life-threatening pulmonary edema can effectively be suppressed by protective, low-pressure PEEP when implemented early enough after exposure to phosgene. However, due to the exploratory nature of this study, the findings may suggest an association between PEEP and protection from pulmonary edema. However, definite conclusions and recommendations cannot be made yet based upon the small sample size and the limited variables examined.

Lung burdens and kinetics of multi-walled carbon nanotubes (Baytubes) are highly dependent on the disaggregation of aerosolized MWCNT.

Previous repeated inhalation exposure studies on rats with multi-walled carbon nanotubes (MWCNT, Baytubes®) suggested that their pulmonary toxicity was predominated by the morphology and density of the aggregated structure. Evidence of any disintegration of these structures in the lung did not exist. The objective of this study was to study as to which extent the formulation of pristine MWCNT as wet-dispersion changes the morphology of assemblage structures in the presence of disintegrated sub-structures. The focus was on the comparative inhalation dosimetry and kinetics of dry- and wet-dispersed Baytubes to better understand the cause of putative differences in pulmonary toxicity originating from pristine and rigorously formulated MWCNT. Rats were nose-only exposed to dry-dispersed and wet-dispersed Baytubes for 6-h at 25-30 mg/m(3). Aerodynamic particle size measurements demonstrate substantial differences in the particle size of dry- (MMAD 2.6 µm) and wet-dispersed (MMAD 0.8 µm) MWCNT. Time-course changes of MWCNT retained in the lung were examined during a post-exposure period of 3 months. Lung burdens were analytically determined in digested lungs using the EC/OC total carbon method. Dosimetry was complemented by light and transmission electron microscopy (TEM) of MWCNT retained in alveolar macrophages (AM). As a result, the initially deposited pulmonary dose of MWCNT was three times higher following wet-dispersed MWCNT at essentially similar inhalation chamber concentrations. The elimination half-time of dry- and wet-dispersed MWCNT was 87 and 46 d, respectively. TEM provided evidence that wet-dispersed MWCNT were inhaled as disintegrated structures with distribution-patterns within the cytoplasm of AMs that differed appreciably from those of dry-dust exposed animals. In summary, this study shows that specialized technical processes to formulate MWCNT may have dramatic consequences on their pulmonary fate and associated toxicity. Such properties can only be revealed by the comparison of pulmonary toxicity with pulmonary (micro-)dosimetry and kinetics.

Derivation of occupational exposure levels (OELs) of low-toxicity isometric biopersistent particles: How can the kinetic lung overload paradigm be used for improved inhalation toxicity study design and OEL-derivation?

BACKGROUND: Convincing evidence suggests that poorly soluble low-toxicity particles (PSP) exert two unifying major modes of action (MoA), in which one appears to be deposition-related acute, whilst the other is retention-related and occurs with particle accumulation in the lung and associated persistent inflammation. Either MoA has its study- and cumulative dose-specific adverse outcome and metric. Modeling procedures were applied to better understand as to which extent protocol variables may predetermine any specific outcome of study. The results from modeled and empirical studies served as basis to derive OELs from modeled and empirically confirmed directions. RESULTS: This analysis demonstrates that the accumulated retained particle displacement volume was the most prominent unifying denominator linking the pulmonary retained volumetric particle dose to inflammogenicity and toxicity. However, conventional study design may not always be appropriate to unequivocally discriminate the surface thermodynamics-related acute adversity from the cumulative retention volume-related chronic adversity. Thus, in the absence of kinetically designed studies, it may become increasingly challenging to differentiate substance-specific deposition-related acute effects from the more chronic retained cumulative dose-related effects. CONCLUSION: It is concluded that the degree of dissolution of particles in the pulmonary environment seems to be generally underestimated with the possibility to attribute to toxicity due to decreased particle size and associated changes in thermodynamics and kinetics of dissolution. Accordingly, acute deposition-related outcomes become an important secondary variable within the pulmonary microenvironment. In turn, lung-overload related chronic adversities seem to be better described by the particle volume metric. This analysis supports the concept that 'self-validating', hypothesis-based computational study design delivers the highest level of unifying information required for the risk characterization of PSP. In demonstrating that the PSP under consideration is truly following the generic PSP-paradigm, this higher level of mechanistic information reduces the potential uncertainty involved with OEL derivation.

Phosgene- and chlorine-induced acute lung injury in rats: comparison of cardiopulmonary function and biomarkers in exhaled breath.

This study compares changes in cardiopulmonary function, selected endpoints in exhaled breath, blood, and bronchoalveolar lavage fluid (BAL) following a single, high-level 30-min nose-only exposure of rats to chlorine and phosgene gas. The time-course of lung injury was systematically examined up to 1-day post-exposure with the objective to identify early diagnostic biomarkers suitable to guide countermeasures to accidental exposures. Chlorine, due to its water solubility, penetrates the lung concentration-dependently whereas the poorly water-soluble phosgene reaches the alveolar region without any appreciable extent of airway injury. Cardiopulmonary endpoints were continually recorded by telemetry and barometric plethysmography for 20h. At several time points blood was collected to evaluate evidence of hemoconcentration, changes in hemostasis, and osteopontin. One day post-exposure, protein, osteopontin, and cytodifferentials were determined in BAL. Nitric oxide (eNO) and eCO2 were non-invasively examined in exhaled breath 5 and 24h post-exposure. Chlorine-exposed rats elaborated a reflexively-induced decreased respiratory rate and bradycardia whereas phosgene-exposed rats developed minimal changes in lung function but a similar magnitude of bradycardia. Despite similar initial changes in cardiac function, the phosgene-exposed rats showed different time-course changes of hemoconcentration and lung weights as compared to chlorine-exposed rats. eNO/eCO2 ratios were most affected in chlorine-exposed rats in the absence of any marked time-related changes. This outcome appears to demonstrate that nociceptive reflexes with changes in cardiopulmonary function resemble typical patterns of mixed airway-alveolar irritation in chlorine-exposed rats and alveolar irritation in phosgene-exposed rats. The degree and time-course of pulmonary injury was reflected best by eNO/eCO2 ratios, hemoconcentration, and protein in BAL. Increased fibrin in blood occurred only in chlorine-exposed rats 1-day post-exposure. Hence, the analysis of NO and CO2 in exhaled breath, including endpoints in blood mirroring changes in the peripheral to pulmonary fluid distribution, seem to be sensitive diagnostic endpoints readily available for early prognostic assessment of severity of injury and efficacy of any chosen countermeasure.

Corticosteroids found ineffective for phosgene-induced acute lung injury in rats.

Various therapeutic regimes have been proposed with limited success for treatment of phosgene-induced acute lung injury (P-ALI). Corticoids were shown to be efficacious against chlorine-induced lung injury but there is still controversy whether this applies also to P-ALI. This study investigates whether different regimen of curatively administered budesonide (BUD, 10 mg/kg bw, i.p. bid; 100 mg/m(3)×30 min, nose-only inhalation), mometasone (MOM, 3 mg/kg bw, i.p. bid) and dexamethasone (DEX, 10, 30 mg/kg bw, i.p. bid), show efficacy to alleviate P-ALI. Efficacy of drugs was judged by nitric oxide (eNO) and carbon dioxide (eCO2) in exhaled air and whether these non-invasive biomarkers are suitable to assess the degree of airway injury (chlorine) relative to alveolar injury (phosgene). P-ALI related analyses included lung function (enhanced pause, Penh), morbidity, increased lung weights, and protein in bronchial alveolar lavage fluid (BALF) one day postexposure. One of the pathophysiological hallmarks of P-ALI was indicated by increased Penh lasting for approximately 20 h postexposure. Following the administration of BUD, this increase could be suppressed; however, without significant improvement in survival and lung edema (increased lung weights and BALF-protein). Collectively, protocols shown to be efficacious for chlorine (Chen et al., 2013) were ineffective and even increased adversity in the P-ALI model. This outcome warrants further study to seek for early biomarkers suitable to differentiate chlorine- and phosgene-induced acute lung injury at yet asymptomatic stage. The patterns of eNO and eCO2 observed following exposure to chlorine and phosgene may be suitable to guide the specialized clinical interventions required for each type of ALI.

Repeated inhalation exposure of rats to an anionic high molecular weight polymer aerosol: application of prediction models to better understand pulmonary effects and modes of action.

Opposed to the wealth of information available for kinetic lung overload-related effects of poorly-soluble, low-toxicity particles (PSP), only limited information is available on biodegradable high molecular weight (HMW) organic polymers (molecular weight >20,000 Da). It is hypothesized that such types of polymers may exert a somewhat similar volume displacement-related mode of action in alveolar macrophages as PSP; however, with a differing biokinetics of the material retained in the lung. This polyurethane polymer was examined in single and 2-/13-week repeated exposure rat inhalation bioassays. The design of studies was adapted to that commonly applied for PSP. Rats were nose-only exposed for 6h/day for the respective study duration, followed by 1-, 2- and 4-week postexposure periods in the single, 2- and 13-week studies, respectively. While the findings in bronchoalveolar lavage (BAL) and histopathology were consistent with those typical of PSP, they appear to be superimposed by pulmonary phospholipidosis and a much faster reversibility of pulmonary inflammation. Kinetic modeling designed to estimate the accumulated lung burden of biopersistent PSP was also suitable to simulate the overload-dependent outcomes of this biodegradable polymer as long as the faster than normal elimination kinetics was observed and an additional 'void space volume' was added to adjust for the phagocytosed additional fraction of pulmonary phospholipids. The changes observed following repeated inhalation exposure appear to be consistent with a retention-related etiopathology (kinetic overload). In summary, this study did not reveal evidence of any polymer-specific pulmonary irritation or parenchymal injury. Taking all findings into account, 7 mg polymer/m(3) (exposure 6h/day, 5-days/week on 13 consecutive weeks) constitutes the point of departure for lower respiratory tract findings that represent a transitional state from effects attributable to an overload-dependent pulmonary inflammation and phospholipidosis. In regard to extrapulmonary toxicity, no effects were found up to the maximum concentration of 107 mg/m(3) examined.

Development of a respiratory sensitization/elicitation protocol of toluene diisocyanate (TDI) in Brown Norway rats to derive an elicitation-based occupational exposure level.

Toluene diisocyanate (TDI), a known human asthmagen, was investigated in skin-sensitized Brown Norway rats for its concentration×time (C×t)-response relationship on elicitation-based endpoints. The major goal of study was to determine the elicitation inhalation threshold dose in sensitized, re-challenged Brown Norway rats, including the associated variables affecting the dosimetry of inhaled TDI-vapor in rats and as to how these differences can be translated to humans. Attempts were made to duplicate at least some traits of human asthma by using skin-sensitized rats which were subjected to single or multiple inhalation-escalation challenge exposures. Two types of dose-escalation protocols were used to determine the elicitation-threshold C×t; one used a variable C (Cvar) and constant t (tconst), the other a constant C (Cconst) and variable t (tvar). The selection of the "minimal irritant" C was based an ancillary pre-studies. Neutrophilic granulocytes (PMNs) in bronchoalveolar lavage fluid (BAL) were considered as the endpoint of choice to integrate the allergic pulmonary inflammation. These were supplemented by physiological measurements characterizing nocturnal asthma-like responses and increased nitric oxide in exhaled breath (eNO). The Cconst×tvar regimen yielded the most conclusive dose-response relationship as long C was high enough to overcome the scrubbing capacity of the upper airways. Based on ancillary pre-studies in naïve rats, the related human-equivalent respiratory tract irritant threshold concentration was estimated to be 0.09ppm. The respective 8-h time-adjusted asthma-related human-equivalent threshold C×t-product (dose), in 'asthmatic' rats, was estimated to be 0.003ppm. Both thresholds are in agreement of the current ACGIH TLV(®) of TDI and published human evidence. In summary, the findings from this animal model suggest that TDI-induced respiratory allergy is likely to be contingent on two interlinked, sequentially occurring mechanisms: first, dermal sensitizing encounters high enough to cause systemic sensitization. Second, when followed by inhalation exposure(s) high enough to initiate and amplify an allergic airway inflammation, then a progression into asthma may occur. This bioassay requires an in-depth knowledge on respiratory tract dosimetry and irritation of the involved test substance to clearly understand the dosimetry causing C- and/or C×t-dependent respiratory tract irritation and eventually asthma.

The metrics of MWCNT-induced pulmonary inflammation are dependent on the selected testing regimen.

Convincing evidence suggests that high-surface-activity nano-materials, such as MWCNT, exert two modes of action (MoA), in which one appears to be related to surface activity/area and occurs concurrent with deposition, and the other is related to cumulative lung burden. Pulmonary inflammation induced by the latter mode appears to be dependent on cumulative volumetric lung burden and on whether the accumulated particle displacement volume within the pool of alveolar macrophages is above or below the kinetic lung overload threshold. However, the relative importance and effect of each MoA are still controversial. In addition, the test protocol variables, which may predetermine the leading MoA, have not yet received increased attention. This study compares the respective outcome of previously published repeated-exposure inhalation studies with MWCNT (Nanocyl and Baytube) in rats. Modeling procedures were performed to compare post hoc the equivalence of empirical and modeled outcomes, including critical protocol variables. This comparison provided compelling evidence that the accumulated retained particle displacement volume was the most prominent unifying denominator linking the pulmonary retained volumetric particle dose to inflammogenicity and toxicity. However, conventional study designs may not always be appropriate to unequivocally dissociate the surface area/activity-related acute adversity from the cumulative retention volume-related adversity. Thus, in the absence of adequately designed studies, it may become increasingly challenging to differentiate substance-specific deposition-related acute effects from the more chronic retained cumulative dose-related effects. In summary, this analysis of existing data supports the conclusion that both the deposition and retention-related effects need to be judiciously dissociated to better understand the MoA of heightened concern. This exercise supports the conclusion that hypothesis-based computational study design delivers the highest degree of scientifically important information and may further reduce the number of experimental animals in repeated-exposure inhalation studies with low-toxicity, biopersistent, poorly soluble granular particles.

Single high-dose dexamethasone and sodium salicylate failed to attenuate phosgene-induced acute lung injury in rats.

Life-threatening acute lung injury potentially occurs following high-level accidental exposures to phosgene gas. This situation was mirrored in rats exposed nose-only at 900-1000 mg phosgene/m(3)min. At this exposure level, previous studies on rats demonstrated sustained reflexively induced cardiopulmonary dysfunction and evidence of vascular fluid redistribution. These findings challenge the currently applied treatment strategies to mitigate the presumed non-cardiogenic lung edema by steroidal or non-steroidal anti-inflammatory drugs. This study investigates whether high doses of curatively administered dexamethasone (DX; 100 mg/kg bw, ip) and sodium salicylate (SS; 200 mg/kg bw, ip), alone or in combination, show efficacy to mitigate the phosgene-induced lung edema. Exhaled nitric oxide (eNO), animal morbidity and mortality, and increased lung weights one day postexposure served as endpoints of lung injury and drug efficacy. When applying this dosing regimen, SS showed minimal (if any) efficacy while DX, alone or in combination with SS, substantially aggravated the emerging lung edema (lung weights) with 40% mortality. The degree of acute lung injury (ALI) was mirrored by increased eNO. Its direct relationship to ALI-severity was evidenced by decreased eNO following NO-synthetase inhibitor administration (aminoguanidine-aerosol) and associated mitigation of ALI. All non-treated phosgene-exposed as well as treated but non-phosgene-exposed rats survived. This experimental evidence suggests that high-dose corticoid treatments may aggravate the pulmonary toxicity of phosgene. Similarly, this outcome supports the supposition that non-inflammatory, cardiogenic and/or neurogenic factors play a role in this type of acute lung injury.

Rat models of acute lung injury: exhaled nitric oxide as a sensitive, noninvasive real-time biomarker of prognosis and efficacy of intervention.

Exhaled nitric oxide (eNO) has received increased attention in clinical settings because this technique is easy to use with instant readout. However, despite the simplicity of eNO in humans, this endpoint has not frequently been used in experimental rat models of septic (endotoxemia) or irritant acute lung injury (ALI). The focus of this study is to adapt this method to rats for studying ALI-related lung disease and whether it can serve as instant, non-invasive biomarker of ALI to study lung toxicity and pharmacological efficacy. Measurements were made in a dynamic flow of sheath air containing the exhaled breath from spontaneously breathing, conscious rats placed into a head-out volume plethysmograph. The quantity of eNO in exhaled breath was adjusted (normalized) to the physiological variables (breathing frequency, concentration of exhaled carbon dioxide) mirroring pulmonary perfusion and ventilation. eNO was examined on the instillation/inhalation exposure day and first post-exposure day in Wistar rats intratracheally instilled with lipopolysaccharide (LPS) or single inhalation exposure to chlorine or phosgene gas. eNO was also examined in a Brown Norway rat asthma model using the asthmagen toluene diisocyanate (TDI). The diagnostic sensitivity of adjusted eNO was superior to the measurements not accounting for the normalization of physiological variables. In all bioassays - whether septic, airway or alveolar irritant or allergic, the adjusted eNO was significantly increased when compared to the concurrent control. The maximum increase of the adjusted eNO occurred following exposure to the airway irritant chlorine. The specificity of adjustment was experimentally verified by decreased eNO following inhalation dosing of the non-selective nitric oxide synthase inhibitor amoniguanidine. In summary, the diagnostic sensitivity of eNO can readily be applied to spontaneously breathing, conscious rats without any intervention or anesthesia. Measurements are definitely improved by accounting for the disease-related changes in exhaled CO2 and breathing frequency. Accordingly, adjusted eNO appears to be a promising methodological improvement for utilizing eNO in inhalation toxicology and pharmacological disease models with fewer animals.

Acute inhalation toxicity of ammonia: revisiting the importance of RD50 and LCT01/50 relationships for setting emergency response guideline values.

This study examined the acute median lethal concentration (LC(50)) and the non-lethal threshold concentration (LC(01)) of ammonia in male and female Wistar rats nose-only exposed at exposure durations of either 1 or 4 h. Additional attributes characterizing the acute toxicity of inhaled ammonia were determined during a post-exposure period of 2 weeks. The objective of this study is to further refine the methodology applied to derive Emergency Response Planning Guideline (ERPG) values on potent sensory irritants in a controlled rat bioassay. In the more susceptible male rats the 1- and 4-h LC(50) (LC(01)) were 12,303 (10,067) and 4923 (4028) mg/m(3), respectively. At sublethal exposure levels the ventilation of rats was about one third of normal breathing. This change in ventilation and inhalation dosimetry was adjusted for Cxt-dependent lethal endpoints whereas sensory irritation-related phenomena were C-dependently adjusted. In summary, the outcome of this study shows that C- and C × t-dependent causes of toxicity need to be appreciated when extrapolating across species with species-specific inhalation dosimetry. It also appears to be indispensable that each exposure metric must be disentangled when translating C × t-dependent lethality and reflexively-induced, sensation-based C-dependent point of departures. For one hour exposure periods, these PODs were derived to be 1500 and 500 ppm, respectively.

Lipopolysaccharide-induced acute lung injury in rats: comparative assessment of intratracheal instillation and aerosol inhalation.

Acute lung injury (ALI) has many possible etiopathologies and is characterized by acute diffuse lung damage with poor prognosis. Lipopolysaccharide (LPS) is widely used as septic model of ALI in pharmacological research. This study compares intratracheal bolus instillation (IT) with dose-adjusted aerosol inhalation (IH) of LPS in Wistar rats using both non-invasive and terminal endpoints. The former comprised exhaled nitric oxide (NOE) and 'enhanced pause' (Penh) both measured in spontaneous breathing conscious rats. Terminal endpoints included lung weights, LDH, protein, total cell counts, and cytodifferentiation in bronchoalveolar lavage (BAL). Measurements were made 1, 3, 7, and 14 days after IT instillation (5 mg LPS/kg body weight) or 6-hour directed-flow nose-only inhalation exposure to respirable LPS-aerosol at 100 mg/m(3) (thoracic dose: 2.6 mgLPS/kg body weight). Controls received saline (IT) or air only (IH). LDH and protein were significantly different from the control in the LPS-IH group (days 1 and 3) with a somewhat inconclusive outcome in the LPS-IT group due to the effects occurring in the control. Total cell counts were equally elevated with similar time-course changes in the LPS-IT and -IH groups. Polymorphonuclear neutrophils (PMNs) were indistinguishable amongst LPS-dosed rats. Again, IT-dosed control rats displayed markedly higher background levels than those dosed by inhalation. Similarly NOE was significantly elevated on post-LPS day 1 as was Penh. In summary, the LPS-aerosol dose delivered by nose-only exposure over 6 h was equally potent as the 2-times higher LPS-IT bolus dose on post-LPS day 1 with somewhat faster recovery thereafter. The climax and discriminatory power of the non-invasive endpoints matched those determined terminally. This supports the conclusion that the pharmacological efficacy and side effects of inhalation pharmaceuticals designed to mitigate ALI can better be identified by LPS-aerosol than by LPS-IT. Non-invasive time-course measurements may deliver apt information both on the efficacious dose as well as the dosing intervals required to maintain the targeted efficacy using a minimum of experimental animals. The outcome of this comparative study supports the conclusion that the inhalation route produces a more uniform type of injury at lower, more meaningful dosages. When designing studies for screening of effective drugs this mode of delivery appears to better approximate the human condition with less dosimetric uncertainty, less experimental variability and better characterization of what was actually delivered to the entire respiratory tract.

Subchronic inhalation toxicity of iron oxide (magnetite, Fe(3) O(4) ) in rats: pulmonary toxicity is determined by the particle kinetics typical of poorly soluble particles.

Wistar rats were nose-only exposed to pigment-sized iron oxide dust (Fe(3) O(4) , magnetite) in a subchronic 13-week inhalation study according to the OECD testing guidelines TG#413 and GD#39. A 4 week pilot study with a 6 month post exposure period served as basis for validating the kinetic modeling approaches utilized to design the subchronic study. Kinetic analyses made during this post exposure period demonstrated that a diminution in particle clearance and lung inflammation occurred at cumulative exposure levels exceeding the lung overload threshold. Animals were exposed 6 h per day, five days per week for 13 consecutive weeks at actual concentrations of 0, 4.7, 16.6 and 52.1 mg m(-3) (mass median aerodynamic diameter ≈1.3 µm, geometric standard deviation = 2). The exposure to iron oxide dust was tolerated without mortality, consistent changes in body weights, food and water consumption or systemic toxicity. While general clinical pathology and urinalysis were unobtrusive, hematology revealed changes of unclear toxicological significance (minimally increased differential neutrophil counts in peripheral blood). Elevations of neutrophils in bronchoalveolar lavage (BAL) appeared to be the most sensitive endpoint of study. Histopathology demonstrated responses to particle deposition in the upper respiratory tract (goblet cell hyper- and/or metaplasia, intraepithelial eosinophilic globules in the nasal passages) and the lower respiratory tract (inflammatory changes in the bronchiolo-alveolar region). Consistent changes suggestive of pulmonary inflammation were evidenced by BAL, histopathology, increased lung and lung-associated-lymph node (LALN) weights at 16.6 and 52.1 mg m(-3) . Increased septal collagenous fibers were observed at 52.1 mg m(-3) . Particle translocation into LALN occurred at exposure levels causing pulmonary inflammation. In summary, the retention kinetics iron oxide reflected that of poorly soluble particles. The empirical no-observed-adverse-effect level (NOAEL) and the lower bound 95% confidence limit on the benchmark concentration (BMCL) obtained by benchmark analysis was 4.7 and 4.4 mg m(-3) , respectively, and supports an OEL (time-adjusted chronic occupational exposure level) of 2 mg m(-3) (alveolar fraction).

Siderite (FeCO3) and magnetite (Fe3O4) overload-dependent pulmonary toxicity is determined by the poorly soluble particle not the iron content.

The two poorly soluble iron containing solid aerosols of siderite (FeCO3) and magnetite (Fe3O4) were compared in a 4-week inhalation study on rats at similar particle mass concentrations of approximately 30 or 100 mg/m³. The particle size distributions were essentially identical (MMAD ≈1.4 µm). The iron-based concentrations were 12 or 38 and 22 or 66 mg Fe/m³ for FeCO3 and Fe3O4, respectively. Modeled and empirically determined iron lung burdens were compared with endpoints suggestive of pulmonary inflammation by determinations in bronchoalveolar lavage (BAL) and oxidative stress in lung tissue during a postexposure period of 3 months. The objective of study was to identify the most germane exposure metrics, that are the concentration of elemental iron (mg Fe/m³), total particle mass (mg PM/m³) or particle volume (µl PM/m³) and their associations with the effects observed. From this analysis it was apparent that the intensity of pulmonary inflammation was clearly dependent on the concentration of particle-mass or -volume and not of iron. Despite its lower iron content, the exposure to FeCO3 caused a more pronounced and sustained inflammation as compared to Fe3O4. Similarly, borderline evidence of increased oxidative stress and inflammation occurred especially following exposure to FeCO3 at moderate lung overload levels. The in situ analysis of 8-oxoguanine in epithelial cells of alveolar and bronchiolar regions supports the conclusion that both FeCO3 and Fe3O4 particles are effectively endocytosed by macrophages as opposed to epithelial cells. Evidence of intracellular or nuclear sources of redox-active iron did not exist. In summary, this mechanistic study supports previous conclusions, namely that the repeated inhalation exposure of rats to highly respirable pigment-type iron oxides cause nonspecific pulmonary inflammation which shows a clear dependence on the particle volume-dependent lung overload rather than any increased dissolution and/or bioavailability of redox-active iron.

Inhaled nitric oxide aggravates phosgene model of acute lung injury.

The principal acute mode of action of inhaled phosgene gas is related to an increase alveolar fluid exudation under pathologic conditions. This paper considers some aspects in modeling phosgene-induced acute lung injury (ALI) in an acute rat bioassay and whether edema formation can be modulated by inhaled nitric oxide (iNO). Protein analysis in bronchoalveolar lavage (BAL) fluid is amongst the most sensitive method to quantify the phosgene-induced non-cardiogenic, pulmonary high-permeability edema following acute inhalation exposure. Maximum concentrations in BAL-protein occur within one day postexposure, typically within a latency period up to about 15 h as a consequence of an increasingly exhausted lymphatic drainage. An almost similar sensitivity was given by the functional endpoint 'enhanced pause (Penh)' when measured by non-invasive whole-body barometric plethysmography over a time period of 20 h. The magnitude of edema formation follows a concentration x time (C¹xt) relationship, although animal model-specific deviations may occur at very short exposure durations (1-20 min) due to a rodent-specific, reflexively induced transient decreased ventilation. This has to be accounted for when simulating accidental exposure scenarios to study the mechanisms involved in pharmacological modulation of fluid transport in this type of ALI. Therefore, a special focus has to be given to the dosimetry of inhaled phosgene, otherwise any change in effect magnitude, as a result of under-dosing of phosgene, may be misconceived as promising therapy. This study demonstrates that accidental exposures can be modeled best in rats by exposure durations of at least 20-30 min. Lung function measurements (Penh) show that pathophysiological effects appear to occur concomitant with the exposure to phosgene; however, its full clinical manifestation requires a gross imbalance of pulmonary fluid clearance. When applying this concept, post-phosgene exposure iNO at 1.5 ppm × 6 h or 15 pm × 20 h led to an aggravation of edema formation while L-NAME, a non-selective inhibitor of nitric oxide synthase, led to attenuation. Ethyl pyruvate, given either prophylactically or therapeutically, was ineffective.

Interrelating the acute and chronic mode of action of inhaled methylenediphenyl diisocyanate (MDI) in rats assisted by computational toxicology.

Polymeric methylenediphenyl diisocyanate (MDI) is a high production volume chemical intermediate consisting of monomeric 4,4'-MDI, its 2,2'- and 2,4'-isomers, and higher oligomeric homologues. The toxicity of pMDI has systematically been investigated in previous regulatory and mechanistic studies. One cornerstone of toxicological risk assessment is to understand the critical Mode of Action (MoA) of inhaled MDI aerosol. This paper compares the no-observed-adverse effect levels (NOAELs) in rats from two published whole-body exposure chronic inhalation bioassays with the lung irritation-based point of departures (PODs) from acute and subacute nose-only inhalation studies. Acute irritation was related to elevated concentrations of protein in bronchoalveolar lavage fluid (short-term studies), whilst the chronic events were characterized by histopathology. In the chronic bioassay the exposure duration was either 6 or 18h/day while in all other studies a 6h/day regimens were applied. The major objective of this paper is to analyze the interrelationship of acute pulmonary irritation and the acute-on-chronic manifestations of pulmonary disease following recurrent chronic inhalation exposure. This included considerations on the most critical metrics of exposure with regard to the acute concentration×exposure duration per day (C×T(day)) and the chronic cumulative dose metrics. In summary, this analysis supports the conclusion that the C×T(day) relative to the acute pulmonary irritation threshold is more decisive for the chronic outcome than the concentration per se or the time-adjusted cumulative dose. For MDI aerosols, the acute threshold C×T(day) was remarkably close to the NOAELs of the chronic inhalation studies, independent on their differing exposure mode and regimens. This evidence is supportive of a simple, direct MoA at the site of initial deposition of aerosol. Accordingly, for chemicals reactive to the endogenous nucleophilic agents contained in the lining fluid of the lung, one unifying essential prerequisite for pulmonary injury appears to be a C×T(day) that exhausts the homeostatic pool of MDI-scavenging agents. In the case that threshold is exceeded, the secondary compensatory chronic response may then cause additional superimposed types of chronic pathologies.

Acute nose-only inhalation exposure of rats to di- and triphosgene relative to phosgene.

Groups of young adult Wistar rats were acutely exposed to trichloromethyl chloroformate (diphosgene) and bis(trichloromethyl) carbonate (triphosgene) vapor atmospheres using a directed-flow nose-only mode of exposure. The exposure duration used was 240 min. The median lethal concentration (LC50) of diphosgene and triphosgene was 13.9 and 41.5 mg/m3, respectively. Based on the molar exposure concentrations, the LC50s of phosgene (previously published), diphosgene, and triphosgene were 0.07, 0.07, and 0.14 mmol/m3, respectively. Although the principal toxic mode of action of the volatile diphosgene was similar to phosgene gas, the vapor phase of triphosgene appeared to be different to that of phosgene and diphosgene based on a more persistent occurrence of signs of respiratory distress and a biphasic onset of mortality. While all substances caused mortality within 1 day postexposure, triphosgene induced a second phase of mortality 11?14 days postexposure. The vapor saturation concentration of triphosgene at ambient temperature is ?100 times its LC50. In summary, triphosgene-induced lung injury patterns are different from that of phosgene and diphosgene. More research is needed to close the substantial data gaps of triphosgene.

Attempts to counteract phosgene-induced acute lung injury by instant high-dose aerosol exposure to hexamethylenetetramine, cysteine or glutathione.

Phosgene is an important high-production-volume intermediate with widespread industrial use. Consistent with other lung irritants causing ALI (acute lung injury), mode-of-action-based countermeasures remain rudimentary. This study was conducted to analyze whether extremely short high-level exposure to phosgene gas could be mitigated using three different inhaled nucleophiles administered by inhalation instantly after exposure to phosgene. Groups of young adult male Wistar rats were acutely exposed to carbonyl chloride (phosgene) using a directed-flow nose-only mode of exposure of 600 mg/m³ for 1.5 min (225 ppm × min). Immediately after exposure to phosgene gas the rats were similarly exposed to three strong nucleophiles with and without antioxidant properties for 5 or 15 min. The following nucleophiles were used: hexamethylenetetramine (HMT), l-cysteine (Cys), and l-glutathione (GSH). The concentration of the aerosol (mass median aerodynamic diameter 1.7-2 µm) was targeted to be in the range of 1 mg/L. Cys and GSH have antioxidant properties in addition. The calculated alveolar molar dosage of phosgene was 9 µmol/kg. At 15-min exposure duration, the respective inhaled dose of HMT, Csy, and GSH were 111, 103, and 46 µmol/kg, respectively. The alveolar dose of drugs was ~10-times lower. The efficacy of treatment was judged by protein concentrations in bronchoalveolar lavage fluid (BALF) collected 1 day post-exposure. In spite of using optimized aerosolization techniques, none of the nucleophiles chosen had any mitigating effect on BALF-protein extravasation. This finding appear to suggest that inhaled phosgene gas acylates instantly nucleophilic moieties at the site of initial deposition and that the resultant reaction products can not be reactivated even following instant inhalation treatment with competing nucleophilic agents. In spite of using maximal technically attainable concentrations, it appears to be experimentally challenging to deliver such nucleophiles to the lower respiratory tract at high dosages.

Brown Norway rat asthma model of diphenylmethane-4,4'-diisocyanate (MDI): determination of the elicitation threshold concentration of after inhalation sensitization.

Occupational exposure to polymeric diphenylmethane-diisocyanate (MDI), a known human asthmagen, can be attributed to two potential routes: the skin and the respiratory tract. While the skin as the route of sensitization was the focus of a previous investigation (Pauluhn, 2008), this paper describes a modified sensitization protocol using a 5-day inhalation exposure (days 0-4) of Brown Norway (BN) rats to two concentration x exposure time (C x t) relationships of 1000, 5000, and 10,000 mg MDI/m³ x min at exposure durations of either 10 or 360-min. Apart from the differences in the induction protocol, all other experimental variables remained identical. This was followed by four 30-min inhalation challenges to 40 mg MDI/m³ on target days 20, 25, 50, and 65. After the last challenge, changes in breathing patterns delayed in onset were recorded and allergic lung inflammation was probed by bronchoalveolar lavage (BAL). In a subsequent study groups of rats were sensitized using the 10-min C x t protocol and challenged 3-times at 40 mg MDI/m³. At the fourth challenge a dose-escalation regimen was used to determine the elicitation threshold on 'asthmatic' rats. Consistent with the skin-sensitization protocol, the most sensitive endpoints characterizing an allergic pulmonary inflammation were again BAL-neutrophils and physiological measurements showing respiratory changes delayed in onset. The dose-escalation challenge yielded an elicitation threshold of 5 mg MDI-aerosol/m³ at 30 min challenge duration. In topically sensitized rats this threshold was estimated to be 3mg/m³. In summary, these data suggest the C x t product of MDI-aerosol that triggers an elicitation response in 'asthmatic' rats is slightly below of that causing acute pulmonary irritation in naïve rats. The high concentration delivered to the respiratory tract during the 10-min exposure period elicited a more vigorous response than the similar C x t at 360 min. Therefore, short high-level exposure patterns appear to bear a higher sensitizing potency than equal C x t products at longer exposure periods. Taking into account the respective differences in exposure intensities, the comparison of elicitation thresholds of BN rats sensitized by inhalation or skin exposure did not demonstrate essential differences.