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Phosgene is a gas under ambient conditions, therefore the primary route of potential human exposure is inhalation. The primary toxic effects in response to both acute and repeated exposures to phosgene are focused on the portal of entry, the respiratory tract. Acute inhalation toxicity studies conclude that, at lethal concentrations, the most common findings are non-cardiogenic pulmonary edema and effects on pulmonary function.

To characterize subchronic effects of phosgene in laboratory animals, Kodavanti et al. (1997) performed an inhalation study (whole body) in male F344 rats with phosgene exposures (6 hr/day) of 0.1 ppm (5 days/wk), 0.2 ppm (5 days/wk), 0.5 ppm (2 days/wk) and 1.0 ppm (1 day/wk) over 4 or 12 weeks. A group of rats was allowed clean air recovery for 4 weeks after 12 weeks of phosgene exposure. This exposure scenario was designed to provide equal C x T product for all concentrations at one particular time point except for 0.1 ppm (50% C x T). Phosgene exposure for 4 or 12 weeks increased lung to body weight ratio and lung displacement volume in a concentration-dependent manner. The increase in lung displacement volume was significant even at 0.1 ppm phosgene at 4 weeks. Light microscopic level histopathology examination of lung was conducted at 0.0, 0.1, 0.2, and 1.0 ppm phosgene following 4 and 12 and 16 weeks (recovery). Small but clearly apparent terminal bronchiolar thickening and inflammation were evident with 0.1 ppm phosgene at both 4 and 12 weeks. At 0.2 ppm phosgene, terminal bronchiolar thickening and inflammation appeared to be more prominent when compared to the 0.1 ppm group and changes in alveolar parenchyma were minimal. At 1.0 ppm, extensive inflammation and thickening of terminal bronchioles as well as alveolar walls were evident. Concentration rather than C x T seems to drive pathology response. Trichrome staining for collagen at the terminal bronchiolar sites indicated a slight increase at 4 weeks and marked increase at 12 weeks in both 0.2 and 1.0 ppm groups (0.5 ppm was not examined), 1.0 ppm being more intense. Whole-lung prolyl hydroxylase activity and hydroxyproline, taken as an index of collagen synthesis, were increased following 1.0 ppm phosgene exposure at 4 as well as 12 weeks, respectively. Desmosine levels, taken as an index of changes in elastin, were increased in the lung after 4 or 12 weeks in the 1.0 ppm phosgene group. Following 4 weeks of air recovery, lung hydroxyproline was further increased in 0.5 and 1.0 ppm phosgene groups. Lung weight also remained significantly higher than the controls; however, desmosine and lung displacement volume in phosgene-exposed animals were similar to controls. Under the conditions described, 0.1 ppm (0.41 mg/m3) was a borderline NOAEC/LOAEC in rats after 4- or 12-week phosgene exposure based on minimal histological effects of low incidences that were fully reversible.

Within this study published by Kodavanti et al. (1997), Selgrade et al. (1995) investigated the effects of repeated phosgene exposures on bacterial infection and natural killer (NK) cell activity. The animals were exposed for 4 or 12 weeks, 6 hr/day, 5 days/wk, to 0.1 ot 0.2 ppm phosgene or 2 days/wk to 0.5 ppm and infected by aerosol with Streptococcus zooepidemicus immediately after the last exposure. An additional group was also infected after 4 weeks of recovery following the 12-week exposure regimens. Bronchoalveolar lavage (BAL) fluid was assessed 0, 6, and 24 hours post-infection for bacteria and inflammatory cells. Differential cell counts in BAL and pulmonary NK activity were also determined in uninfected rats 18 h after the last exposure. All phosgene exposures impaired clearance of bacteria from the lungs and caused an increase in polymorphonuclear leukocytes (PMNs) in BAL of infected rats. Effects in the 0.5 ppm exposure group were greatest, and were significantly different from those in the 0.2 ppm exposure group, although the product of concentration x time was the same. BAL cell counts and bacterial clearance were normal in rats assessed 4 weeks after the 12-week phosgene exposures. Bacterial clearance and the PMN response to infection following repeated exposure were similar to those observed after a single exposure; that is, for theses endpoints, effects due to repetitive exposure were neither additive nor attenuated. In contrast, NK activity was suppressed only at the 0.5 ppm level, and the magnitude of suppression was much less than that following acute exposure, suggesting that attenuation of this effect did occur with repeated exposure. The data indicate that susceptibility to streptococcal infection is a sensitive endpoint for phosgene toxicity following subchronic exposure. This finding is not unexpected since the alveolar lining fluids contain a panel of antimicrobial proteins and peptides (e.g. defensine). Phosgene exposure affects transiently the homeostasis of pulmonary surfactant. Accordingly, the

diminished response to a bacterial inocculum following exposure is consistent with the primary mode of action of phosgene, i.e. transient destabilization of blood-air barrier function. Under the conditions described the LOAEC was 0.1 ppm (0.41 mg/m3) in rats after 4- or 12-week phosgene exposure.

In another study examination of the bronchoalveolar lavage (BAL) fluid for protein as the most sensitive parameter of phosgene toxicity demonstrated that in contrast to single exposure to 0.2 ppm (6 hr/day), repeated exposure to 0.1 or 0.2 ppm (6 hr/day, 5 days/week, for 4, 8 or 12 weeks) showed no effect (when determined 1 day post exposure). BAL fluid protein returned completely to background concentrations even during continued exposure for 4 weeks or longer (Hatch et al., 2001).

In the context of a review of the German MAK value of phosgene two recent acute inhalation studies in rats and dogs were taken into account for the determination of a new occupational exposure limit (OEL) value (Greim, 2008). In the first acute inhalation study in male Wistar rats, the temporal course of indicators in the bronchoalveolar lavage (BAL) fluid and histopathological changes after 30- or 240-minute exposure to phosgene gas (directed-flow nose-only) were pursued over recovery periods of 4 or 12 weeks (Pauluhn, 2006b). In 30-min exposed rats the concentrations were 0.94, 2.02, 3.89, 7.35, and 15.36 mg/m³, which relate to C × t products of 28.2, 60.6, 116.7, 220.5, and 460.8 mg/m³ × min. In 240-min exposed rats the concentrations were 0.20, 0.39, 0.79, 1.57, and 4.2 mg/m³, which relate C × t products of 47.0, 92.9, 188.6, 376, and 1008 mg/m³ × min. (Remark: test conc. 4.2 mg/m3 not mentioned in the study report of Pauluhn (2006b) but in the publication of Pauluhn (Inhalation Toxicology 18: 595-607, 2006)).Mortality did not occur at any C × t product. The most pronounced changes were related to C × t-dependent increases in the following markers in BAL: protein, soluble collagen, polymorphonuclear leukocytes (PMN) counts, and alveolar macrophages with foamy appearance. These indicators were maximal on the first postexposure day, while total cell counts and alveolar macrophages containing increased phospholipids reached their climax around postexposure day 3. At 1008 mg/m³ × min the most sensitive indicators in BAL, that is, protein, PMN, and collagen, resolved within 2 weeks, whereas at lower C × t products they reached the level of the control by post-exposure day 7. At 1008 mg/m³ × min (day 28), histopathology revealed a minimal to slight hypercellularity in terminal bronchioles with focal peribronchiolar inflammatory infiltrates and focal septal thickening. At lower C × t products (day 84) the rats from all groups were indistinguishable and Sirius red-stained lungs did not provide evidence of late-onset sequelae, such as fibrotic changes or collagen deposition. At similar C × t products the changes in BAL were slightly less pronounced using 30-min exposure periods when compared to 240-min exposure periods. In summary, the phosgene-induced transmucosal permeability caused a C × t-dependent increase of several BAL indicators, of which those of protein, PMN, and soluble collagen were most pronounced. Exposure intensities up to 116.7 mg/m³ × min did not cause changes different from those observed in controls, while at 188.6 mg/m³ × min distinct differences to the control existed. Despite the extensively increased airway permeability, histopathology did not provide evidence of lung tissue remodeling or irreversible sequelae.

To better understand the human relevance of phosgene-induced changes in BAL fluid protein observed in acutely exposed rats,groups of Beagle dogs were similarly exposed for 30 minutes to phosgene using a head-only mode of exposure (Pauluhn, 2006c). The actual exposure concentrations were 9, 16.5, and 35 mg/m³, with resultant C × t products of 270, 495, and 1050 mg/m³ × min. In rats, a C × t product of 270 mg/m³ × min caused a significant elevation of protein in the bronchoalveolar lavage (BAL) fluid, while the non-lethal threshold concentration (LCt01) was estimated to be 1075 mg/m³ × min. The endpoints examined in dogs focused on changes in BAL, lung weights, arterial blood gases, and lung histopathology approximately 24 hour post-exposure. Mortality did not occur at any C × t product. Increased lung weights and elevations in protein, soluble collagen, and polymorphonuclear leukocyte (PMN) counts in BAL were observed at 1050 mg/m³ × min with borderline changes at 495 mg/m³ × min. Following exposure to 1050 mg/m³ × min, the analysis of arterial blood gases provided evidence of a significantly decreased arterial pO2. Histopathology revealed a mild, although distinctive, inflammatory response at the bronchoalveolar level at 495 mg/m³ × min, whereas serofibrinous exudates and edema were observed at 1050 mg/m³×min. The magnitude of effects correlated with the individual dogs’ respiratory minute volume and breathing patterns (panting). Collectively, phosgene-induced indicators of acute lung injury appeared to be characterized best by protein in BAL fluid. With regard to both the inhaled dose and the associated increase of protein in BAL, the responses obtained in dogs appear to be more similar to humans. In contrast, elevations in BAL protein occurred in rats at three-fold lower concentrations when compared to dogs. The results of this study demonstrate that the magnitude of elevations of plasma exudate in BAL fluid following acute exposure to the pulmonary irritant phosgene is markedly more pronounced in rats when compared to the dog which is considered more human-like than rats. This is believed to be associated with the higher ventilation of small rodents and with rodent-specific sensory bronchopulmonary defense reflexes.

In summary, the minimal histological changes at the terminal bronchioles observed in rats at 0.1 ppm showed no time-dependent increase between a 4- or 12-week phosgene exposure (Kodavanti et al., 1997). After subchronic exposure of rats to phosgene the clearance of bacteria pointed neither to adaptive nor to additive effects in comparison to acutely exposed animals (Selgrade et al., 1995). Thus, there is no evidence for an increase of severity of effect over time and it is confirmed that the inhalation hazard of phosgene is based on its acute toxicity. Therefore, acute studies evaluating sensitive parameters can be used for the derivation of an occupational exposure limit (OEL) value for phosgene (Greim, 2008). In acute inhalation studies in rats (exposure duration 30 and 240 min) the comparative analysis of the most sensitive endpoint protein concentration in BAL fluid showed a linear dependence of C x t at double logarithmic delineation (Pauluhn, 2006b). This summarizing analysis proves that phosgene-induced early pulmonary changes at single exposure are determined by the inhaled phosgene dose (C x t), and not by the concentration alone. Since the anatomy and physiology as well as the ventilation pattern of the dog is more similar to those of man than those of the rat, an additional acute inhalation study in dogs was performed by Pauluhn (2006c). In this study a single 30-min exposure revealed neither significant changes in BAL fluid nor adverse histological lung findings at a C x t product of 270 mg/m3 x min. Regarding the validity of the Haber rule over wide concentration areas and exposure times at single exposure, this would correspond to a concentration of 0.14 ppm (0.56 mg/m3) for a 8-hr exposure (Greim, 2008).

Based on the 8-hr concentration of 0.14 ppm without adverse effect calculated for the dog and taking into consideration the mode of action, a MAK value of 0.1 ppm (0.41 mg/m3) was established according to the "preferred-value-approach" (Greim, 2008).

Since than the EU Scientific Committee on Occupational Exposure Limits (SCOEL) recommended an OEL of 0.4 mg/m3 referring to an 8-hour exposure period. A ceiling limit value of 2.0 mg/m3 was recommended for phosgene. The justification of these OELs was based on the MAK documentation on phosgene (Greim, 2008).