Institute of Toxicology, Bayer AG, Building 514, 42096 Wuppertal, Germany
Received May 15, 2000; accepted August 7, 2000
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ABSTRACT |
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Key Words: isocyanate aerosol inhalation; pulmonary edema; aerosol of HDI-homopolymer; HDI-isocyanurate; surfactant; polyurethane coating.
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INTRODUCTION |
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The use pattern of this product makes it possible that workers are inhaling this material in aerosol form. However, due to the aerosolization of a complex system of chemically reactive agents, reported health effects may not necessarily be causally related to HDI polyisocyanates alone. Moreover, in this context, it is important to recognize that the content of the more volatile HDI monomer (vapor pressure 1.4 103 kPa at 25°C) has decreased over the past decades, i.e., results obtained from earlier studies may have been confounded by the purging or evaporation of HDI monomer from the nonvolatile HDI-homopolymer liquid. Appreciable amounts of HDI has been shown to be associated with the aerosol fraction of HDI polyisocyanates (Rando and Poovey, 1999
). Alexandersson et al. (1987) found evidence of small airway disease in auto spray painters, but no sensitization was found. On the other hand, Vandenplas et al. (1993) described positive asthmatic reactions in sensitized subjects after exposure to HDI homopolymers.
The objective of this study is to analyze the concentration dependence and time dependence on changes in the bronchoalveolar lavage fluid (BALF) of rats as a result of single inhalation exposure of 6 h to aerosolized HDI-IC during a postexposure period of 1 week. BALF was analyzed for end points indicating noncytotoxic and/or cytotoxic noxious effects on the bronchial epithelial barrier function, including an impairment of the vascular endothelium. An increased extravasation of plasma constituents was addressed by analysis of the angiotensin-converting enzyme (ACE) and total protein. A possible involvement of pulmonary surfactant has indirectly been addressed by the determination of phospholipids in BALF and BAL cells (BALC). Additional end points were considered to address the function of type II pneumocytes (alkaline phosphatase), cell injury and lysis (lactate dehydrogenase), and lysosomal instability (acid phosphatase). All end points were determined directly after cessation of a single 6-h exposure period and following postexposure periods of 3 h and 1, 3, and 7 days to determine the onset and progression of effects related to direct cellular injury and/or interactions with pulmonary surfactant and ensuing inflammatory or compensatory response.
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MATERIALS AND METHODS |
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Animals, diet, and housing conditions.
Specific-pathogen-free female Wistar rats of the strain Hsd Cpb:WU (SPF) were purchased from Harlan Winkelmann GmbH, Borchen, Germany. The choice of strain and gender was based on experience obtained from previous studies (Pauluhn et al., 1999; Pauluhn, 2000
). At the commencement of the study the rats were approximately 2 months old. Animals were quarantined for at least 5 days prior to being placed on study. Animals were placed in polycarbonate cages (one per cage) containing bedding material (low-dust wood shavings) and were provided Altromin 1324 feed and water ad libitum except during exposure. The light cycle was automatically controlled in the animal holding room to provide 12 h of fluorescent light and 12 h of darkness each 24 h. Temperature and relative humidity were continually monitored, with daily means in the range of 22°C and 4060%, respectively. All experiments and procedures described were performed in compliance with Good Laboratory Practice (GLP) requirements (OECD, 1983), taking into account the European Union (EU) animal welfare regulations (European Community Directive 86/609, 1986
).
Experimental design.
Three studies are addressed in this paper: determination of the acute median lethal toxic potency (LC50) of aerosolized HDI-IC in relation to evaporated HDI on rats according to the OECD No. 403 (Organization for Economic Cooperation and Development, 1981) and OPPTS 870.1300 testing protocols (single 4-h exposure; United States Environmental Protection Agency, 1998
), and concentration dependence and time course of end points in bronchoalveolar lavage following single 6-h exposure of rats to aerosolized HDI-IC.
Acute median lethal toxic potency (LC50).
At the end of the acclimatization period, rats were randomly assigned to the respective exposure group, each consisting of 5 male and 5 female rats per group. In the HDI-IC study, some exposures were made using 10 rats per group and gender. The rats were exposed nose-only inhalation to the HDI vapor or the HDI-IC aerosol in a single exposure of 4 h on day 0 followed by postexposure periods of 14 (HDI-IC) days or 4 weeks (HDI) due to the long duration of signs. Body weights were recorded before exposure, on days 3 and 7, and weekly thereafter. Clinical signs were observed twice daily. All animals were sacrificed at the time of death or at the end of the postexposure period.
Concentration response and time course of bronchoalveolar lavage.
At the end of the acclimatization period, the rats were randomly assigned to the exposure groups, each consisting of 30 female rats. The rats were exposed nose-only to actual breathing zone concentrations of 0 (conditioned dry air), 3.9 ± 0.5, 15.9 ± 0.6, 54.3 ± 10.0, and 118.1 ± 8.4 mg HDI-IC/m3 air (57 samples per exposure; ± represents the standard deviation, SD) in a single exposure of 6 h. This duration of exposure allows a direct comparison with results from repeated exposure 3- and 13-week inhalation studies with this test agent. Dosimetrically adjusted, these concentrations were considered to be equivalent to 6, 24, 81, and 177 mg/m3 of a 4-h exposure period, i.e., the highest concentration slightly exceeds the LC01 of 163 mg/m3 (Fig. 2; for definition of LC01 see "Statistical Analyses"). Clinical observations were made daily. Body weights were recorded before exposure and prior to each serial sacrifice. After complete exsanguination, the excised lungs of the animals were weighed, then lavaged as described below. Lavage samples were taken directly after cessation of exposure (0 h) and following postexposure time periods of 3 h, 1, 3, and 7 days.
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All exposure atmospheres were characterized using the modified nitro-reagent derivatization technique of Dunlap et al. (1976). Details have been described previously (Pauluhn et al., 1995; 1999
). Additionally, filter analyses (Sartorius glass fiber filters) were taken. Chamber air was sampled from the vicinity of the breathing zone of the rats at least one sample per hour for filter analyses or one sample per exposure for nitro-reagent analyses. For particle-size analyses, a low-pressure critical orifice AERAS stainless steel cascade impactor was used (HAUKE, 4810 Gmunden, Austria). The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated as described previously (Pauluhn, 1994
). In the LC50 study the MMAD was in the range of 2.42.9 µm (GSD
1.7); in the time-course study the MMAD was 1.72.0 µm (GSD
1.5). The total gravimetric mass concentration measured by cascade impactor analyses, filter, and/or nitro-reagent analyses provided virtually identical results. Therefore, no distinction of results obtained by the various techniques was made. Because of the greater number of samples available, concentrations refer to gravimetric analysis. Further details of the exposure methodology and its validation have been published elsewhere (Pauluhn, 1994
; Pauluhn et al., 1999
).
HDI vapor.
Dry conditioned air was bubbled through the HDI liquid (ca. 90 ml) contained in a glass bubbler (kept in a water bath at 45°C). This atmosphere was subsequently diluted with dry, conditioned air to attain the targeted concentration. The stability of the test atmosphere was monitored continuously using a total hydrocarbon analyzer equipped with a flame ionization detector (Compur, Munich, Germany). Five groups of rats were nose-only exposed, as described for HDI-IC, to the following actual concentrations using the nitro-reagent derivatization method: 0 (air control), 55, 107, 120, and 151 mg HDI/m3 air. The mean recovery of nominally evaporated HDI (weight difference before and after exposure divided by the total volume of air per exposure) was 87%. As vapor atmospheres were generated at temperatures higher than in the inhalation chamber, the presence of condensation aerosol was examined by a real-time aerosol laser particle velocimeter (TSI APS 3300, TSI Inc., St. Paul, MN). In the 107, 120 and 151 mg HDI/m3 exposure groups, the total mass of aerosol detected by this piece of equipment was 6, 15, and 22 mg/m3, respectively.
Bronchoalveolar lavage.
Shortly after the animal was exsanguinated by transecting the aorta, the diaphragm was incised and the lungs were allowed to collapse. The excised lungs of the animals were then lavaged twice with 5 ml saline (kept at 37°C) per rat, and the 2 washings were combined. This number of washes was chosen based on the work of Henderson et al. (1985), who showed that most of the cells and protein are recovered in the first 2 washes. More details of the lavage technique have been published elsewhere (Pauluhn et al., 1999). Samples of bronchoalveolar lavage fluid were analyzed for factors indicative of an inflammatory response, which proved useful in context with isocyanate inhalation studies (Pauluhn et al., 1995
, 1999
). Additional end points were considered addressing markers of instability of or damage to the alveolar capillary barrier. Phosphatidylcholine in BALF and BALC was addressed, as it may serve as proxy of changes of surfactant-phospholipid homeostasis. The role of pulmonary surfactant has been discussed elsewhere (Hook, 1991
; Kodavanti and Mehendale 1990
; Reasor, 1981
). Within the acellular supernatant of BALF, the following indicators were assessed (Henderson, 1989
; Henderson and Belinsky, 1993
):
Angiotensin-converting enzyme (ACE), an enzyme contained in the luminal vascular endothelium of the lung that hydrolyzes angiotensin I to angiotensin II (Cushman and Cheung, 1971), was determined with a method largely consistent with that of Horiuchi et al. (1982) and described in detail elsewhere (Pauluhn et al., 1999
).
-GT was determined according to Naftalin et al. (1969) using Sigma Diagnostic Procedure No. 545 (1986). The cellular content of the lavage fluid was removed by centrifugation at 200 x g (10 min at 4°C). Total cell numbers were counted electronically (Schärfe-System, Casy 1, Reutlingen, Germany). Differential counts were not made because previous studies with other isocyanate aerosols have shown that changes in cell count were caused by an increase in alveolar macrophages rather than inflammatory cells (Pauluhn et al., 1999
). The remaining parameters were determined using commercially available reagents as published previously (Pauluhn et al., 1999
).
Statistical analyses.
Data were analyzed by a one-way analysis of variance followed by a Tukey-Kramer post hoc test. For all tests the criterion for statistical significance was set at p < 0.05. Asterisks in figures denote statistically significant differences: * indicates p < 0.05 and ** indicates p < 0.01. Each group consisted of 6 rats per serial sacrifice. For each sacrifice, relative data were calculated by division of group means and standard deviations of treatment groups by the mean of rats of the control group sacrificed at the same time point. This was made to allow a better appreciation of the time- and concentration-related changes. Data in figures were expressed relative to the data of the respective control group. The median lethal concentration (LC50) and its confidence interval were calculated according to Rosiello et al. (1977). The regression parameters of the linear probability mortalitylog concentration relationship were used to calculate the LC01. The 1% response (LC01) was chosen because it is considered to give values reasonably close to experimentally observed LC0 values (AEGL, 1999).
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RESULTS |
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Time Course of Pulmonary Response of Rats Exposed for 6 Hours to HDI-IC
Rats exposed for 6 h to 54.3 mg/m3 and above elaborated concentration-dependent signs, most of them related to respiratory tract irritation such as irregular and labored breathing pattern, bradypnea, and nostrils with red encrustations. Rats exposed to 54.3 mg/m3 displayed signs up to the second, and in the 118.1 mg/m3 exposure group, up to the fifth postexposure day. Necropsy findings of rats exposed to 118.1 mg/m3 provided evidence of macroscopic alterations such as distended lungs, edema, hydrothorax, consolidation, and pulmonary hemorrhages. On postexposure day 7, lung-associated lymph nodes appeared to be enlarged. One out of 30 rats exposed to this concentration succumbed the night after exposure. Necropsy findings of rats exposed to 54.3 mg/m3 were less pronounced and were related mainly to focal pulmonary hemorrhages and less-collapsed lungs. A marked, but transient decrease of body weights was observed in rats exposed to 54.3 mg/m3 and above (data not shown). Wet lung weights were statistically significantly increased in rats exposed to 15.9 mg/m3 and above, with maximum effects 3 h and 1 day postexposure (Fig. 3). Rats exposed to 3.9 mg/m3 did not experience any statistically significant increase of lung weights.
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At all time points the average recovery of the pooled lavage fluid instilled into the lung was high ( 85%), except in the 118.1 mg/m3 group directly after cessation of exposure (10% lower than the average; data not shown). Serial sacrifices of rats exposed for 6 h to 15.9 mg HDI-IC/m3 and above revealed that most end points examined in bronchoalveolar lavage showed a remarkable time-related increase from 3 h after cessation of exposure to postexposure day 1 and, in some cases, also postexposure day 3. Changes considered to be related to pulmonary irritation returned almost completely to the level of the control group on day 7. Those changes apparently related to tissue restoration or compensatory response, e.g., tissue GSH or the increased lysosomal activity of BALC were still statistically significantly increased on day 7.
The relative comparison of effects suggests that the most sensitive marker indicative of a dysfunction of the air-blood barrier was characterized by a marked concentration-dependent increase of ACE activity and total protein in BALF, which peaked in parallel on postexposure day 1 (Fig. 4). The activity of LDH in BALF was statistically significantly increased in groups exposed to 54.3 mg HDI-IC/m3 and above (Fig. 4
). At these high exposure levels, the time course of changes of protein and ACE coincided, whereas that of LDH was different following exposure to 118.1 mg/m3. The coexisting statistically significant increase of intracellular phospholipids in relation to that of intracellular acid phosphatase suggests that elevated amounts of phospholipids, possibly originating from surfactant, were phagocytized by alveolar macrophages (Fig. 5
). The somewhat upregulated lysosomal activity appears to be in line with elevated catabolism of phagocytized phospholipids. The uptake of intracellular phospholipids appears to occur in a concentration-related, somewhat protracted fashion, i.e., intracellular phospholipids were less elevated on the day of exposure and markedly elevated on the first and third postexposure days. Despite this coincidence, the acid phosphatase in BALC did not appear to increase proportionally with the engulfment of phospholipids (day 3), and the subsequent restoration of lysosomal function appeared to be contingent upon the extent of cellular loading. The concentration phospholipids in BALF showed a similar time course of changes; however, the relative magnitude of changes was less pronounced (peak response on day 3 approximately 5 times the control value) and less concentration dependent (data not shown). Alkaline phosphatase activity in BALF displayed maximum effect on day 1 (approximately 3 times the control) without showing any concentration-dependent increase between the 15.9 and 118.1 mg/m3 exposure groups (data not shown).
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DISCUSSION |
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It could be speculated that the approximately 4 times higher acute toxic lethal potency of HDI vapor is related to the greater number of reactive NCO groups per molecule (HDI: molecular mass, 168; content of NCO: 50%; HDI-IC: molecular mass of isocyanuraten = 3, 504; content of NCO: 25%). However, experimental evidence obtained with other volatile isocyanates supports the view that the pathomechanism causing predominant damage within the respiratory tract is more dependent on the location of highest deposition. Especially for volatile isocyanates, it is believed that water solubility and chemical reactivity appear to be more decisive for the outcome of study than the mere content of reactive isocyanate groups per molecule. As reported previously, the ratios of 4-h LC50s in rats of n-:iso-propyl isocyanate was 1:3, whereas that of n-:iso-:tertiary-butyl isocyanate was 1:
4:
30, demonstrating that changes in reactivity have marked impact on acute inhalation toxicity (Pauluhn, 1988
). Moreover, volatile isocyanates with a high acute lethal toxic potency showed a biphasic type of mortality, whereas intermediate to lower potency ones demonstrated a more monophasic, i.e., early type of mortality due to pulmonary irritation.
The particle size of the aerosolized HDI-IC was adequate to penetrate the lower respiratory tract of rats. Therefore, the acute 6-h study with subsequently performed serial sacrifices for bronchoalveolar lavage focused on the investigation of the time course of acute lung injury occurring in the bronchoalveolar region of the lung. Based on the results of this single 6-h exposure study, the most salient changes observed were related to a transient disturbance of the air/blood barrier function. After exposure up to 15.9 mg/m3, this dysfunction was characterized by an ephemeral permeability of total protein and ACE. Appreciably increased activities of the marker of cytotoxicity LDH could only be detected following exposure to high concentrations of aerosol, i.e., 54.3 mg/m3 and above. The apparent sequence of effects occurring on the first and third postexposure days appears to be consistent with affected surfactant homeostasis. The concentration dependence of protein and LDH in BALF and phospholipids in BALC obtained on day 1 is illustrated in Figure 7. Among other possibilities, alterations in surface tension on the alveolar surface may have contributed to increased transudation of fluid and solutes from the capillaries (Albert et al., 1979
; Nieman, 1985
). Qualitatively similar findings were described to occur with polymeric MDI aerosol after acute inhalation exposure, and experimental evidence suggests that partially decreased sulfhydryl levels by pharmacological intervention made the lung more susceptible to the edemagenic potency of respirable MDI aerosol (Pauluhn, 2000
). Likewise, the measurements of GSH in lung tissue in this study also appear to suggest that pulmonary levels of GSH may be a modulating factor of susceptibility.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Alexandersson, R., Hedenstierna, G., Plato, N., and Kolmodin-Hedman, B. (1987). Exposure, lung function, and symptoms in car painters exposed to hexamethylenediisocyanate and biuret modified hexamethylenediisocyanate. Arch. Environ. Health 42, 367373.[ISI][Medline]
AEGL (1999). Standing Operating Procedures of the National Advisory Committee on Acute Exposure Guideline Levels for Hazardous Substances. (Draft) April 30, 1999.
Cushman, D. W., and Cheung, H. S. (1971). Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem. Pharmacol. 20, 16371648.[ISI]
Day, B. J., Carlson, G. P., and DeNicola, D. B. (1990). -Glutamyltranspeptidase in the rat bronchoalveolar lavage fluid as a probe for 4-ipomeanol and
-naphthylthiourea-induced pneumotoxicity. J. Pharmacol. Methods 24, 18.[ISI][Medline]
Dinsdale, D., Green, J. A., Manson, M. M., and Lee, M. J. (1992). The ultrastructural immunolocalization of -glutamyltranspeptidase in rat lung: correlation with the histochemical demonstration of enzyme activity. Histochem. J. 24, 144152.[ISI][Medline]
European Community Directive 86/609/EEC (1986). Guideline of the Council dated November 24, 1986 on the Reconciliation of Legal and Administrative Regulations of the Member Countries for the Protection of Animals used for Studies and other Scientific Purposes. Journal of the European Community, Legal Specifications L 358, 29.
Dunlap, K. L., Sandridge, R. L., Keller, J. (1976). Determination of isocyanates in working atmosphere by high performance liquid chromatography. Anal. Chem. 48, 497499.[ISI]
Ferguson, J. S., Schaper, M., and Alarie, Y. (1987). Pulmonary effects of a polyisocyanate aerosol: hexamethylene diisocyanate trimer (HDIt) or desmodur-N (Des-N). Toxicol. Appl. Pharmacol. 89, 332346.[ISI][Medline]
Greenberg, M. M., and Foureman, G. L. (1995). Derivation of the inhalation reference concentration for hexamethylene diisocyanate. Toxic Subst. Mech. 14, 151167.
Henderson, R. F., Benson, J. M., Hahn, F. F., Hobbs, C. H., Jones, R. K., Mauderly, J. L., McClellan, R. O., and Pickrell, J. A. (1985). New approaches for the evaluation of pulmonary toxicity: bronchoalveolar lavage fluid analysis. Fundam. Appl. Toxicol. 5, 451458.[ISI][Medline]
Henderson, R. F. (1989). Bronchoalveolar lavage: a tool for assessing the health status of the lung. In Concepts in Inhalation Toxicology, (R. O. McClellan and R. F. Henderson, Eds.), pp. 415444. Hemisphere, New York.
Henderson, R. F., and Belinsky, A. (1993). Biological markers of respiratory tract exposure. In Toxicology of the Lung, (D. E. Gardner, J. D. Crapo, and R. O. McClellan, Eds.), pp. 253282. Raven Press, New York.
Hook, G. E. R. (1991). Alveolar proteinosis and phospholipidosis of the lungs. Toxicol. Pathol. 19, 482513.[ISI][Medline]
Horiuchi, M., Fujimura, K-I., Terashima, T., and Iso, T. (1982). Method for the determination of angiotensin-converting enzyme in blood and tissue by high-performance liquid chromatography. J. Chromatogr. 233, 123130.[Medline]
Janko, M., McCarthy, K., Fajer, M., and van Raalte, J. (1992). Occupational exposure to 1,6-hexamethylene diisocyanate-based polyisocyanates in the state of Oregon, 19801990. Am. Ind. Hyg. Assoc. J. 53, 331338.[ISI][Medline]
Kelly, F. J. (1999). Glutathione: in defence of the lung. Food Chem. Toxicol. 37, 963966.[ISI][Medline]
Kodavanti, U. P., and Mehendale, H. M. (1990). Cationic Amphiphilic Drugs and Phospholipid Storage Disorder. Toxicol. Rev. 42, 327354.
Naftalin, L, Sexton, M., Whitaker, J. F., and Tracy, D. (1969). A routine procedure for estimation serum -glutamyltranspeptidase activity. Clin. Chim. Acta 26, 293296.[ISI][Medline]
Nieman, G. F. (1985). Current concepts of lung-fluid balance. Respir. Care 30, 10621076.
Organization for Economic Cooperation and Development (OECD)GLP. (1983). Publication of the German version of the OECD Principles of Good Laboratory Practice (GLP), Bundesanzeiger 35, No. 42a. March 2, 1983.
Organization for Economic Cooperation and Development (OECD). (1981). Guideline for Testing of Chemicals No. 403. "Acute Inhalation Toxicity", adopted May 12, 1981.
Pauluhn, J. (1988). A mechanistic approach to assess the inhalation toxicity and hazard of methylisocyanate and related aliphatic isocyanates. In Assessment of Inhalation Hazards Integration and Extrapolation Using Diverse Data, (U. Mohr, D. V. Bates, D. L. Dungworth, P. N. Lee, R. O. McClellan, F. J. C. Roe, Eds.), pp. 119128. ILSI Monographs, Springer-Verlag Heidelberg,
Pauluhn, J. (1994). Validation of an improved nose-only exposure system for rodents. J. Appl. Toxicol. 14, 5562.[ISI][Medline]
Pauluhn, J. (2000). Acute inhalation toxicity of polymeric diphenyl-methane-4,4`-diisocyanate (MDI) in rats: time course of changes in bronchoalveolar lavage. Arch. Toxicol. 74, 257269.[ISI][Medline]
Pauluhn, J., Emura, M., Mohr, U., Popp, A., and Rosenbruch, M. (1999). Two-week inhalation toxicity of polymeric diphenyl-methane-4,4`-diisocyanate (PMDI) in rats: analysis of biochemical and morphological markers of early pulmonary response. Inhal. Toxicol. 11, 11431163.[ISI][Medline]
Pauluhn, J., Rüngeler, W., and Mohr, U. (1995). Phenyl isocyanate-induced asthma in rats following a 2-week exposure period. Fundam. Appl. Toxicol. 24, 217228.[ISI][Medline]
Pauluhn, J., and Mohr, U. (1999). Repeated 4-week inhalation exposure of rats: effect of low-, intermediate, and high-humidity chamber atmospheres. Exp. Toxicol. Pathol. 51, 178187.[ISI][Medline]
Rando, R. J., and Poovey, H. G. (1999). Development of a dichotomous vapor/aerosol sampler for HDI-derived total reactive isocyanate group. AIHA J. 60, 737746.
Reasor, M. J. (1981). Drug-induced lipidosis and the alveolar macrophage. Toxicology 20, 133. Review.[ISI][Medline]
Reuzel, P. G. J., Kuper, C. F., Feron, V. J., Appelman, L. M., and Loser, E. (1994). Acute, subacute, and subchronic inhalation toxicity studies of respirable polymeric methylene diphenyl diisocyanate (polymeric MDI) aerosol in rats. Fundam. Appl. Toxicol. 22, 186194.[ISI][Medline]
Rosiello, A. P., Essignmann, J. M., and Wogan, G. N. (1977). Rapid and accurate determination of the median lethal dose (LD50) and its error with small computer. J. Toxicol. Environ. Health 3, 797809.[ISI][Medline]
Takayama, M., Itoh, S., Nagasaki, T., and Tanimizu, I. (1977). A new enzymatic method for determination of serum choline-containing phospholipids. Clin. Chim. Acta 79, 9398.[ISI][Medline]
United States Environmental Protection Agency (1998). US-EPA Health Effects Test Guidelines 870.1300Acute Inhalation Toxicity. United States Environmental Protection Agency, Office of Prevention, Pesticides and Toxic Substances, EPA 712C-98-193, August 1998.
Valentini, J. E., Wong, K.-L., and Alarie, Y. (1983). Single-tracer technique to evaluate pulmonary edema and its application to detect the effect of hexamethylene diisocyanate trimer aerosol exposures. Toxicol. Appl. Pharmacol. 69, 461470.[ISI][Medline]
Van Klaveren, R. J., Hoet, P. H. M., Pype, J., Demedts, M., and Nemery, B. (1997). Increase in -glutamyltransferase by glutathione depletion in rat type II pneumocytes. Free Radic. Biol. Med. 22, 525534.[ISI][Medline]
Vandenplas, O., Cartier, A., Lesage, J., Cloutier, Y., Perreault, G., Grammer, L. C., Shaughnessy, M. A., and Malo, J.-L. (1993). Prepolymers of hexamethylene diisocyanate as a cause of occupational asthma. J. Allergy Clin. Immunol. 91, 850861.[ISI][Medline]
Weyel, D. A., Rodney, B. S., and Alarie, Y. (1982). Sensory irritation, pulmonary irritation, and acute lethality of a polymeric isocyanate and sensory irritation of 2,6-toluene diisocyanate. Toxicol. Appl. Pharmacol. 64, 423430.[ISI][Medline]
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