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Repeated dose toxicity: inhalation

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sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Due to limited test design study is only acceptable for the assessment of lung effects and the discussion on the mode of action.
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
Pulmonary structural and extracellular matrix alterations in Fischer 344 rats following subchronic phosgene exposure
Kodavanti UP, Costa DL, Giri SN, Starcher B & Hatch GE
Bibliographic source:
Fund. Appl. Toxicol. 37: 54-63

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Method: Repeated Inhalation Toxicity
The purpose of the study was to elucidate potential long-term effects on collagen and elastin metabolism during pulmonary injury/recovery and obtain information about the concentration x time (C x T) behavior of low levels of phosgene.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:

Test animals

Fischer 344
Details on test animals or test system and environmental conditions:
Source: Charles River Breeding Labs
Age at study initiation: 60 days
Weight at study initiation: 260-300 g
Housing: temperature and humidity controlled; AAALAC-approved animal facilities with 12 hr dark/light cycle.
Diet: certified rat chow ad libitum

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure:
whole body
other: nitrogen
Details on inhalation exposure:
Rats were housed in large 2.0 m3 Hazelton 2000 (0.0, 0.1, 0.2, and 0.5 ppm groups) and small 0.3 m3 Rochester inhalation chambers (0.0 and 1.0 ppm groups) (approved by AAALAC) during phosgene exposure. Animals housed in Hazelton chambers remained in the chamber during nonexposure periods; however, animals housed in Rochester inhalation chambers were relocated to the animal colony. No significant differences in body weight gain (large chambers vs small chambers; 4 weeks, 251.5 ± 17.9 vs 244.7 ± 10.5; 12 weeks, 319.2 ± 11.9 vs 309.7 ± 13.3; 16 weeks, 355.3 ±23.5 vs 347.1 ± 14.5) or in any other parameters occurred between two different control groups housed in two different settings. Following 12 weeks of phosgene exposure all recovery group animals were housed in animal colony for 4 weeks of recovery period. Animals were randomly assigned to different groups using a computerbased weight-matched randomization program. A mass-flow controller (Tylan, Torrance, CA) metered phosgene (300 ppm in nitrogen; Matheson Gas Products, Morrow, GA), with an airflow of 1.0 m3/min for Hazelton chambers and 0.32 mVmin for Rochester inhalation chambers. The gas mixture passed downward through the cages holding the test animals and was exhausted at the bottom into a water scrubber. Temperature and humidity in the chambers as well as in the animal colony were (mean ± SD) 23.0 ± 4°C and 50 ± 10%, respectively. Control animals were placed simultaneously in an identical chamber and allowed to breath filtered air.

Phosgene concentrations in the chambers were determined with Hewlett-Packard 5880 and 5840 gas chromatographs. Methane (5%) in argon (P-
5) was used as the carrier phase. For Model 5880, a fused silica capillary column, SPB-1, was used (Supelco Inc., Bellefonte, PA) with a flow rate
of 30.0 ml/min. The column temperature was maintained at 35°C. For Model 5840, a 10% SP-2100, 80/100 Supelcoport SS 1/8" column (Supelco,
Bellefonte, PA) was used with a flow rate of 30 ml/min. Column temperature was maintained at 40°C during operation. Prior to the start of phosgene
exposure, an appropriate representative sampling port was determined by analyzing samples collected at various locations within the chamber. During exposure, phosgene concentrations were monitored once every 20 min from a representative sampling port. The 12-week average chamber phosgene concentrations were (mean ± SD (range)) for 0.1 ppm = 0.101 ± 0.002 (0.098-0.113), 0.2 ppm = 0.201 ± 0.002 (0.196-0.207), 0.5 ppm = 0.505 ± 0.008 (0.495-0.536), and 1.0 ppm = 0.976 ± 0.03 (0.912-1.009).

Rats were exposed to either air or phosgene, 6 hr/day at 0.1 (5 days/week), 0.2 (5 days/week), 0.5 (2 days/week), or 1.0 ppm (1 day/week) in order to derive equal C X T product for one particular time point except in the 0.1 ppm group (50% C X T). At the end of 4 or 12 weeks of phosgene exposure, or 4 weeks of clean air recovery after 12 weeks of phosgene exposure, rats were killed and the lungs analyzed.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Hewlett Packard 5880 and 5840 gas chromatographs; during exposure, phosgene concentrations were monitored once every 20 min from a representative sampling port.
Duration of treatment / exposure:
4 and 12 weeks exposure plus 4 weeks recovery
Frequency of treatment:
0.1 and 0.2 ppm: 6 hr/day, 5 days per week
0.5 ppm: 6 hr/day, 2 days per week
1.0 ppm: 6 hr/day, 1 day per week.
Doses / concentrationsopen allclose all
Doses / Concentrations:
0.1, 0.2, 0.5 and 1.0 ppm
nominal conc.
Doses / Concentrations:
0.101, 0.201, 0.505, 0.976 ppm
other: 12-week average chamber phosgene concentrations
No. of animals per sex per dose:
8 male for all groups (2 control groups, large chambers (n=8), small chambers (n=4))
Control animals:
yes, sham-exposed
Details on study design:
Rats were anesthetized with urediane (1.0 g/ml/kg body wt), the abdomen was opened, blood was removed by exsanguination of the dorsal aorta, and the trachea was cannulated. The left lung was used for histopathology and the right lung was used for biochemical analysis. The right lung was tied, and then the lung lobes were clipped off and weighed. Following lung weight measurements, the lobes were quickly frozen in liquid nitrogen and stored at -80°C until analyzed. The trachea with the left lung attached was suspended in a petri dish containing distilled water and inflated to 25 cm water transpulmonary pressure with 2.5% glutaraldehyde/0.16 M cacodylate buffer. Thirty min postinflation, the lung was removed and the displacement volume was obtained. The fixed tissues were then stored in 10% buffered formalin until further processing.

Positive control:


Sacrifice and pathology:
Midsagittal sections (4 µm) of the lung tissue were prepared from paraffin blocks and mounted on glass microscope slides. Slides were stained with Masson's Trichrome stain for identification of collagen fibers and counter stained with hematoxylin and eosin. Lung sections were examined under a light microscope. Histopathological evaluation of these lung tissues was done in a blinded manner for exposure group designation (Experimental Pathology Laboratory, Research Triangle Park, NC).
Other examinations:
The right caudal lobe was used for hydroxyproline and desmosine analysis and the rest of the lobes were used for prolylhydroxylase assay. The right lung lobes (except a caudal lobe) were thawed and homogenized in cold 20 mM Tris-HCl buffer, pH 7.6. Prolyl hydroxylase activity was measured as the enzyme-catalyzed release of tritiated water from L-[3,4-3H]proline-labeled procollagen (Gin el ah, 1983). For hydroxyproline and desmosine analysis the caudal lobes were lyophilized and then hydrolyzed in 6 M hydrochloric acid at 110°C for 16 hr. The hydrolyzate was filtered through 0.45 µm Teflon filters. A portion of the filtrate was then neutralized using 6 M sodium hydroxide to a pH of 7-7.5 and the samples were diluted for calorimetric detection of hydroxyproline as described (Bergman and Loxley, 1961). An aliquot of filtrate was dried under nitrogen. The residue was resuspended in water and dried in order to completely eliminate hydrochloric acid from the sample. The dried samples were then suspended in water and analyzed for desmosine by radioimmunoassay (Starcher, 1982).
A multivariate analysis of variance (MANOVA) (SAS 516; SAS Institute, Cary, NC) was used. According to the study protocol, if significant (p < 0.1) multivanate time, exposure, or exposure by time interaction effects were found, further univariate analysis using the same analysis model as that used in the MANOVA were performed. Corrected pairwise comparisons were used as subtests to evaluate differences between exposure groups. The Type I error rate was set at p = 0.05 for significance. Control values (air chambers) for large and small chambers were pooled because no statistical differences were noted between the animals exposed in two chambers in any of the parameters measured at any time. Data represents the mean ± SE of 10-12 controls or 8 exposed rats.

Results and discussion

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Executive summary:

In summary, terminal bronchiolar and lung volume displacement changes occurred at very low phosgene concentrations (0.1 ppm). Phosgene concentration, rather than C x T product appeared to drive toxic responses. The changes induced by phosgene (except of collagen) following 4 weeks were not further amplified at 12 weeks despite continued exposure. Phosgene-induced alterations of matrix were only partially reversible after 4 weeks of clean air exposure.