Phosgene

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Reference 2

Reference Type:

publication

Title:

Acute nose-only exposure of rats to phosgene. Part I: concentration × time dependence of LC50s, nonlethal-threshold concentrations, and analysis of breathing patterns

Author:

Pauluhn J

Year:

2006

Bibliographic source:

Inhal. Toxicol. 18: 423-435

Reference 3

Reference Type:

publication

Title:

Workshop Summary: Phosgene-induced pulmonary toxicity revisited - Appraisal of early and late markers of pulmonary injury from animal models with emphasis on human significance

Author:

Pauluhn J et al.

Year:

2007

Bibliographic source:

Inhal. Toxicol. 19: 789-810

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

- shorter durations of exposure were used in addition to the standard 4 hour exposure.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Deviations:

yes

Remarks:

- shorter durations of exposure were used in addition to the standard 4 hour exposure.

GLP compliance:

yes

Test type:

standard acute method

Test material

Test material information

Constituent 1

Reference substance name:

Phosgene

EC Number:

200-870-3

EC Name:

Phosgene

Cas Number:

75-44-5

Molecular formula:

CCl2O

IUPAC Name:

phosgene

Details on test material:

Phosgene (carbonyl chloride, CAS no. 75-44-5), with certified gas standards of 17.7, 45.2, 70.3, or 968 ppm in synthetic air, was from Praxair GmbH, Hattingen, Germany, with certified stability for the duration of study. The gas was contained in 10-L cylinders at 150 bar. The conversion factor for phosgene is 1 ppm = 4.1 mg/m3.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
Source/supplier: Harlan-Winkelmann GmbH, Borchen
Strain: Wistar Hsd Cpb: WU (SPF)
Weight: 184 to 209 g male 160 to 180 g female
Age: young adult
Feeding: Animals were provided standard fixed formula diet (KLIBA 3883 pellets maintenance diet for rats and mice) and municipal tap water in drinking bottles. Both food and water were available ad libitum except during exposure.

ENVIRONMENTAL CONDITIONS:
Animals were housed in polycarbonate cages (1 rat per cage), containing bedding material (low-dust wood shavings). 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 40–60%, 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, 1986).

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

For this study, different starting concentrations of phosgene in synthetic air were used in order to minimize potential dilution errors. Controlled flows of phosgene were discharged from the cylinder and was either metered by precision pumps (Telab gas dosing pump) or by flowmeters into a continuous flow of conditioned dry air. The respective target concentration was achieved by dilution cascades prior to entering the directed flow nose-only chamber. The test atmosphere was then forced through openings in the inner concentric cylinder of the chamber, directly toward the rats’ breathing zone. This directed-flow arrangement minimizes rebreathing of exhaled test atmosphere. The aluminum inhalation chamber used consisted of 2 segments with a total of 40 exposure ports at the perimeter location. Each segment of this chamber had the following dimensions: inner diameter= 14 cm, outer diameter=35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 L). Details of this modular chamber and its validation with regard to spatial homogeneity have been published elsewhere (Pauluhn, 1994). All airflows were monitored and adjusted continuously by means of calibrated flow controllers. A soap-bubble meter was used to monitor the accuracy of the flow meters. The supply airflow was 30 L/min to maintain an airflow rate of 0.75 L/min per exposure port. The ratio between supply and exhaust air was selected so that 90% of the supplied air was extracted either via the exhaust air location and partially also via sampling ports. The slight positive balance between the air volume supplied and extracted ensured that no passive influx of air into the exposure chamber occurred (via exposure restrainers or other apertures). The slight positive balance provides also adequate dead-space ventilation of the exposure restrainers. The pressure difference between the inner inhalation chamber and the exposure zone was 0.02 cm H2O (Pauluhn, 1994). A train of gas scrubbing devices was used for the exhausted air: 20% aqueous solution of sodium hydroxide and activated charcoal. Temperature and humidity sensors were located at the exposure location of the inhalation chamber and data were recorded online. Temperature and humidity values were approximately 21°C and 8%, respectively. Controlled low-humidity inhalation studies did not show any confounding effect due to low humidity (Pauluhn & Mohr, 1999). The exposure system was accommodated in an adequately ventilated enclosure. Room air and in the chemical fume hoods accommodating the exposure chamber was monitored continuously with a paper tape monitor to detect instantly concentrations exceeding the current workplace standards of phosgene. Staff operating the exposure equipment wore full respiratory gear with external air supply.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

10 - 240 min

Concentrations:

Actual concentrations (mg/m3): 166.5, 181.6, 212.0, 250.9 (10 min. exposure); 51.3, 54.5, 67.7, 86.9 (30 min. exposure); 29.9, 39.4, 47.6 (60 min. exposure); 9.0, 11.1 (240 min. exposure).

No. of animals per sex per dose:

5

Control animals:

other: 5 male and 5 female control animals were exposed against air over 60 min.

Details on study design:

In this study the concentration × exposure time (C × t) relationship of phosgene was examined in rats using a directed-flow nose-only exposure design and exposure durations of concern for accidental exposures (10 to 240 min) followed by a post-exposure period of 2 weeks. Exposure atmospheres were characterized by several independent analytical procedures and compared with nominal concentrations. Supplemental physiological data were generated to better understand the (C × t) relationship of phosgene. This analysis included an estimation of nonlethal threshold concentrations (LC01), that is, the highest calculated level that does not cause lethality. By default, one-third of the LC50 is used to calculate the LC01 as defined by NRC (2001).

At the end of the acclimatization period rats were randomly assigned to the respective exposure groups, each consisting of 5 male and 5 female rats per group. In exposures focusing on LC50/LC01 estimations, rats were exposed nose-only by inhalation to the phosgene gas at single exposure periods of either 10, 30, 60, or 240 min on day 0, followed by a post-exposure period of 2 weeks. Body weights were recorded before exposure, on days 3, 7, and 14. Clinical signs were observed twice daily. All animals were sacrificed and gross-pathological examinated at the time found dead or at the end of the post-exposure period. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract.

Statistics:

- Necropsy findings: If specific findings occur from the respiratory tract of surviving rats they are evaluated statistically using the pair-wise Fisher test after the R x C chi-squared test. The Fisher test was only performed if differences occurred between groups in the R x C chi-squared test or if a frequency value of < 5 was calculated. This procedure was performed in accordance with Gad and Weil (1982). For calculation of the unilateral p value a symmetrical distribution was assumed (p unilateral = (p bilateral)/2).
- Body weights: Means and single standard deviations of body weights are calculated. Mean body weights are also depicted graphically as a function of time. Since in acute studies individual group means may differ prior to commencement of the first exposure, the body weight gain was statistically evaluated for each group. For these evaluations a one-way ANOVA (vide infra) is used.
- Calculation of the LC50: If calculation of a median lethal concentration (LC50) is possible, it is performed by computer (PC) according to the method of Rosiello et al. (1977) as modified by Pauluhn (1983). This method is based on the maximum-likelihood method of Bliss (1938). If only 2 pairs of values with greater than 0% lethality and less than 100% are available then the first linear approximation is based on these values and a x2-homogeneity test is not performed. In this case the interpolated concentration at 50% lethality is designated the approximate LC50. Additionally, the moving average interpolation according to Schaper et al. (1994) is used for calculation, if applicable.
- Analysis of variance (ANOVA): This parametric method checks for normal distribution of data by comparing the median and mean. The groups are compared at a confidence level of (1-α) =95% (p =0.05). The test for the between-group homogeneity of the variance employed Box's test if more than 2 study groups were compared with each other.

Results and discussion

Effect levels

Effect levels 1

Sex:

male/female

Dose descriptor:

other: approximate LC50

Effect level:

8.6 mg/m³ air (analytical)

Exp. duration:

240 min

Remarks on result:

other: estimated lethality threshold concentration (LC01) = 5.3 mg/m3

Effect levels 2

Sex:

male/female

Dose descriptor:

LC50

Effect level:

31.3 mg/m³ air (analytical)

95% CL:

28 - 35

Exp. duration:

60 min

Remarks on result:

other: estimated lethality threshold concentration (LC01) = 21.1 mg/m3

Effect levels 3

Sex:

male/female

Dose descriptor:

LC50

Effect level:

54.5 mg/m³ air (analytical)

95% CL:

48 - 62

Exp. duration:

30 min

Remarks on result:

other: estimated lethality threshold concentration (LC01) = 29.2 mg/m3

Effect levels 4

Sex:

male/female

Dose descriptor:

LC50

Effect level:

253.3 mg/m³ air (analytical)

95% CL:

194 - 331

Exp. duration:

10 min

Remarks on result:

other: estimated lethality threshold concentration (LC01) = 105.3 mg/m3

Any other information on results incl. tables

Table 1: Summary of acute inhalation toxicity of male and female rats - single nose-only exposure to phosgene gas

Duration of Exposure (min)	Analytical concentration (mg/m3)	Corresponding C x t product (mg/m3 x min)	Toxicological results	Onset and duration of signs	Occurrence of Mortality
Males
60	0 (Control)	---	0 / 0 / 5	---	---
10	166.5	1664.6	2 / 5 / 5	0d – 14d	24h
10	181.6	1816.3	0 / 5 / 5	0d – 12d	---
10	212.0	2119.7	1 / 5 / 5	0d – 8d	24h
10	250.9	2509.2	3 / 5 / 5	0d – 9d	24h
30	51.3	1537.5	3 / 5 / 5	0d – 8d	24h
30	54.5	1635.9	3 / 5 / 5	0d – 9d	24h
30	67.7	2029.5	5 / 5 / 5	0d	24h
30	86.9	2607.6	5 / 5 / 5	0d	24h
60	29.9	1795.8	2 / 5 / 5	0d – 10d	24h
60	39.4	2361.6	5 / 5 / 5	0d	24h
60	47.6	2853.6	5 / 5 / 5	0d	24h
240	9.0	2164.8	5 / 5 / 5	0d – 1d	24h, 2d
240	11.1	2656.8	5 / 5 / 5	0d	24h
Females
60	0 (Control)	---	0 / 0 / 5	---	---
10	166.5	1664.6	1 / 5 / 5	0d – 10d	24h
10	181.6	1816.3	0 / 5 / 5	0d – 11d	---
10	212.0	2119.7	1 / 5 / 5	0d – 8d	24h
10	250.9	2509.2	3 / 5 / 5	0d – 10d	24h
30	51.3	1537.5	1 / 5 / 5	0d – 11d	24h
30	54.5	1635.9	1 / 5 / 5	0d – 9d	24h
30	67.7	2029.5	5 / 5 / 5	0d -1d	24h
30	86.9	2607.6	4 / 5 / 5	0d – 8d	24h
60	29.9	1795.8	2 / 5 / 5	0d – 7d	24h
60	39.4	2361.6	4 / 5 / 5	0d – 8d	24h
60	47.6	2853.6	5 / 5 / 5	0d	24h
240	9.0	2164.8	1 / 5 / 5	0d – 10d	24h
240	11.1	2656.8	4 / 5 / 5	0d – 8d	24h, 2d

Toxicological results:

number of dead animals / number of animals with signs after cessation of exposure / number of animals exposed

 

The directed-flow nose-only mode of exposure utilized in this study provides the most controlled means of exposure of small laboratory animals. Each group consisted of 5 male and 5 female young adult Wistar rats. The exposure durations used ranged from 10 to 240 minutes and corresponding C x t products bracketing a range from 1538 - 2854 mg/m3x min. With few exceptions, mortality occurred within 24 hours after exposure. LC50 values for 10, 30, 60, and 240 min were 253.3, 54.5, 31.3, and 8.6 mg/m3,respectively. The corresponding Cn x t products were 2532, 1635, 1878, and 2052 mg/m3x min. In this range of exposure durations, the exponent n was found to be ≈0.9 for both median lethal (LC50) as well as the lethal threshold (LC01) effects. The ratios of LC50/LC01 were 2.4, 1.9, 1.5, and 1.6 for 10, 30, 60, and 240 minutes, respectively. Due to the apparent rat-specific changes in breathing patterns at very short durations of exposure, a generic ratio of 1.5 appears to be most representative.

 

Experimental data appear to suggest that the 10-min LC50 and LC01 values were higher than predicted by the data obtained at 30, 60, and 240-min. This appears to suggest that breathing patterns of rats decrease transiently during the initial exposure period making the rat more resistant due to an apparent decrease in respiratory ventilation. This hypothesis was verified/refuted by exploratory respiratory function measurements.These examinations show unequivocally that with the commencement of exposure respiratory changes rapid in onset occurred and that these changes regressed during continued exposure to phosgene after attainment of their nadir approximately 10-min of exposure. These changes are best characterized by the breathing pattern related endpoints AP, AT, ET/IT and to some extent also TV and MV. Borderline, although distinct, changes occurred at 1.2 mg/m3 and above which did not show evidence of recovery during the 30-min post-exposure period at 47.6 mg/m3and above. Collectively, these data show that for very short exposure periods (≈10-min) and concentrations exceeding 13.3 mg/m3 the alveolar exposures intensity might be lower than predicted due to decreased in ventilation. Therefore, deviations from the C x t relationship at short durations of exposure are inherently associated with a rodent-specific, ventilation related "underdosing" of animals rather than changes in toxic mechanisms. Therefore, short-term exposure indices (<30 min) calculated based on longer exposure periods (30 - 240 min) are conservative per se as the fixed outcome C x t products increased with increasing duration of exposure.

 

In all exposure groups of both sexes clinical signs of the respiratory tract (mainly bradypnea, labored breathing patterns, irregular breathing patterns, stridor, breathing sounds, nasal discharge (serous) and salivation) were observed after phosgene exposure. Statistically significant and concentration-dependent effects of body weight gains were observed in all exposure groups. In all rats that succumbed during the course of study an increased incidence of macroscopic alterations of the respiratory tract were found. These were characterized by a white foamy discharge from the nose, a less collapsed and discolorated (mainly dark-red) lung, hydrothorax, and trachea with white foamy content. Surviving rats did not show macroscopic alterations considered to be of pathodiagnostic significance.

Applicant's summary and conclusion

Executive summary:

The directed-flow nose-only mode of exposure utilized in this study provides the most controlled means of exposure of small laboratory animals. Each group consisted of 5 male and 5 female young adult Wistar rats. The exposure durations used ranged from 10 to 240 minutes and corresponding C x t products bracketing a range from 1538 - 2854 mg/m3x min. With few exceptions, mortality occurred within 24 hours after exposure. LC50 values for 10, 30, 60, and 240 min were 253.3, 54.5, 31.3, and 8.6 mg/m3,respectively. The corresponding Cn x t products were 2532, 1635, 1878, and 2052 mg/m3 x min. In this range of exposure durations, the exponent n was found to be ≈ 0.9 for both median lethal (LC50) as well as the lethal threshold (LC01) effects. The ratios of LC50/LC01 were 2.4, 1.9, 1.5, and 1.6 for 10, 30, 60, and 240 minutes, respectively. Due to the apparent rat-specific changes in breathing patterns at very short durations of exposure, a generic ratio of 1.5 appears to be most representative. In all exposure groups of both sexes clinical signs of the respiratory tract (mainly bradypnea, labored breathing patterns, irregular breathing patterns, stridor, breathing sounds, nasal discharge (serous) and salivation) were observed after phosgene exposure. Statistically significant and concentration-dependent effects of body weight gains were observed in all exposure groups. In all rats that succumbed during the course of study an increased incidence of macroscopic alterations of the respiratory tract were found. These were characterized by a white foamy discharge from the nose, a less collapsed and discolorated (mainly dark-red) lung, hydrothorax, and trachea with white foamy content. Surviving rats did not show macroscopic alterations considered to be of pathodiagnostic significance.