Benzoic acid, 2-chloro-5-isocyanato-, 1,1-dimethyl-2-oxo-2-(2-propen-1-yloxy)ethyl ester

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-en-1-yl 2-(2-chloro-5-isocyanatobenzoyloxy)-2-methylpropanoate

EC Number:

695-953-8

Cas Number:

204918-22-9

Molecular formula:

C15H14ClNO5

IUPAC Name:

prop-2-en-1-yl 2-(2-chloro-5-isocyanatobenzoyloxy)-2-methylpropanoate

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd. Biotechnology & Animal Breeding Division, Wölferstrasse 4, CH-4414 Füllinsdorf / Switzerland
- Age at study initiation: Males, 9 weeks.
Females, 11 weeks.
- Weight at study initiation: Males, Group 1: 244.0 - 261.5 g. Group 2: 239.5 - 257.9 g.
Females, Group 1: 195.1 - 205.5 g. Group 2: 200.0 - 224.6 g.
- Housing: Groups of 5, same sex.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 23°C
- Humidity: 45 - 88 %
- Air changes: 10 - 15/hour
- Photoperiod: 12 hours light / 12 hours night.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

CHAMBER DESCRIPTION
Inhalation exposure system:
Inhalation exposures were performed using a system similar to that originally described by Sachsse et al (1973, 1976). The animals were confined separately in restraint tubes which were positioned radially around the nose-only, flow past exposure chamber. The design of this chamber is based upon the fluid dynamic modelling of the test atmosphere flow. It ensures a uniform test item distribution, provides a constant stream of ‘fresh’ test item to each animal, and precludes re-breathing the exhaled air.

GENERATION OF TEST ATMOSPHERE
Test atmosphere generation:
For each dose group, the test atmosphere was generated from liquefied test item using a glass nebulizer with a stainless steel nozzle and inlet. The test item was liquefied by warming it in the glass nebulizer kept in a water-bath at approximately 80°C. Both the test item inside the glass nebulizer and the water bath were continuously stirred with a magnetic stirrer during aerosol generation. Foe aerosol generation and dilution, compressed filtered air maintained at ambient temperature was fed into the nebulizer. Each generated aerosol was diluted I order to achieve the respective target aerosol concentration of 1 or 5 mg/l air.


TEST ATMOSPHERE
Exposure System Monitoring:
Test atmosphere samples were collected directly from the the feed tube in the breathing zone of the animals, at an empty port of the exposure chamber.

Nominal Concentrations Determination:
For each dose group, the nominal concentration was determined by weighing the glass nebulizer containing the liquefied test item before and after the respective exposure to determine the quantity of test item used. Then the weight used during the 4 hours of exposure was divided by the total airflow volume to give the nominal concentration.

Gravimetric Concentration Determination:
Samples from the test atmosphere were collected four times during the exposure for Group 1 and six times during exposure of Group 2. Each sample filter was weighed before and after sampling. After weighing, each filter of Group 2 was put into a light protected glass vial and covered with 20ml of methanol, as these filters were chemically analysed. During exposure of Group 1, aerosol samples assigned to chemical analysis were taken separately from those assigned to gravimetric determination of aerosol concentration, in order to facilitate methanol coverage of the filter samples assigned to chemical analysis immediately after sampling.

Analytical Concentration Determination:
Samples from the test atmosphere were collected four times during the exposure for Group1 and six times during exposure for Group 2. For Group 1, the samples assigned to chemical analysis were taken separately from those assigned to gravimetric determination. For Group 2 the samples used for gravimetric determination were chemically analysed. After weighing the filters were put into light protected vials and covered with 20g of methanol to minimise loss of test item by evaporation from the filter. The samples were analysed by HPLC with UV detection using a method supplied by the Sponsor.

Particle Size Distribution and MMAD:
The particle size distribution was determined twice during the exposure of Group 1 and three times during the exposure of Group 2. Representative samples of the test atmosphere were drawn through the impactor with a flow rate of 2.7l/min and the particles deposited according to their aerodynamic size onto the impactor slips, on each stage of the impactor. To obtain the mass deposited on each stage of the impactor, the aluminium slips were weighedbefore and after sampling.
The total mass (µg) deposited in the impactor was then calculated by adding together the mass deposited on each impactor stage.
As the effective Cut-off Diameter (ECD) represent the lower size limit of the particles collected on each stage, the cumulative percent less than the indicated size was tabulated as a function of the ECD.
This data was used to calculate he mass median aerodynamic (MMAD) and geometric standard deviation (GSD). The target range for the MMAD was 1 to 4 µm.

Oxygen Concentration:
The oxygen concentration was continuously monitored and recorded for the duration of each exposure period. The results were reported at 30 minute intervals from the start to the end of exposure.

Relative humidity / temperature:
The relative humidity and temperature were continuously monitored and recorded for the duration of each exposure period. The results were reported at 30 minute intervals from the start to the end of exposure.

Airflow rate:
The exposure airflow rate was adjusted before each exposure and monitored indirectly through the aerosol generation and dilution systems. The airflow rates for aerosol generation and dilution were recorded nine times during each inhalation exposure period (i.e. 30 min intervals form start to end of exposure).
For Group 1, the total airflow was maintained at 24.6 l/min, i.e. 4.6 l/min for aerosol generation in the glass nebulizer in addition to 20.0 l/min for dilution. For Group 2, the total airflow was maintained at 23.6 l/min, i.e. 5.6 l/min for aerosol generation in addition to 18.0 l/min for dilution. The airflow rate of the test atmosphere per animal port was 1.03 l/min during the exposure period of Group 1 and 0.98 l/min during that of Group 2.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

ca. 4 h

Concentrations:

The target aerosol concentration for the exposure of Group 2 (i.e. first exposure) was 5 mg/l air for 4 hours. The target aerosol concentration for Group 1 (i.e. second exposure) was chosen to find a concentration inducing no or low mortality.

Table 1
Administered Test Atmosphere Concentration

Group Concentration of Test Item (mg/l air)
Nominal Gravimetric Analytical
1 2.114 1.316 ± 0.061, n=4 1.238 ± 0.022, n=4A
2 6.755 5.455 ± 0.503, n=6 5.162 ± 0.532, n=6A

A: Mean atmosphere concentration and standard deviation of test item (as received from the Sponsor) per litre air obtained from n filter samples chemically analysed for the active ingredient of the test item.

No. of animals per sex per dose:

5

Details on study design:

- Duration of observation period following administration:
14 days

- Frequency of observations and weighing:
Mortality: Mortality was checked once daily during the acclimatisation phase, once before exposure on the day of exposure, once per hour during exposure,
at least once after exposure on test day 1, and twice daily during the remainder of the observation period. Total of 15 days.
Clinical signs: Clinical signs were recorded once per hour during exposure, once after test day 1, and once daily thereaftre.
Body weights: Bdy weights were rcorded on test days (before exposure), 4, 8 and 15 (day of necropsy).

- Necropsy of survivors performed:
yes
Animals surviving to the end of the test period were sacrificed on day 15.
Animals which died spontaneously during the exposure or observation period were necropsied when found dead.

All animals were necropsied and abnormalities recorded. The lungs, trachea, larynx and the head containg the nasopharyngeal tissues were collected from all animals and fixed in neutral phosphate buffered 4% formaldehyde solution. The lungs were instilled with the fixative at a hydrostaic pressure of 30cm H2O.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

>= 1.238 - <= 5.162 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

All males of Group 1and one of ten animals of Group 2 (female no. 10) survived the exposure and subsequent 14 day observation period, and were sacrificed as scheduled on test day 15. The deaths of four males (nos. 1, 3, 4 and 5) and one female (no. 8) of Group 2, were noticed between 3 hours and 3 hours 45 minutes after beginning of the 4-hour exposure period, and one male (no. 2) and two females (nos. 6 and 7) died within 2.5 hours after the end of the exposure. Early in the morning on the day afterwards (test day 2), a further female (no. 9) and Group 2 was found dead.

Table 2
Mortality observed
Male & Female Males Females
Group 1 (1.238 mg/l air) 0 % 0 % 0 %
Group 2 (5.162 mg/l air) 90 % 100 % 80 %

Clinical signs:

other: Group 1: Slight to moderate breath sounds (rales) (0m/3f), a decrease in spontaneous activity with slight severity in male animals and slight to moderate severity in female animals (5m/5f), ruffles fur with slight to moderate severity in male animals and

Body weight:

Group 1:
Slight to moderate breath sounds (rales) (0m/3f), a decrease in spontaneous activity with slight severity in male animals and slight to moderate severity in female animals (5m/5f), ruffles fur with slight to moderate severity in male animals and slight to marked severity in female animals (5m/5f), and hunched posture (0m/5f).
All findings were first noticed approximately 4 hours after the beginning of exposure, i.e. outside the exposure tubes immediately after the end of exposure. The findings of rales and hunched posture disappeared within two days, that of decreased spontaneous activity and ruffled fur within three days after exposure. As from two days after the exposure (test day 3) until termination of the study on test day 15 all male animals and from three days after the exposure (test day 4) all female animals were free from clinical signs.

Group 2:
Tachypnea (0m/4f), slight rales (0m/2f), a slight to marked decrease in activity with regards to gait/motility (1m/4f), and ruffled fur with slight severity in one male animal and slight to moderate severity in female animals (1m/2f)).
All findings were first noticed approximately 4 hours after the beginning of exposure, i.e. outside the exposure tubes immediately after the end of exposure. By then four males and one female had already died. In the only survivor of this group (female no. 10), the findings of decreased activity, and ruffled fur disappeared within four days, that of tachypnea within five days after the exposure, whilst slight rales persisted until the scheduled day of necropsy on day 15. In view of 90% mortality in Group 2, the observation of the one female survivor was terminated on day 15, although the clinical signs had not fully cleared by then.

Gross pathology:

Group 1:
Examination of each animal on the scheduled day of necropsy (test day 15) did not reveal any macroscopical findings.
Group 2:
Necropsy revealed the following findings in the lungs of the nine animals which died during and/or within 1 day after the exposure. Incompletely collapsed (5m/4f), dark red discoloured (5m/4f), several or many grey white foci of 2 – 3 mm diameter (3m/3f) and foamy fluid released from the bronchi (2m/4f). In the only survivor of this group (female no.10), the findings of alopecia in th head region was noted. The findings in the lungs of the premature deaths were attributed to the treatment with the test item. The finding of alopecia in the female survivor was considered to be secondary to adhesion of some of the test aerosol to animal hair in the head region, and not to represent a sign of toxicity.

Applicant's summary and conclusion

Interpretation of results:

harmful

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The LC50 of CA 2814 B (Intermediate of CGA 276854) obtained in this study was estimated to be between 1.238 and 5.162 mg/l air.

Executive summary:

 

Groups of five male and five female albino rats (Hanlbm:WIST(SPF)) were exposed by nose-only, flow-past inhalation to the test item at mean concentrations of 1.238 or 5.126 mg/l air. At 1.238 mg/l air, the mean mass median aerodynamic diameter (MMAD) was 4.16 µm the geometric standard deviation (GSD) 2.76. At 5.162 mg/l air, the MMAD and GSD was 7.62 µm and 3.67, considerably exceeding MMAD target range of 1 to 4 µm recommended for acute inhalation toxicity testing. 

All animals were observed for clinical signs and mortality during and/or following the inhalation exposure, i.e. until premature death or over a 15 day observation period. Body weights were recorded prior to exposure on test day 1, and during the observation period on test days 4, 8 and 15. On day 15, all animals were sacrificed and necropsied. 

 

The ranges of temperature, relative humidity, oxygen content and airflow measured during the exposures were satisfactory for a study of this type. 

 

There were no deaths in Group 1 (1.238 mg/l air). In Group 2 (5.162 mg/l air) 9 of 10 animals (5 males and 4 females) died towards the end of exposure or within one day afterwards. The principle clinical signs consisted of effects on breathing, decreased activity and ruffled fur seen at both dose levels, and hunched posture at the low-dose level. The effects on breathing were reflected by the findings of breath sounds (rales) at both dose levels, in addition to tachypnea at the high-dose level. All clinical signs were first noticed immediately after the end of exposure and disappeared in Group 1 within two to three days afterwards. In the only survivor of Group 2, the finding of breathing sounds (rales) persisted until the scheduled day of necropsy (day 15). Marginal to slight, transient body weight losses were evident in two female animals of Group 1, and a marked, transient body weight loss in the female survivor of Group 2. The clinical signs and transient losses of body weight were attributed to treatment with the test item. Macroscopic changes in the premature deaths, such as incompletely collapsed and dark red discoloured lungs, grey white foci in the lungs and/or foamy fluid released from the bronchi were also attributed to the treatment with the test item. 

 

High biological effectiveness of the test aerosol of Group 2 was evident from the 90% mortality and the necropsy findings of foamy fluid released from the bronchi and several/may white foci in the lungs obtained in this group. Therefore, the large MMAD measured for the test aerosol of Group 2, exceeding the recommended target range, was toxicologically irrelevant. 

 

In conclusion, the LC50 of CA 2814 B (Intermediate of CGA 276854) obtained in this study was estimated to be between 1.238 and 5.162 mg/l air.