Registration Dossier

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:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Principles of method if other than guideline:
Deviation: Test article was fed into the nebulizer from a glass resevoir, not from a syringe pump, in order to facilitate pre-warming of the test article thus reducing its viscosity and faciliating aerosol generation. Mentioning of the syringe pump was an unintended false statement in the protocol.
GLP compliance:
yes (incl. certificate)
Test type:
fixed concentration procedure

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd. Biotechnology & Animal Breeding Division. 4414 Füllinsdorf / Switzerland
- Age at study initiation: Males: 9 weeks. Females: 10 weeks
- Weight at study initiation: Males: 255.9 - 287.1 g. Females: 205.4 - 225.8 g
- Fasting period before study: None
- Housing: Groups of 5
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Approxiately 19° C - 20° C
- Humidity (%): 43 - 66 %
- Air changes (per hr): 10 - 15/hour
- Photoperiod: 12/12 hours artificial fluorscent light. Music played during the light and dark period.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Inhalation exposure system.
The animals were confined separately in restraint tubes which were positioned radially around the exposure chamber. The design ensures a uniform test article distribution, provides a constant stream of “fresh” test article to each animal, and precludes rebreathing the exhaled air.

- Test atmosphere generation:
The test article was warmed in a nebulizer and a glass reservoir supplying the nebulizer, both kept in a water bath at approximately 60°C.
Compressed filtered air, pre-heated by passage through a copper spiral, which was placed in an approximately 95°C polyethyleneglycol bath, was fed into the nebulizer at 7.5 litres/minute. Then the generated aerosol was diluted using compressed filtered air in order to achieve the target concentration of 5 mg test article per litre air. The diluent air (12L/min) was pre-heated by passage through a copper spiral which was places in another approximately 95°C polyethyleneglycol bath. The aerosol dilution system was connected to the exposure chamber which was kept at ambient
temperature.

- Method of holding animals in test chamber: Restraint tubes

- Source and rate of air: Compressed filtered air including test article: 7.5 L/min
Diluent air: 12 L/min

- Method of particle size determination: MMAD - Target range 1 to 4 µm. Refer to Table 1.

- Temperature, humidity, pressure in air chamber: Refer to Table 2.

TEST ATMOSPHERE
- Brief description of analytical method used: HPLC with UV detection
- Samples taken from breathing zone: Yes

CLASS METHOD
- Rationale for the selection of the starting concentration: Target concentration for the exposure was 5 mg/L air (actual concentration of
respirable substances) for 4 hours (OECD 403 "Acute Inhalation Toxicity").
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
ca. 4 h
Concentrations:
Target: 5 mg/L air
Nominal: 6.440 mg/L air
Analytical: 5.313 mg/L air (± 0.139, n = 4)
Gravimetric: 5.447 mg/L air (± 0.147, n = 4)
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days

CLINICAL OBSERVATIONS
- Mortality: Mortality checked once per hour during exposure, once after exposure on test day 1, and twice daily during the observation period of the test.

- Clinical Signs: Clinical signs checked once per hour during exposure period, once after
exposure on test day 1, and once daily during the remainder of the
observation period.

- Body weights: Body weights were recorded on test days 1 (before exposure), 4, 8 and 15
(day of necropsy).


- Necropsy of survivors performed: yes
- Necropsy: Animals were examined macroscopically and any abnormalities were recorded. The lungs, trachea, larynx and the head containing the nasopharyngeal tissues
were collected from all animals and fixed in neutral phosphate buffered 4%
formaldehyde solution. In addition the thyroid gland of female no. 9 was collected
and fixed in neutral phosphate buffer 4 %formaldehyde solution due to
macroscopic change. The lungs were instilled with the fixative at a hydrostatic pressure of 30 cm H2O.
Statistics:
The LOGIT-Model was not used as only one group ws exposed and there were no deaths.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
ca. 5.313 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No spontaneous deaths occurred in this study. All animals were sacrificed as scheduled on test day 15.
Clinical signs:
other: The following clinical signs were recorded during and/or after the inhalation exposure in all animals. Slight (males) / slight to moderate (females) salivation, slight to moderate decrease in spontaneous activity, slightly to markedly ruffled fur, and hu
Body weight:
There were slight transient losses in body weight in one of five males (no. 3, -5.6 %) and all of five female animals (mean loss in all females -3.0 %) from test day 1 (prior to exposure) to test day 4 (three days after exposure). During the remainder of the 15-day observation period all male and all but one female animals (except female no. 9 (gained body weight normally. In female no. 9, the body weight gain remained retarded until test day 8 (seven days after exposure) re-increasing thereafter.
Gross pathology:
Macroscopical findings:
Both sides of the thyroid gland were thickened in female no. 9. At the present stage, this finding was not attributed to treatment with the test article, as there was only one incidence. Examination of each other animal on the scheduled day of necropsy (test day 15) did not reveal any macroscpoical findings.
Other findings:
There were no deaths. Clinical signs consisted of salivation, decreased spontaneous activity, ruffled fur and hunched posture in all animals and were considered to be treatment related. Clinical signs were seen from approximately 1 hour after the beginning of exposure and disappeared within 2 to 3 days after exposure. Slight, transient losses in body weight were evident in one male and all female animals followed by a transient retardation in body weight gain in one of the females were considered to be treatment related. There were no macroscopical pathology findings attributable to treatment.

Exposure Conditions:
The nominal, analytical and gravimetric test atmosphere concentrations (means ± SD) the Mass Median Aerodynamic Diameter (MMAD) and its Geometric Standard Deviation (GSD) are summarised in Table 1.

The mean test atmosphere concentration determined by chemical analysis compared well (-2.5%) with the gravimetrically determined mean concentration, and was slightly lower (-17.5%) than the nominal aerosol concentration. Accumulation of some of the test article in the exposure system may have contributed to the slight difference between the analytical/gravimetric and the nominal concentrations.
The particle size (expressed as mean MMAD of two impactor samples) of the generated aerosol was well within the target range of 1 to 4 µm, so that deposition of the particles can be assumed to have occurred in both, the upper and the lower respiratory tract. Hence, the particle size distribution and MMAD obtained were considered to be appropriate for acute inhalation toxicity testing.
The ranges of temperature, relative humidity and oxygen concentration measured during the exposure and indicated in Table 2.


Any other information on results incl. tables

Table 1:

 

Achieved Concentrations (mg/L air)

± Standard Deviation

MMAD (µm) and [GSD]

Group

Nominal

Gravimetric

Analytical

 

1

6.440

5.447± 0.147

(n = 4)

5.313± 0.139

(n = 4)

2.63

[2.42]

 

Table 2:

Group

Temperature (°C)

Rel. Humidity (% RH)

O2 Concentration (Vol %)

1

22.8 ± 0.37

n = 9

7.3 ± 0.11

n = 8

20.9 ± 0.03

N = 9

 

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
The LC 50 of CA 2219 A (Intermediate of CGA 276854) obtained in this study, was estimated to be greater than 5.313 mg/l air (chemically determined mean concentration of undiluted formulated test article in the test atmosphere).
Executive summary:

The purpose of this acute 4-hour inhalation toxicity study was to assess the acute inhalation toxicity of CA 2219 A (Intermediate of CGA 276854) when administered to rats for a single continuous 4-hour period at target concentration of 5 mg/l air. 

 

Groups of five male and 5 female Albino Wistar rats were exposed by nose only, flow past inhalation to the test article at a mean concentration of 5.313 mg/L air (s.d. ± 0.139, n = 4), and a mean mass median aerodynamic diameter (MMAD) of 2.63 µm and geometric standard deviation (GSD) of 2.42 (mean of two impactor samples). All animals were observed for clinical signs and mortality during and following the inhalation exposure, i.e. 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 (day of necropsy). All animals underwent necropsy and any gross macroscopical changes were recorded. 

 

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

 

There were no deaths. Clinical signs consisted of slight to moderate salivation, a slight to moderate decrease in spontaneous activity, slight to markedly ruffled fur and hunched posture in all animals. The finding of salivation was restricted to the exposure period, the other clinical signs disappeared within 2 to 3 days afterwards. Slight, transient losses in body weight were evident in one male and all female animals from test day 1 to 4, and were followed by retarded body weight gain in one of the females until test day 8. There were no macroscopical pathology findings attributed to treatment. 

 

In conclusion, the LC 50 of CA 2219 A (Intermediate of CGA 276854) obtained in this study, was estimated to be greater than 5.313 mg/l air (chemically determined mean concentration of undiluted formulated test article in the test atmosphere). –

 

The clinical signs observed in all animals and the transient effect on body weight development in one male and all female animals were attributed to treatment with the test article.