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Toxicological information

Acute Toxicity: inhalation

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Administrative data

acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 403) performed under GLP, only 10% formulation of the registered substance was tested.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 403 (Acute Inhalation Toxicity)
according to guideline
EU Method B.2 (Acute Toxicity (Inhalation))
according to guideline
EPA OPPTS 870.1300 (Acute inhalation toxicity)
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:

Test material

Constituent 1
Test material form:
solid - liquid: suspension
dispersion containing micronized ETH50 (nano form)
Details on test material:
Please refer to "Confidential details on test material".

Test animals

Details on test animals or test system and environmental conditions:
- Source: RCC Ltd Laboratory Animal Services, 4414 Füllinsdorf, Switzerland
- Age at study initiation: about 9-10 weeks old
- Weight at study initiation: males 226-262 g, females 186-223 g
- Housing: the animals were kept in groups of 5 rats /sex in Makrolon® type-IV cages with wire mesh tops and standard softwood bedding
- Diet: free access to certified standard diet (Kliba-Nafag 3433)
- Water: Tap water was offered ad libitum
- Acclimation period: At least 5 days to the laboratory environment

- Temperature: 22 ± 3 °C
- Humidity: 33 - 63 %
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
flow-past exposure
other: purified water
Details on inhalation exposure:
Inhalation Exposure System
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.

Test Atmosphere Generation
Both, the aqueous placebo dilution prepared for Group 1 and the aqueous test item dilution prepared for Group 2 were generated in ambient conditions using a 1000 mL cyclone glass atomiser with a stainless steel injector but without a baffle in the path of the air/liquid mixture. The atomiser was connected to an appropriate reservoir containing the aqueous placebo or test item dilution. At the bottom of the atomiser a hole and drainage pipe were in place, in order to prevent separation, accumulation and/or drying up of placebo or test item components inside the atomiser and consequently blockage of the atomiser. The aqueous placebo or test item dilution inside the reservoir was stirred with a magnetic stirrer during the respective aerosol generation. The placebo aerosol (assigned to Group 1) was generated from the aqueous placebo dilution at similar settings as those used for the generation of the test aerosol (assigned to Group 2) and on both occasions the atomiser was operated at maximum throughput of placebo or test item dilution. Therefore, the aerosol concentrations administered to the animals
were considered to represent the highest technically achievable aerosol concentration levels suitable for acute inhalation toxicity testing in the rat.

Exposure System Monitoring
The concentration of the aqueous placebo and test item dilutions determined gravimetrically and/or by chemical analysis, the particle size distribution determined gravimetrically, temperature, relative humidity and oxygen concentration were measured on test atmosphere samples collected directly from the aerosol delivery tube in the breathing zone of the animals, at an empty port of the exposure chamber. This approach was chosen in order to obtain representative samples of what was delivered to the animals. Airflow rates were determined for the recording of temperature, relative humidity and oxygen concentration and during the collection of samples for the determination of test aerosol concentration using a dry-test meter and a pressure gauge, calibrated with a reference dry-test meter. Sampling airflow rates during the collection of impactor samples were determined using a calibrated pressure gauge.

Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
Remarks on duration:
single exposure
A concentration of 4.976 mg/L air was attained during the 4 hour exposure period and was considered to represent the highest technically achievable aerosol concentration level
No. of animals per sex per dose:
15 animals per sex in 2 dose groups (test item group and placebo control group), subdivided into 3 satellite groups each:

1) sacrifice at approximately 14 hours post end of exposure for broncho-alveolar lavage fluid (BALF) and plasma sampling (5 animals per sex and treatment)
2) interim pathology at approximately 24 hours post end of exposure (5 animals per sex and treatment)
3) pathology at 14 days post exposure (5 animals per sex and treatment)
Control animals:
Details on study design:
- Duration of observation period following administration: 14 days (satelite group 3)
- Frequency of observations and weighing:
Mortality was checked at least once daily during the acclimatization period, once before exposure on the day of exposure (test day 1), at approximately 1, 2, 3 and 4 hours after exposure start, approximately 1 hour after the end of exposure on test day 1, and at least twice daily during the remainder of the observation period.
In each dose group, clinical signs were recorded at approximately 1, 2, 3 and 4 hours after exposure start A and approximately 1 hour after the end of exposure on the respective test day 1. In addition, clinical signs were recorded once daily on test days 2 to 15. Observations included, but were not limited to changes in behaviour, somatomotor activity, body position, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, e.g. tremors or convulsions, reactivity to handling or sensory stimuli, altered strength, alteration of the skin, fur, nose, eyes and mucous membranes.
In each dose group, body weights were recorded on the respective test day 1 (before exposure) in all animals, and on the respective test days 4, 8 and 15 in the animals assigned to sacrifice on test day 15.

- Necropsy of survivors performed: yes
- Other examinations performed:
Their terminal body weights and lung weights were recorded and the lung to terminal body weight ratios determined.
Broncho-Alveolar Lavage Fluid (BALF):
The lungs of each animal were washed six times with approximately 4 mL physiological saline at room temperature by slow instillation and withdrawal of fluid with thoracic massage. After centrifugation, aliquots of the cell suspension were taken for total and differential cell counts.
TNF-alpha and IL-6 levels were determined in duplicate in the supernatant of BALF using ELISA. Determination of total protein was performed.

Histotechnique / Histopathology:
From all animals (excluding those used for BALF and plasma investigations) the lungs (sections from two lobes) and tracheobronchial lymph nodes were processed, embedded in paraffin, cut at a nominal thickness of 2-4 micrometers, stained with hematoxylin and eosin, and histopathologically examined using a light microscope.
PROBIT analysis for calculation of LC50 was not used, as there were no deaths and only one group exposed to placebo dilution and one group exposed to test item dilution were used.
Statistical tests adopted for the comparison of some of the data from Group 1 (Placebo Dilution) with the respective data from Group 2 (Test Item Dilution) are stated on the corresponding summary tables.

Results and discussion

Effect levels
Dose descriptor:
Effect level:
> 4.976 mg/L air
Based on:
Exp. duration:
4 h
Remarks on result:
other: Mean aerosol concentration of the aqueous dilution of FAT 65’080/F (containing approximately 10% of its active ingredient, ETH50)
No premature deaths occurred in this study. All animals were sacrificed as scheduled on test
days 2 (day of interim sacrifice) or 15 (terminal sacrifice).
Clinical signs:
other: There were no clinical signs attributable to treatment with the aqueous placebo dilution (Group 1) or test item dilution (Group 2) during the present study period. The only finding noted was hair loss in both forelegs in two female animals of Group 2 duri
Body weight:
Possible effects of treatment on body weight were not evaluated in the animals assigned to sacrifice on test day 2 at approximately 14 or 24 hours post end of exposure. Intergroup comparison of body weight in the animals assigned to terminal pathology at 14 days post exposure (test day 15), revealed statistically significantly lower body weight in female animals of Group 2 (Test Item Dilution) than in those of Group 1 (Placebo Dilution) on test day 4. A trend similar to the one in female animals, but statistically not significant, was also seen in male body weights on this day. However, this, possibly test item related, effect on body weight was only transient and considered to be minor in degree, as no body weight loss was seen in any
animal during any interval from weight recording to weight recording.
Gross pathology:
Macroscopic pathology findings attributable to treatment with the test item dilution were not
Other findings:
In BALF collected at approximately 14 hours post end of exposure total cell count (mainly macrophage and neutrophil numbers), TNFα and total protein levels were considerably higher in both sexes of Group 2 than in Group 1, whilst total protein levels in plasma did not distinguish the two groups. The changes in BALF were consistent with the histopathology findings of granulocytic infiltration in alveolar wall and lumen, diffuse alveolar histiocytosis and alveolar lining cell activation seen in all animals of Group 2 assigned to interim pathology at approximately 24 hours post end of exposure (test day 2). By test day 15, these histopathology findings were no longer evident in Group 2. They were never seen in Group 1.
In addition, on test day 2, lung weights and lung to terminal body weight ratios were moderately higher in both sexes of Group 2 than in Group 1. By test day 15, the mean lung weight was only slightly higher in males of Group 2 than in Group 1 and was unaffected in females. Lung to body weight ratios did no longer differ significantly between Groups 1 and 2.

Any other information on results incl. tables

The MMADs obtained from two gravimetric measurements of particle size distribution during each exposure were marginally higher in Group 2 as compared to Group 1 and almost identical within each group. The latter led to the conclusion that the particle size of the generated aerosol was quite stable throughout each exposure period.

All MMADs were also within the target range of 1 to 4 μm (and the GSD was in the target range of 1.5 to 3.0), so that deposition of the particles can be assumed to have occurred in both, the upper and the lower respiratory tract, in each dose group. Hence, the particle size distribution and MMADs obtained were considered to be appropriate for acute inhalation toxicity testing.

Temperature and oxygen concentration of the generated atmospheres were quite stable and at acceptable levels during both inhalation exposures.

Applicant's summary and conclusion