Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 9 to July 14, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): LAT (Bacillus licheniformis BML 612)(GICC M05003242)
- Substance type: UVCB
- Physical state: liquid
- Lot/batch No.: 20088051
- Expiration date of the lot/batch: At lest stable until 1 May 2011
- Stability under test conditions: The test material and dilutions of 50% and 25% is stable for at least 5 hours at room temperature
- Storage condition of test material: minus 20 degrees of C, stable for at least 90 days

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Harlan UK Ltd., Oxon, UK.
- Housing: Five animals of same sex per cage, (polypropylene cage)
- Weight at time of dosing: between 202-231 g (females), 244- 264 g (males)
- Housing: In animal room with control of temperature and humidity
- Diet: Standard diet ad libitum
- Water: Tap water ad libitum
- Acclimatization period: At least 5 days
- Temperature (°C): 19-25°C
- Humidity : 30-70 %

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Radleys, Saffron Walden, Essex, UK
- Exposure chamber volume: 30 L, flow rate 45 L/min providing 90 air changes per hour at an oxygen concentration of 20.8%.
- Method of holding animals in test chamber: Snout only
- Source and rate of air: Compressed air was supplied by an oil free compressor and passed through a water trap and respiratory quality filters before introduced to the nebuliser, the aerosol generator at a flow rate of 10 L /min.
- Method of conditioning air: The nebuliser was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test material under pressure, and to a metered compressed air supply.
- System of generating particulates/aerosols: The test material was aerosolised using a glass concentric jet nebuliser located on the top of the exposure chamber.
- Method of particle size determination: Gravimetric analysis using a marble cascade impactor at 90, 150 and 225 min after start of exposure. The material collected on the stages of the sampler was weighed to determine the particle size distribution in the atmosphere.
- Treatment of exhaust air: Through a high efficiency filter to a metered exhaust system
- Temperature, humidity, pressure in air chamber: Temperature and relative humidity were measured in the animals breathing zone and recorded every 30 minutes during the 4 hour exposure period; 17-19 degrees of C during exposure, 63-100% humidity during exposure, normal pressure of the atmosphere

TEST ATMOSPHERE
- Brief description of analytical method used: Seventeen air samples of 2 L during the 4 hour exposure period were taken, at 2 L/min, through weighed glass fibre filters. The collected material was weighed to determine the concentration of test material in the exposure chamber.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: 73.1% inhalable (< 4 um)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): MMAD=2.12 µm, GSD=2.82 µm

Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
>= 4 h
Concentrations:
4.96 mg/L (mean achieved atmosphere concentration of test item as received)
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations for clinical signs of effect: Hourly intervals during exposure, immediately and one hour after termination of exposure and subsequently once daily in the 14-day observation period. Weighing: Prior to treatment on the day of exposure and on Days 7 and 14.
- Necropsy of survivors performed: yes

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 4.96 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality.
Clinical signs:
Clinical signs of hunched posture and piloerection were observed following exposure. This is commonly noted in animals due to the method of restraint. During and following exposure, the rats exhibited a wet appearance also commonly seen due to the method of restraint. In addition increased respiratory rate was noted in all animals during exposure, on removal from the chamber and one hour post-exposure. Animals recovered quickly to appear normal from the first day post-exposure.
Body weight:
Normal body weight development was noted during the study.
Gross pathology:
No abnormalities.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In a group of ten rats no deaths occured following 4 hours of inhalation of alpha-amylase at a mean achieved atmosphere concentration of 4.96 mg/L, based on test material as received. It is therefore considered that the LC50 for alpha-amylase is greater than 4.96 mg/L.
Executive summary:

In accordance with OECD guideline No. 403, a Limit Test was performed with one group of rats consisting of 5 females and 5 males.

The animals were exposed by snout only exposure for 4 hours to air containing aerosolised alpha-amylase at a concentration of 4.96 mg/L (test material as received).

Particle size measurements revealed that the respirable fraction (% of aerosol mass < 4 µm) was 73.1%. The mass median aerodynamic diameter was 2.7 µm.

The animals were observed for clinical signs at hourly intervals during exposure immediately and one hour after termination of exposure and subsequently once daily in the 14-day observation period. After the observation period, the animals were sacrificed and examined pathologically.

No deaths occured during the study period. Clinical signs of hunched posture and piloerection were observed following exposure. This is commonly noted in animals due to the method of restraint. During and following exposure, the rats exhibited a wet appearance also commonly seen due to the method of restraint. In addition increased respiratory rate was noted in all animals during exposure, on removal from the chamber and one hour post-exposure. Animals recovered quickly to appear normal from the first day post-exposure.

No effect was observed on the body weight, and the pathological examination revealed no abnormalities.

 

Clinical signs of hunched posture and piloerection were observed following exposure. This is commonly noted in animals due to the method of restraint. During and following exposure, the rats exhibited a wet appearance also commonly seen due to the method of restraint. In addition increased respiratory rate was noted in all animals during exposure, on removal from the chamber and one hour post-exposure. Animals recovered quickly to appear normal from the first day post-exposure. No effect was observed on the body weight and necropsy revealed no abnormalities.

In conclusion, no deaths occured in rats after 4 hours of inhalation of a mean achieved atmosphere concentration of alpha-amylase of 4.96 mg/L. LC50 for alpha-amylase is therefore considered to be greater than 4.96 mg/L.