Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 September to 04 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 436 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
2009
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on July 10, 2012 / signed on September 07, 2012)
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Pale yellow liquid
- Storage condition of test material: Approximately 4 °C in the dark under nitrogen

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 8-12 weeks
- Weight at study initiation: 200-350 g
- Housing: Animals were housed in groups of three by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes
- Diet: Food (Harlan 2014C Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: Fifteen changes per hour
- Photoperiod: 12 h dark / 12 h light

N-LIFE DATES: 13 September to 04 October 2012

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
1.93 µm
Geometric standard deviation (GSD):
2.65
Remark on MMAD/GSD:
Inhalable fraction (% < 4 µm) = 77.4
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was aerosolised using a glass concentric jet nebuliser (Radleys, Saffron Walden, Essex, UK) located at the top of the exposure chamber. The nebuliser was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and to a metered compressed air supply.
- Exposure chamber volume: Cylindrical exposure chamber had a volume of approximately 30 litres (dimensions: 28 cm diameter x 50 cm high).
- On the day of exposure each rat was acclimatised (for approximately 2 hours) to a tapered polycarbonate restraining tube.
- Method of holding animals in test chamber: During the exposure, each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.
- Source and rate of air: Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the nebuliser. Chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.
- Method of particle size determination: The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK).
- Treatment of exhaust air: The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure.
- Temperature and humidity in air chamber: Temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every thirty minutes throughout the four-hour exposure period.
- Exposure Chamber Oxygen Concentration: Oxygen levels within the exposure chamber were measured by an electronic oxygen analyser (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the four-hour exposure period. The test atmosphere was generated to contain at least 19% oxygen.
- Exposure Chamber Atmosphere Concentration:
Prior to the inhalation phase of the study, the non-volatile component of the test item was determined by adding a small, known amount of test item to glass fibre filters and recording their weights. The filters were then dried in a desiccator between 19 and 21°C for approximately 24 hours and then weighed again. The difference in the two weights was taken as the volatile content of the test item and the non-volatile component was calculated as a percentage. The mean non-volatile component of the batch used during the formal exposure was found to be 96.1 % (n=10).
The test atmosphere was sampled at regular intervals during the exposure period. A weighed glass fibre filter was placed in a filter holder and temporarily sealed in a vacant port of the exposure chamber in the animals’ breathing zone. A known quantity of the exposure chamber atmosphere was drawn through the filter using a vacuum pump.
After sampling, the filter was dried, under similar conditions as those previously described, and weighed again 24 hours later. The difference in the pre and post sampling weights, divided by the volume of atmosphere sampled, was the chamber concentration in terms of non-volatile component.
Based on the results of the preliminary work, these figures were adjusted to obtain a true figure for the test item concentration in the chamber.
The nominal chamber concentration was calculated by dividing the mass of test item disseminated into the chamber by the total volume of air that flowed through the chamber during the exposure.
The nominal concentration was 353 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was extremely difficult, even when generating an aerosol from a formulation of the test item.
- Pretreatment: Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterisation period test item input rates were varied to achieve the required atmospheric concentrations.
- Following an appropriate equilibration period a single group of six rats (three males and three females) was exposed to an atmosphere of the test item for a period of four hours. A target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 106 % of target and no deaths occurred, no further levels were required.

TEST ATMOSPHERE
-Homogeneity of the test atmosphere within the chamber was not specifically determined during this study. Chambers of the same design (ADG Developments Ltd, Hitchin, Herts, UK) have been fully validated and shown to produce evenly distributed atmospheres in the animals’ breathing zone with a wide variety of test items (Green J D et al, 1984).
- Samples taken from breathing zone: Yes

TEST ATMOSPHERE
- Particle size distribution: See table 7.2.2/2
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 1.93 µm / 2.65
Analytical verification of test atmosphere concentrations:
no
Duration of exposure:
4 h
Concentrations:
Target concentration: 5 mg/L
Mean achieved atmosphere concentration: 5.31 ± 0.22 mg/L
Nominal concentration: 18.8 mg/L
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for fourteen days. Any evidence of overt toxicity was recorded at each observation.
- Frequency of weighing: Individual bodyweights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14.
- Necropsy of survivors performed: Yes, At the end of the fourteen day observation period all animals were killed by intravenous overdose of sodium pentobarbitone. All animals, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.
Statistics:
None

Results and discussion

Preliminary study:
Not applicable
Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.31 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: no mortality was observed
Mortality:
No mortality was observed.
Clinical signs:
- Signs of hunched posture, pilo-erection and red/brown staining around the eyes and snout are commonly seen in animals for short periods on removal from the chamber following 4-hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. These observations are considered to be associated with the restraint procedure and, in isolation, are not indicative of toxicity.
- In addition to the observations considered to be due to the restraint procedure, increased respiratory rate was noted in all animals during exposure, on removal from the chamber and one hour after removal.
- One day after exposure, all animals exhibited increased respiratory rate, hunched posture and pilo-erection. Animals recovered to appear normal from Days 8 to 10 post-exposure.
Body weight:
- Two males and two female animals exhibited slight bodyweight losses on the first day post-exposure. Reasonable bodyweight development was noted in all male animals during the remainder of the recovery period. In contrast, two female animals exhibited bodyweight losses from Days 1 to 3 post-exposure.
- Bodyweight gains were noted in all female animals for the remainder of the recovery period.
Gross pathology:
No macroscopic abnormalities were detected amongst animals at necropsy.
Other findings:
None

Any other information on results incl. tables

Table 7.2.2/1: Exposure Chamber Concentration

The test atmosphere was sampled seventeen times during the exposure period and the actual concentration of the test item calculated. The mean values obtained were:

Atmosphere Concentration

Mean Achieved (mg/L) 

Standard Deviation 

Nominal (mg/L) 

5.31

0.22

18.8

 

The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.

The theoretical chamber equilibration time (T99) was 3 minutes (Silver, 1946).

Table 7.2.2/2: Particle Size Distribution

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (μm)

Inhalable Fraction

(% <4 μm)

Geometric Standard Deviation

5.31

1.93

77.4

2.65

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the inhalation LC50 for ST 01 C 11 is higher than 5.31 mg/L for 4 h in rats and therefore it is not classified according to Regulation (EC) No. 1272/2008 (CLP) and to GHS.
Executive summary:

In an acute inhalation toxicity study performed according to OECD Guideline 436, groups (3/sex/dose) of RccHanTM: WIST strain rats were exposed (nose only) to aerosol atmosphere of ST 01 C 11 at concentration of 5.31 mg/L (mean achieved) for 4 h. Animals were observed for mortality, clinical signs and body weight for 14 days and at the end of the study the animals were sacrificed for macroscopic examination.

 

No mortality was observed. Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. Isolated occurrences of red/brown staining around the eyes or snout were also noted. Animals recovered to appear normal from Days 8 to 10 post-exposure. Two male and two female animals exhibited slight bodyweight losses on the first day post-exposure. Reasonable bodyweight development was noted in all male animals during the remainder of the recovery period. In contrast, two female animals exhibited bodyweight losses from Days 1 to 3 post-exposure. Bodyweight gains were noted in all female animals for the remainder of the recovery period. No macroscopic abnormalities were detected amongst animals at necropsy.

LC50 (4 hours) combined > 5.31 mg/L

 

Under the test conditions, the inhalation LC50 for ST 01 C 11 is higher than 5.31 mg/L for 4 h in rats and therefore it is not classified according to Regulation (EC) No. 1272/2008 (CLP) and to GHS.