Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 November to 24 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 403 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
UK GLP Compliance Programme (inspected on 12-14 March 2014 / signed on 12 May 2014)
Test type:
traditional method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Clear colorless liquid
- Storage Conditions: ca. 4 °C in the dark under nitrogen
Specific details on test material used for the study:
- Purity test date: 29 January 2015

Test animals

Species:
rat
Strain:
other: RccHan : WIST strain
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 up to five 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: 15 per hour
- Photoperiod: 12 h dark / 12 h light

N-LIFE DATES: 04 November to 24 December 2014

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
2.91 µm
Geometric standard deviation (GSD):
2.12
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was aerosolized using a glass concentric jet nebulizer (Radleys, Saffron Walden, Essex, UK) located at the top of the exposure chamber.
- Exposure chamber volume: Cylindrical exposure chamber had a volume of approximately 30 L (dimensions: 28 cm diameter x 50 cm high).
- One day prior to the day of exposure each rat was acclimatized (for approximately 2 h) to a tapered polycarbonate restraining tube.
- Method of holding animals in test chamber: During the exposure period, 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: 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 (35, 93 and 215 minutes of exposure respectively) 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.
- 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. Temperature and humidity in air chamber were 20 °C and 5-6%, respectively.
- Exposure chamber oxygen concentration: Oxygen levels within the exposure chamber were measured by an electronic oxygen analyzer (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 (actual 20.8-20.9%).
- Exposure Chamber Atmosphere Concentration: Prior to the start of the study, the non-volatile component of the test item was determined by adding a small, known amount of test item to glass fiber filters and recording their weights. The filters were then dried in a desiccator between 20 and 22 °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 non-volatile component of the batch used during the study was found to be approximately 0.39 % (n=3). It was therefore considered that chemical analysis should be employed in order to determine test atmosphere concentrations.
The test atmosphere was sampled nine times (3, 30, 61, 90, 122, 150, 182, 210 and 235 minutes of exposure respectively) during the exposure period. The sampling procedure involved 2 L of test atmosphere being drawn through a glass impinger containing Methanol (each made up to 80 mL).
The nominal chamber concentration was calculated by dividing the mass of test item used by the total volume of air passed through the chamber.
The nominal concentration was 398 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was moderately difficult.
- Pretreatment: Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterization period test item input rates were varied to achieve the required atmospheric conditions.
- Following an appropriate equilibration period a single group of ten rats (five males and five 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 135 % of target, the standard deviation of the test atmosphere concentrations was within acceptable limits and no deaths occurred, no further levels were required.

TEST ATMOSPHERE
- Brief description of analytical method used: Test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique.
- Samples taken from breathing zone: Yes
- Particle size distribution: See table 7.2.2/1
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 2.91 µm / 2.12
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Nominal concentration: 26.8 mg/L
Mean achieved atmosphere concentration: 6.74 mg/L
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: 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 up to 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 14 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:
> 6.74 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality was observed.
Clinical signs:
- Signs of hunched posture and pilo-erection 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, the following observations were detected:
- During exposure, no significant observations were apparent. On removal from the chamber and one hour post-exposure, all animals exhibited increased respiratory rate.
- One day after exposure, all animals exhibited increased respiratory rate and hunched posture. Observations gradually receded over the recovery period such that animals appeared normal from Days 4 to 6 post-exposure.
Body weight:
All males and two female animals exhibited body weight losses on the first day post-exposure. With the exception of one female animal which showed no body weight gain from Days 1 to 3 post-exposure and another female animal which exhibited a slight body weight loss from Days 3 to 7 post-exposure, body weight gains were noted in all animals during the remainder of the recovery period.
Gross pathology:
No macroscopic abnormalities were detected at necropsy amongst six animals at the end of the 14 day recovery period. However, four other animals exhibited dark patches on the lungs.
Other findings:
None

Any other information on results incl. tables

Exposure Chamber Concentration

The actual concentration of the test item was measured off-line by gas chromatography (GC). The test atmospheres were sampled after theoretical chamber equilibration and then at approximately half-hourly intervals during the exposure period.

The concentration of the test item was shown to be stable and the mean values obtained were:

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

6.74

1.10

26.8

 

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

Particle Size Distribution

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (μm)

Inhalable Fraction

(% <4 μm)

Geometric Standard Deviation

6.74

2.91

66.6

2.12

Table 7.2.2/1: Exposure Chamber Atmosphere Concentrations

Duration of Exposure (minutes)

 

Volume of Air Sampled (L)

 

Chamber Flow Rate (L/min)

 

Atmosphere Concentration (mg/L)

 

3

2

60

7.05

30

2

60

6.94

61

2

60

6.24

90

2

60

5.52

122

2

60

6.17

150

2

60

9.09

182

2

60

5.55

210

2

60

6.62

235

2

60

7.46

 

Mean achieved atmosphere concentration (mg/L) = 6.74

Standard deviation = 1.10

Nominal concentration:

Test item used (g)

407

Air Flow (L/min)

60

Total Generation Time (mins)

253*

Nominal Concentration (mg/L)

26.8

* = The test atmosphere was generated for a total of 13 minutes prior to animal insertion to ensure test item concentration was being achieved. 

Table 7.2.2/2:Particle Size Distribution

Cascade Impactor Data

Impactor Stage

Number

Cut Point

(μm)

Amount Collected (mg) per Sample Number

Mean Amount Collected (mg)

1

2

3

3

8.4

0.05

0.04

0.06

0.05

4

7.3

0.19

0.19

0.20

0.19

5

3.6

0.43

0.40

0.35

0.39

6

1.3

0.65

0.56

0.60

0.60

7

0.94

0.14

0.07

0.08

0.10

8

0.43

0.03

0.01

0.03

0.02

Back-up Filter

<0.43

0.06

0.04

0.00

0.03

Total Mean Amount of Test Item Collected

1.38

 

Calculation

 

Cut Point

(μm)

Log10

Cut Point

Mean Cumulative Amount Less Than Cut Point

mg

%

Probit

8.4

0.924

1.33

96.4

6.80

7.3

0.863

1.14

82.6

5.94

3.6

0.556

0.75

54.3

5.11

1.3

0.114

0.15

10.9

3.77

0.94

-0.027

0.05

3.62

3.20

0.43

-0.367

0.03

2.17

2.98

 

Results

Mean Mass Median Aerodynamic Diameter (MMAD) = 2.91 μm

Geometric Standard Deviation (GSD) = 2.12

Predicted amount less than 4 μm = 66.6 %

Table 7.2.2/3: Mortality Data

 

Mean

Achieved

Atmosphere

Concentration

(mg/L)

Sex

Deaths

During

Exposure

Deaths

Post

Exposure

(1 Hour)

Deaths During Day of Observation

Total

Deaths

1

2

3

4

5

6

7

8-14

6.74

Male

0

0

0

0

0

0

0

0

0

0

0/10

Female

0

0

0

0

0

0

0

0

0

0

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the inhalation LC50 for ST 11 C 13 is higher than 6.74 mg/mL for 4 h in rats therefore it is not classified according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In an acute inhalation toxicity study performed according to OECD Guideline 403, groups (5/sex/dose) of RccHan : WIST strain rats were exposed (nose only) to aerosol atmosphere of ST 11 C 13 at concentration of 6.74 mg/mL (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 during the study. Observations noted during the study in all animals included increased respiratory rate, hunched posture, pilo-erection and wet fur. All animals recovered to appear normal from Days 4 to 6 post-exposure. All males and two female animals exhibited body weight losses on the first day post-exposure. With the exception of one female animal which showed no body weight gain from Days 1 to 3 post-exposure and another female animal which exhibited a slight body weight loss from Days 3 to 7 post-exposure, body weight gains were noted in all animals during the remainder of the recovery period. No macroscopic abnormalities were detected at necropsy amongst six animals at the end of the fourteen day recovery period. However, four other animals exhibited dark patches on the lungs.

 

Under the test conditions, the inhalation LC50 for ST 11 C 13 is higher than 6.74 mg/mL for 4 h in rats therefore it is not classified according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.