Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2010- 04-08 to 2010-07-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
The age of the preliminary investigation animals may have been 6 weeks, and not 7 to 8 weeks as per protocol.
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: from Charles River (UK) Ltd
- Age at study initiation: 7-8 weeks
- Weight at study initiation: in the range of 308 to 341 g for males and 220 to 274 g for females.
- Fasting period before study: no data
- Housing: the animals were housed inside a restricted entry rodent facility, one animal per cage during the preliminary investigation and five of one sex per cage during the main study.
The cages were made of a polycarbonate body with a stainless steel mesh lid. Wood shavings were used as bedding and were sterilised by autoclaving and changed at appropriate intervals each week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): within the range of 19 to 23°C
- Humidity (%): within the range of 40 to 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.

IN-LIFE DATES: no data

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
3.4 µm
Geometric standard deviation (GSD):
2.72
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow through nose only chamber. This system was an aluminium alloy construction comprising a base unit, one animal exposure section and a top section.
- Method of holding animals in test chamber: Rats were held in polycarbonate tubes with their snouts protruding from the end of the tubes into the exposure chamber.
- Source and rate of air: From in-house compressed air system(breathing quality), Generator flow: 25 L/minute
- System of generating particulates/aerosols: Turn Table Dust Feed mechanism designed to produce and maintain atmospheres of dust by suspending material in a stream of dry air. Test article flowed from a hopper, assisted by agitation, into a concentric groove in the turntable. As the turntable rotated, the dust passed under the uplift tube which draws up the material using the negative pressure supplied using the venturi principle. The material is carried in the air stream and into the chamber.The concentration of dust in the air may be altered by modifying the speed of rotation of the turntable plate. The Turn Table Dust Feed mechanism was attached to an aerosol conditioning chamber for the delivery of the powder and the aerosol passed through it into the top of the exposure chamber.
- Temperature, humidity, pressure in air chamber:
Temperature was measured from the breathing zone of the animals via an unused exposure port using an electronic thermometer. The mean chamber temperatures were 20.5 and 20.1°C for the control and dosed groups respectively.
Chamber Relative Humidity was measured using an electronic hygrometer inserted into the breathing zone of the animal via an unused exposure port. The mean chamber relative humidity were 21.4 and 21.2% for the control and dosed groups respectively.
Pressure in air chamber was not reported.
- Air flow rate: Air flow from Turn Table Dust Feed mechanism was 36 L/min
- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 8-stage cascade impactor (Marple 298). Samples were collected twice during exposure. The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, was calculated by this difference. The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than < 0.42, 0.76, 1.27, 2.86, 4.9, 8.0, 12.08 and 17.39 µm (aerodynamic diameter) was calculated. From this data, the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated.
- Treatment of exhaust air: Extract airflow was drawn at a flow rate of 40 L/minute.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration of the generated atmosphere was measured gravimetrically during the exposure by pulling a suitable, known volume of test atmosphere in the exposure chamber, through a glass fibre filters. Sampling was performed eight times during the exposure. Samples were collected from a vacant animal exposure port (animals breathing zone). The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.

- Samples taken from breathing zone: yes, samples were collected from a vacant animal exposure port.

VEHICLE (if applicable)
- none
Analytical verification of test atmosphere concentrations:
yes
Remarks:
Table 7.2.2/1
Duration of exposure:
ca. 4 h
Concentrations:
Target concentration: 5.0 mg/L
Mean achieved atmosphere concentration: 5.11 mg/L ; Standard deviation: 0.76
No. of animals per sex per dose:
Five/sex/dose
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed intermittently for signs of reaction to the test substance during exposure and at least twice daily throughout the observation period. The weight of each animal was recorded on Days 1 (prior to dosing), 2, 3, 4, 8 and 15.
- Necropsy of survivors performed: yes, animals were killed by an intraperitoneal overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a macroscopic examination which consisted of opening the cranial, thoracic and abdominal cavities.
- Other examinations performed: Clinical signs were recorded during exposure, immediately after exposure and then at 1-hour and 2-hours post-exposure. Throughout the study, all cages were checked at least twice daily, once in the morning and again towards the end of the normal working day, for dead or moribund animals. The lungs (including the larynx and trachea) were dissected free, weighed and the weights recorded.
Statistics:
None

Results and discussion

Preliminary study:
A preliminary exposure, with 1 male and 1 female rat, was conducted at the target (Limit test) concentration of 5 mg/L for a period of 4-hours in order to assess the likely response of rats to the test substance. As a concentration of 5.63 mg/L was tolerated by the preliminary investigation animals, the main study test animals were also exposed to the target concentration of 5 mg/L for a period of 4-hours. Control animals received a single 4-hour exposure air only.
Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC0
Effect level:
>= 5.11 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
There were no unscheduled deaths.
Clinical signs:
Immediately following exposure all test animals had reduced body temperature, whilst 7/10, 5/10 and 5/10 animals had irregular breathing, hunched posture and post dose salivation staining, respectively. In addition chin rubbing, unsteady gait and partially closed eyelids were recorded in 4/10, 3/10 and 2/10 animals, respectively. These signs generally persisted to 1 and 2-hours post-exposure, however with decreasing incidence, and were no longer present from Day 2 onwards.
Ungroomed coat and noisy breathing/râles were recorded at 2-hours post-exposure in 10/10 and 6/10 animals respectively, and persisted up to Days 3 and 5 respectively, however with decreasing incidence over time. In addition gasping and underactive behaviour were noted in 3/10 and 1/10 animals respectively at 2-hours post-exposure, whilst distended abdomen was recorded in one male and one female; these signs were not present at Day 2, with the exception of one male which had a distended abdomen and was gasping up to Day 2/3.
Piloerection, recorded in all test animals, was generally observed from 2-hours post-exposure, and persisted up to Day 4.
Body weight:
Bodyweight loss was recorded for all treated animals on Day 2 (7 to15% of pre-exposure Day 1 weights). On Day 3, one treated male had lost further weight (-19% of pre-exposure Day 1 weight), however all other treated animals had gained weight between Days 2 and 3. Subsequently, from Day 4 post-exposure all treated animals gained weight during the remainder of the observation period.
Gross pathology:
There were no macroscopic findings in the animals.
Other findings:
- Organ weights: Mean lung weights were slightly higher in treated animals (1.09X and 1.14X control, in males and females, respectively)

Any other information on results incl. tables

Table 7.2.2/1: Clinical signs in male animals

 

 

Number showing signs

Males

Signs

Time in hours

 

 

During exposure

IAE

1.0

2.0

Day 2

Day 3

Day 4

 

 

 

 

 

 

 

 

 

Control

Wet fur

0

2

0

0

0

0

0

 

 

 

 

 

 

 

 

 

Treated

Salivation

1

0

0

0

0

0

0

 

Wet fur

0

2

2

0

0

0

0

 

Chin rubbing

0

2

0

0

0

0

0

 

Post salivation staining

0

3

0

0

0

0

0

 

Reduced body temperature

0

5

0

0

0

0

0

 

Irregular breathing

0

2

0

1

0

0

0

 

Partially closed eyelids (both)

0

2

3

0

0

0

0

 

Unsteady gait

0

1

0

0

0

0

0

 

Piloerection

0

0

0

4

5

5

1

 

Ungroomed

0

0

0

5

1

1

0

 

Rales/noisy breathing

0

0

0

2

2

1

0

 

Gasping

0

0

0

1

1

1

0

 

Distended abdomen

0

0

0

1

1

0

0

 

Brown staining snout

0

0

0

0

1

1

0

IAE:    Immediately after exposure

Table 7.2.2/2: Clinical signs in female animals

 

 

Number showing signs

Females

Signs

Time in hours

 

 

During exposure

IAE

1.0

2.0

Day 2

Day 3

Day 4

Day 5

 

 

 

 

 

 

 

 

 

 

Control

Wet fur

0

3

0

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

Treated

Wet fur

0

3

2

1

0

0

0

0

 

Irregular breathing

5

5

5

1

0

0

0

0

 

Hunched posture

0

5

3

0

0

0

0

0

 

Chin rubbing

0

2

0

0

0

0

0

0

 

Reduced body temperature

0

5

2

0

0

0

0

0

 

Post salivation staining

0

2

3

0

0

0

0

0

 

Piloerection

0

1

2

3

4

5

1

0

 

Test substance staining

0

2

0

0

0

0

0

0

 

Gasping

0

1

0

2

0

0

0

0

 

Unsteady gait

0

2

2

0

0

0

0

0

 

Partially closed eyelids (both)

0

0

2

0

0

0

0

0

 

Ungroomed

0

0

0

5

2

2

0

0

 

Rales/noisy breathing

0

0

0

4

3

2

2

1

 

Brown staining snout

0

0

0

3

4

2

0

0

 

Underactive

0

0

0

1

0

0

0

0

 

Distended abdomen

0

0

0

1

0

0

0

0

 

 

 

 

 

 

 

 

 

 

IAE:    Immediately after exposure

Table 7.2.2/3: Male lung weights

Males

Animal

Lung weight (g)

 

number

 

 

 

 

Control

1

1.69

 

2

1.64

 

3

1.48

 

4

2.08

 

5

1.68

 

Mean

1.71

 

SD

0.22

 

 

 

Treated

11

1.94

 

12

1.92

 

13

1.84

 

14

1.98

 

15

1.67

 

Mean

1.87

 

SD

0.12

Table 7.2.2/3: Female lung weights

Females

Animal number

Lung weight (g)

 

 

 

Control

6

1.42

 

7

1.22

 

8

1.24

 

9

1.41

 

10

1.55

 

Mean

1.37

 

SD

0.14

 

 

 

Treated

16

1.62

 

17

1.65

 

18

1.48

 

19

1.51

 

20

1.53

 

Mean

1.56

 

SD

0.07

 

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The 4 hour inhalation LC0 value of the test substance was equal or higher to 5.11 mg/L.
Executive summary:

In an acute inhalation toxicity study, groups of five male and five female Sprague-Dawley rats were exposed, nose-only, to test substance concentration of 0 (control) or 5 mg/L. The mean Mass Median Aerodynamic Diameter (MMAD) value for exposure was within the guideline expected range (3.4 µm). This study was performed according to OECD Guideline 403 and with Good Laboratory Practices.

A preliminary exposure, with 1 male and 1 female rat, was conducted at the target (Limit test) concentration of 5 mg/L for a period of 4-hours in order to assess the likely response of rats to the test substance. As a concentration of 5.63 mg/L was tolerated by the preliminary investigation animals, the main study test animals were also exposed to the target concentration of 5 mg/L (concentration achieved:5.11 mg/L) for a period of 4-hours. Control animals received a single 4-hour exposure to air only.

No deaths were observed.

Immediately following exposure all test animals had reduced body temperature, whilst 7/10, 5/10 and 5/10 animals had irregular breathing, hunched posture and post dose salivation staining, respectively. In addition chin rubbing, unsteady gait and partially closed eyelids were recorded in 4/10, 3/10 and 2/10 animals, respectively. These signs generally persisted to 1 and 2-hours post-exposure, however with decreasing incidence, and were no longer present from Day 2 onwards.

Ungroomed coat and noisy breathing/râles were recorded at 2-hours post-exposure dose in 10/10 and 6/10 animals respectively, and persisted up to Days 3 and 5 respectively, however with decreasing incidence over time. In addition gasping and underactive behaviour were noted in 3/10 and 1/10 animals respectively at 2-hours post-exposure, whilst distended abdomen was recorded in one male and one female; these signs were not present at Day 2, with the exception of one male which had a distended abdomen and was gasping up to Day 2/3. Piloerection, recorded in all test animals, was generally observed from 2-hours post-exposure, and persisted up to Day 4.

Mean lung weights were slightly higher in treated animals (1.09X and 1.14X control, in male and females, respectively).

The macroscopic examination performed fourteen days after exposure did not reveal any treatment-related changes. The nature and incidence of all the findings were consistent with the commonly seen background macroscopic changes.

The 4 -hour inhalation LC50 was found to be greater than 5.11 mg/L (ie 5110 mg/m3)