Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23 September 2013 (Start of in-life phase) to 20 December 2013 (GLP compliance statement)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Food and Agricultural Materials Inspection Centre (FAMIC), 12 Nohsan, Notification No. 8147, April 2011; including the most recent partial revisions
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material : Lithium cryolite
- Molecular formula : Li3AlF6
- Molecular weight : 162
- Physical state: White powder
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Health inspection: At least prior to exposure. It was ensured that the animals were healthy and without any abnormality that might affect the study integrity.
- Age at study initiation: Young adult animals were selected (approximately 10-11 weeks old).
- Weight at study initiation: Animals used within the study were of approximately the same age and body weight variation did not exceed +/- 20% of the sex mean.
- Fasting period before study: none
- Housing: Group housing of five animals per sex per cage in labelled Makrolon cages (type IV; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
Animal husbandry on the Day of exposure : The animals were moved to the inhalation area to in order to perform the exposure. During the exposure, there was no access to food and water. After exposure, the animals were returned their cages which were placed in a fume cupboard for a short time period to allow test substance remnants to evaporate. A sheet of filter paper was used to cover the bedding material to prevent suffocation in case of bad health condition and in order to recover and to aid the clinical observations. The sheet was removed and before the end of the exposure day, the animals were returned the animal room.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) except during exposure to the test substance.
- Water (e.g. ad libitum): Free access to tap water except during exposure to the test substance.
- Acclimation period: Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures.
There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Environmental controls for the animal room were set to maintain 18 to 24°C
- Humidity (%): a relative humidity of 40 to 70%
- Air changes (per hr): approximately 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): a 12-hour light/12-hour dark cycle
Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.


IN-LIFE DATES: From: 23 September 2013 To: 17 October 2013

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure chamber:
The design of the exposure chamber is based on the flow past nose-only inhalation chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983). The chamber consisted of three animal sections with eight animal ports each. Each animal port had its own atmosphere inlet and exhaust outlet. The animals were placed in restraining tubes and connected to the animal ports. The number of animal sections and number of open inlets were adapted to the air flow in such a way that at each animal port the theoretical air flow was at least 1 L/min, which ensures an adequate oxygen supply to the animals. The main inlet of the test atmosphere was located at the top section and the main outlet was located at the bottom section. The direction of the flow of the test atmosphere guaranteed a freshly generated atmosphere for each individual animal.
All components of the exposure chamber in contact with the test material were made of stainless steel, glass, rubber or plastic. To avoid exposure of the personnel and contamination of the laboratory the exposure chamber was placed in a fume hood, which maintained at a slight negative pressure.

- Test atmosphere generation :
Administering the test substance to a stream of pressurized air using a combination of a spiral feeder and micronizing jet mill (Bernstein, D.N., Aerosols, pp 721- 723, 1984) generated an aerosol. The rotation speed of the feeder was varied to obtain the desired exposure concentration. The aerosol was passed through a series of cyclones, allowing larger particles to settle. The primary aerosol was diluted with pressurized air before it entered the exposure chamber. The mean total airflows were 91 and 98 L/min. for the 1 and 0.5 mg/L exposure groups respectively.
From the exposure chamber the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood.

- Method of holding animals in test chamber:
see section "details on test animals and environmental conditions".

- Method of particle size determination:
The particle size distribution was characterized twice during each exposure period. The samples were drawn (2 L/min) from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. The samples were collected with an 8 stage Marple personal cascade impactor containing fiber glass filters (SKC 225-713, fiber glass, SKC Omega Specialty Division, Chelmsford, MA, USA) and a fiber glass back-up filter (SEC-290-F1, Westech, Upper Stondon, Bedfordshire, England). Amounts of test substance collected were measured gravimetrically. Subsequently the Mass Median Aerodynamic Diameter (MMAD) and the Geometric Standard Deviation (GSD) were determined.

- Temperature, humidity, pressure in air chamber:
The temperature and relative humidity were measured with a humidity and temperature indicator (E+E Elektronik, Engerwitzdorf, Austria) and were recorded after the animals were placed in the experimental set-up and at 30 minute intervals after initiation of the exposure. The probe was inserted in a tube mounted in one of the free animal ports of the middle section of the exposure chamber.
The temperature of the atmosphere during the exposures was between 20.7 and 21.8°C and relative humidity was between 27 and 39%. These conditions were considered appropriate for this relatively short 4 hours exposure duration.


TEST ATMOSPHERE
- Brief description of analytical method used:
A total of 16 and 21 representative samples were taken for determination of the actual concentration during exposure at 1 and 0.5 mg/L, respectively. Samples were drawn from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber.
Samples were drawn through a glass fiber filter (type SF 92 vorfilter, Schleicher & Schuell, Dassel, Germany). The collected amount of the test substance in the air sample was measured gravimetrically. Sample volumes were measured by means of a dry gas meter (type G 1.6, Actaris Meterfabriek B.V., Dordrecht, The Netherlands).
Subsequently the time-weighted mean concentrations with the standard deviations were calculated.
It was considered during the trial generations that the opacity of the test atmosphere could not be reliably monitored by means of an aerosol monitoring system. An indication of the stability of the test atmosphere was obtained from the concentration measurements, which were equally distributed over time.

- Samples taken from breathing zone: yes

VEHICLE
No vehicle used except air.

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: see section "Any other information on results incl. tables"
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice. At 1 mg/L, the MMAD was 2.9 µm (gsd 2.0) and 2.6 µm (gsd 2.0). At 0.5 mg/L, the MMAD was 3.6 µm (gsd 1.9) and 3.2 µm (gsd 1.8).
CLASS METHOD
- Rationale for the selection of the starting concentration: Target concentrations were based on the cut off concentration values specified in the UN and EC classification guidelines. Five animals of each sex were exposed in a limit test for 4 hours to a target concentration of the test substance of 1 mg/L. Based on the results, five females (identified as most sensitive sex) were exposed to 0.5 mg/L.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
gravimetrically measurement
Duration of exposure:
4 h
Concentrations:
0.5 and 1 mg/L.

The nominal concentration was calculated by dividing the amount of test substance used by the volume of pressurized air (average air flow times exposure time) entering the exposure chamber used for exposure of the animals. Due to the small volume of the exposure chamber the equilibrium time was negligible. The volume of air was calculated from the average air flow (measured by means of thermal mass flow meters and was recorded regularly, preferably in 30 minute intervals) and the exposure time.
No. of animals per sex per dose:
5 animals of each sex were exposed to a target concentration of the test substance of 1 mg/L.
5 females (identified as most sensitive sex) were exposed to 0.5 mg/L.
Control animals:
no
Details on study design:
- Treatment:
Prior to each exposure, both eyes of each rat were instilled with Opthosan (AST Farma BV, Oudewater, The Netherlands) to protect the eyes against potential irritation by the test substance. Prior to exposure the animals were restrained in polycarbonate restraining tubes; these tubes were connected to the exposure chamber. Sixteen minutes after the last animal was placed the generation of the test atmosphere was started. The exposure time was 4 hours.

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing:
Mortality/Viability: Twice daily. The time of death was recorded as precisely as possible.
Body weights: Days 1 (pre-administration), 2, 4, 8 and 15 and at death (if found dead or sacrificed after Day 1).

- Necropsy of survivors performed: yes
The moribund animals and animals surviving to the end of the observation period were sacrificed by an intraperitoneal injection with Euthasol ® (AST Farma BV, Oudewater, The Netherlands). All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded. Particular attention was given to any changes in the respiratory tract.

- Other examinations performed:
clinical signs during exposure: three times during exposure for mortality, behavioural signs of distress and effects on respiration.
clinical signs after exposure : On Day 1, one and three hours after exposure and once daily thereafter until Day 15. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 4: grading slight (1) to very severe (4)
Maximum grade 3: grading slight (1) to severe (3)
Maximum grade 1: presence is scored (1).

- Electronic capture data :
Observations/measurements in the study were recorded electronically using the following programs:
REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Environmental monitoring.
TOXDATA version 8.0 (WIL Research Europe B.V., ‘s-Hertogenbosch, The Netherlands): Mortality / Clinical signs / Body weights. Clinical signs during exposure or not defined in TOXDATA and body weights of decedent animals were recorded manually.
Statistics:
No statistical analysis was performed.

Results and discussion

Preliminary study:
No preliminary study has been performed.
Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 0.5 - < 1 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: only females were exposed at 0.5 mg/L because identified as the most sensitive sex at 1 mg/L
Mortality:
At 1 mg/L, two females were found dead on Days 3 and 4. Two males and three females were sacrificed for ethical reasons on days 4 and 5.
At 0.5 mg/L, one female was sacrificed on day 5. No further mortality occurred.
Clinical signs:
other: At 1 mg/L, slow breathing was noted among the animals during exposure. After exposure, lethargy, flat posture, hunched posture, slow breathing, laboured respiration, shallow respiration, piloerection, chromodacryorrhoea, ptosis and or hypothermia were not
Body weight:
Body weight loss was noted among all surviving animals during the first week post-exposure. All animals regained weight during the second week.
Gross pathology:
Macroscopic post mortem examination revealed abnormalities of the thymes (reduced in size) of one female (0.5 mg/L) sacrificed for ethical reasons during the study. No other findings were noted in any of the animals.

Any other information on results incl. tables

List of deviations

- protocol deviations : deviations from the maximum level for daily mean relative humidity occurred.

Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.

- standard operating procedures deviations : none.

Test atmosphere characterization : Concentration

For the exposure to 1 mg/L, the time-weighted mean actual concentration was 1.1 ± 0.05 mg/L. The nominal concentration (amount of test substance used divided by the volume of pressurized air used) was 8.2 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 13%.

For the exposure to 0.5 mg/L, the time-weighted mean actual concentration was 0.52 ± 0.01 mg/L. The nominal concentration (amount of test substance used divided by the volume of pressurized air used) was 0.47 mg/L. The generation efficiency (ratio of actual and nominal concentration) was 111%.

The concentration measurements distributed over time showed that the substance was sufficiently stable.

1 mg/L exposure group

Start of generation of test atmosphere:

08:50

 

End of generation of test atmosphere:

12:50

 

Time

(hh:mm)

Action

Sample volume

(L)

Mass sampled (mg)

Concentration

(mg/L)

 

 

 

% of total

exposure

time

 

 

 

Weight

concentration

(mg/L)

8:50

start of generation

n.a.

n.a.

n.a.

 

0.0

 

n.a.

8:52

start of sampling

5

6.37

1.274

 

0.8

 

0.011

9:02

start of sampling

5

6.19

1.238

 

4.2

 

0.052

9:12

start of sampling

5

6.73

1.346

 

4.2

 

0.056

9:19

start of sampling

5

5.00

1.000

 

2.9

 

0.029

9:34

start of sampling

5

4.30

0.860

 

6.2

 

0.054

9:44

start of sampling

5

6.43

1.286

 

4.2

 

0.054

10:12

start of sampling

5

5.68

1.136

 

11.7

 

0.133

10:32

start of sampling

5

6.03

1.206

 

8.3

 

0.101

11:07

start of sampling

5

6.50

1.300

 

14.6

 

0.190

11:16

start of sampling

5

5.92

1.184

 

3.8

 

0.044

11:26

start of sampling

5

3.32

0.664

 

4.2

 

0.028

11:31

start of sampling

5

5.30

1.060

 

2.1

 

0.022

11:51

start of sampling

5

4.84

0.968

 

8.3

 

0.081

12:13

start of sampling

5

6.32

1.264

 

9.2

 

0.116

12:29

start of sampling

5

5.30

1.060

 

6.7

 

0.071

12:40

start of sampling

5

4.98

0.996

 

4.6

 

0.046

12:50

end of generation

n.a.

n.a.

0.996

 

4.2

 

0.041

 

 

 

Time-weighted mean concentration

1.126

 

 

 

 

 

Standard deviation

0.046

 

 

 

 

 

n

16

 

 

0.5 mg/L exposure group

Start of generation of test atmosphere:

08:41

 

End of generation of test atmosphere:

12:41

 

Time

(hh:mm)

Action

Sample volume

(L)

Mass sampled (mg)

Concentration

(mg/L)

 

 

 

% of total

exposure

time

 

 

 

Weight

concentration

(mg/L)

8:41

start of generation

n.a.

n.a.

n.a.

 

0.0

 

n.a.

8:44

start of sampling

5

3.07

0.614

 

1.3

 

0.008

8:50

start of sampling

5

4.46

0.892

 

2.5

 

0.022

8:55

start of sampling

5

2.43

0.486

 

2.1

 

0.010

9:05

start of sampling

5

1.81

0.362

 

4.2

 

0.015

9:11

start of sampling

5

2.22

0.444

 

2.5

 

0.011

9:18

start of sampling

5

3.10

0.620

 

2.9

 

0.018

9:24

start of sampling

5

2.34

0.468

 

2.5

 

0.012

9:35

start of sampling

5

2.27

0.454

 

4.6

 

0.021

9:47

start of sampling

5

2.03

0.406

 

5.0

 

0.020

9:52

start of sampling

5

2.08

0.416

 

2.1

 

0.009

9:58

start of sampling

5

2.88

0.576

 

2.5

 

0.014

10:17

start of sampling

5

2.56

0.512

 

7.9

 

0.041

10:36

start of sampling

6

3.47

0.578

 

7.9

 

0.046

10:44

start of sampling

5

2.54

0.508

 

3.3

 

0.017

10:58

start of sampling

5

2.56

0.512

 

5.8

 

0.030

11:20

start of sampling

5

2.62

0.524

 

9.2

 

0.048

11:39

start of sampling

5

2.72

0.544

 

7.9

 

0.043

11:46

start of sampling

5

2.41

0.482

 

2.9

 

0.014

12:04

start of sampling

5

2.63

0.526

 

7.5

 

0.039

12:17

start of sampling

5

2.39

0.478

 

5.4

 

0.026

12:29

start of sampling

5

2.81

0.562

 

5.0

 

0.028

12:41

end of generation

n.a.

n.a.

0.562

 

5.0

 

0.028

 

 

Time-weighted mean concentration

0.520

 

 

 

 

 

Standard deviation

0.013

 

 

 

 

 

n

21

 

1)    Assumed concentration, based on the last sample.

2)    n.a.= not applicable

Test atmosphere characterization: Particle size

The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice. At 1 mg/L, the MMAD was 2.9 µm (gsd 2.0) and 2.6 µm (gsd 2.0). At 1 mg/L, the MMAD was 3.6 µm (gsd 1.9) and 3.2 µm (gsd 1.8).

1 mg/L exposure group

 

 

 

Start of generation of test atmosphere:

08:34

End of generation of test atmosphere:

12:50

Sampling speed (L/min):

2

measurement 1:

Sampling time:

09:52

Sample volume (L):

7

 

Stage

Cut point

(mm)

Mass sampled

(mg)

Relative mass

(%)

Cumulative mass

(% of total sampled)

1

21.0

0.03

0.47

99.53

2

15.0

0.07

1.09

98.45

3

10.0

0.08

1.24

97.20

4

6.0

0.65

10.09

87.11

5

3.5

1.33

20.65

66.46

6

2.0

2.81

43.63

22.83

7

0.9

1.03

15.99

6.83

8

0.5

0.44

6.83

0.00

Back up

0.25

0.00

0.00

0.00

MMAD1(mm):

2.9

gsd2:

2.0

1 Mass Median Aerodynamic Diameter

2 Geometric standard deviation

 

measurement 2:

Sampling time:

11:42

Sample volume (L):

7

 

Stage

Cut point

(mm)

Mass sampled

(mg)

Relative mass

(%)

Cumulative mass

(% of total sampled)

1

21.0

0.03

0.49

99.51

2

15.0

0.03

0.49

99.02

3

10.0

0.02

0.33

98.69

4

6.0

0.48

7.83

90.86

5

3.5

2.15

35.07

55.79

6

2.0

2.23

36.38

19.41

7

0.9

0.74

12.07

7.34

8

0.5

0.43

7.01

0.33

Back up

0.25

0.02

0.33

0.00

MMAD1(mm):

2.6

gsd2:

2.0

1 Mass Median Aerodynamic Diameter

2 Geometric standard deviation

 

0.5 mg/L exposure group

 

 

Start of generation of test atmosphere:

08:41

End of generation of test atmosphere:

12:41

Sampling speed (L/min):

2

measurement 1:

Sampling time:

10:02

Sample volume (L):

16

 

Stage

Cut point

(mm)

Mass sampled

(mg)

Relative mass

(%)

Cumulative mass

(% of total sampled)

1

21.0

0.08

1.94

98.06

2

15.0

0.08

1.94

96.12

3

10.0

0.14

3.40

92.72

4

6.0

0.31

7.52

85.19

5

3.5

2.02

49.03

36.17

6

2.0

0.82

19.90

16.26

7

0.9

0.54

13.11

3.16

8

0.5

0.13

3.16

0.00

Back up

0.25

0.00

0.00

0.00

MMAD1(mm):

3.6

gsd2:

1.9

1 Mass Median Aerodynamic Diameter

2 Geometric standard deviation

 

measurement 2:

Sampling time:

11:53

Sample volume (L):

16

 

Stage

Cut point

(mm)

Mass sampled

(mg)

Relative mass

(%)

Cumulative mass

(% of total sampled)

1

21.0

0.00

0.00

100.00

2

15.0

0.02

0.53

99.47

3

10.0

0.02

0.53

98.94

4

6.0

0.30

7.98

90.96

5

3.5

2.39

63.56

27.39

6

2.0

0.61

16.22

11.17

7

0.9

0.29

7.71

3.46

8

0.5

0.13

3.46

0.00

Back up

0.25

0.00

0.00

0.00

MMAD1(mm):

3.2

gsd2:

1.8

1 Mass Median Aerodynamic Diameter

2 Geometric standard deviation

Applicant's summary and conclusion

Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
The inhalatory LC50, 4h value of LITHIUM CRYOLITE in Wistar rats was established to be within the range of 0.5 – 1 mg/L.
Executive summary:

The acute inhalation toxicity of Lithium cryolite in the rat was investigated according to the OECD Testing Guideline 403 and under GLP.

 

Lithium cryolite was administered as an aerosol by inhalation for 4 hours to one group of five male and five female Wistar rats at 1 mg/L. Based on the results, one group of five females was dosed at 0.5 mg/L. Animals were subjected to daily observations and determination of body weights on Days 1, 2, 4, 8 and 15 and at death. Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15).

 

For the exposure to 1 mg/L, the time-weighted mean actual concentration was 1.1 ± 0.05 mg/L. For the exposure to 0.5 mg/L, the time-weighted mean actual concentration was 0.52 ± 0.01 mg/L. The concentration measurements distributed over time showed that the substance was sufficiently stable.

 

The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice. At 1 mg/L, the MMAD was 2.9 µm (gsd 2.0) and 2.6 µm (gsd 2.0). At 0.5 mg/L, the MMAD was 3.6 µm (gsd 1.9) and 3.2 µm (gsd 1.8).

 

At 1 mg/L, two females were found dead on Days 3 and 4. Two males and three females were sacrificed for ethical reasons on days 4 and 5. At 0.5 mg/L, one female was sacrificed on day 5. No further mortality occurred.

 

At 1 mg/L, slow breathing was noted among the animals during exposure. After exposure, lethargy, flat posture, hunched posture, slow breathing, laboured respiration, shallow respiration, piloerection, chromodacryorrhoea, ptosis and or hypothermia were noted among the animals. The surviving animals recovered from the symptoms between Days 7 and 9. At 0.5 mg/L, no clinical signs were seen during exposure. After exposure, abnormal posture, hunched posture, piloerection, dehydration, lean appearance and/or ptosis were seen among the animals. The surviving animals had recovered from the symptoms by Day 11.

 

Body weight loss was noted among all surviving animals during the first week post-exposure. All animals regained weight during the second week.

 

Macroscopic post mortem examination of revealed abnormalities of the thymes (reduced in size) of one female (0.5 mg/L) sacrificed for ethical reasons during the study. No other findings were noted in any of the animals.

 

Based on the above observations, the inhalatory LC50, 4h value of LITHIUM CRYOLITE in Wistar rats was established to be within the range of 0.5 – 1 mg/L.

 

Based on these results, the substance should be classified as toxic if inhaled according to the CLP and the UN GHS.