Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Feb 2014 - 19 Mar 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: males 242-267 g; females 170-199 g
- Housing: singly housing in suspended stainless steel perforated bottom caging; enrichment (e.g., toy) was placed in each cage; litter paper was placed beneath the cage and was changed at least three times per week
- Diet: Harlan Teklad Global 16% Protein Rodent Diet® #2016, ad libitum, except during the exposure
- Water: Filtered tap water was supplied ad libitum, except during exposure
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 45-55
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose Only Inhalation Chamber, ADG Developments LTD
- Exposure chamber volume: 28 L
- Method of holding animals in test chamber: Animals were individually housed in polycarbonate holding tubes which seal to the chamber with an “O” ring during exposure.
- Source and rate of air: Approx. 60.0 liters per minute (Lpm) of filtered generator air was supplied by an air compressor (Powerex Model: SES050822).
- Method of conditioning air: Mass Flow Controller (Omega, Model # FMA-5527)
- System of generating particulates/aerosols: The test substance was aerosolized using a Jet-Mill (Fluid Energy Jet-Mill Serial #J2724E). Prior to aerosolization, the test substance was ground in a coffee mill (Cuisinart, Model #DCG-20N).
- Method of particle size determination: An eight-stage 1 ACFM Andersen Ambient Particle Sizing Sampler was used to assess the particle size distribution of the test atmosphere.
- Temperature, humidity, pressure in air chamber: The exposure chamber temperature and relative humidity range during exposure were 20ºC and 22-24%, respectively.

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric samples were withdrawn at 6 intervals from the breathing zone of the animals. Samples were collected using 37 mm glass fiber filters (WhatmanTM GF/B) in a filter holder attached by ¼ inch Tygon® tubing to a vacuum pump (GAST, Model #1531-107B-G557X). Filter papers were weighed before and after collection to determine the mass collected. This value was divided by the total volume of air sampled to determine the chamber concentration. The collections were carried out for 1 minute at airflows of 4.0 Lpm. Sample airflows were measured using a Mass Flow Controller (Aalborg, Model #GFC-17).
- Samples taken from breathing zone: yes

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: An eight-stage 1 ACFM Andersen Ambient Particle Sizing Sampler was used to assess the particle size distribution of the test atmosphere. Samples were withdrawn from the breathing zone of the animals at two intervals. The filter paper collection stages were weighed before and after sampling to determine the mass collected upon each stage.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were determined graphically using two-cycle logarithmic probit axes.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
5.1 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 and weighing: All animals were observed for mortality during the exposure period. The animals were examined for signs of gross toxicity, and behavioral changes upon removal from the exposure tube and at least once daily thereafter for 14 days or until death occurred. Individual body weights of the animals were recorded prior to test substance exposure (initial) and again on Days 1, 3, 7, and 14 (terminal) or after death.
- Necropsy of survivors performed: yes. Surviving rats were euthanized via CO2 inhalation on Day 14. Gross necropsies were performed on all decedents and euthanized animals. Tissues and organs of the thoracic and abdominal cavities were examined.
- Other examinations performed: Observations included gross evaluation of skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, and coma.
Statistics:
Statistical analysis was limited to the calculation of the mean and standard deviation.

Results and discussion

Effect levelsopen allclose all
Key result
Sex:
female
Dose descriptor:
LC50
Effect level:
< 5.1 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Key result
Sex:
male
Dose descriptor:
LC50
Effect level:
> 5.1 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.1 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
One male and three females died within seven days of exposure to the test atmosphere. Prior to death, the animals were hypoactive and exhibited abnormal respiration and/or ano-genital staining.
Clinical signs:
other:
Body weight:
Although all animals lost weight through Day 3, the animals gained body weight during Days 7 to 14. However, two of the survivors did not surpass their initial body weights by the end of the 14-day observation period.
Gross pathology:
Gross necropsy of the decedents revealed discoloration of the lungs, distention of the stomach, discoloration of the liver and/or distention of the intestines. No gross abnormalities were noted for any of the surviving animals when necropsied at the conclusion of the 14-day observation period.

Any other information on results incl. tables

TABLE 1: GRAVIMETRIC CHAMBER CONCENTRATIONS

Sample Number

Time of Sample (hour)

Mass Collected (mg)

Airflow Sampled (Lpm)

Collection Time (min)

Chamber Concentration (mg/L)

1

0.5

22.2

4

1

5.55

2

1

17.4

4

1

4.35

3

2

20.6

4

1

5.15

4

2.5

21.1

4

1

5.28

5

3.5

20.6

4

1

5.15

6

3.75

20.5

4

1

5.13

Average ± Standard Deviation

5.10±0.40

 

TABLE 2: SUMMARY OF PARTICLE SIZE DISTRIBUTION

Sample No.

Time of Sample (hours)

Collection Time (minutes)

Mass Median Aerodynamic Diameter1

(µm)

Geometric Standard Deviation

1

1.5

1

2.80

2.32

2

3

1

3.06

2.51

Average

2.93

2.42

 1This figure is an estimation based on graphic analysis of the particle size distribution as measured with a 1 ACFM Andersen Ambient Sizing Sampler.

 

TABLE 3: INDIVIDUAL BODY WEIGHTS AND MORTALITY (5.10 mg/L)

Animal No.

Sex

Body Weight (g)

Mortality

Initial

Day 1

Day 3

Day 7

Day 14

Day

Weight (g)

3301

M

253

215

215

237

279

E

-

3302

M

267

229

201

216

236

E

-

3303

M

242

211

201

223

238

E

-

3304

M

248

211

192

-

-

7

178

3305

M

265

230

240

269

298

E

-

3306

F

193

169

155

168

197

E

-

3307

F

199

172

158

-

-

5

139

3308

F

179

157

-

-

-

2

149

3309

F

196

171

171

-

-

6

169

3310

F

170

163

167

173

183

E

-

E - Euthanized via CO2inhalation after weighing on Day 14

 

TABLE 4: INDIVIDUAL CAGE-SIDE OBSERVATIONS (5.10 mg/L)

Animal                                                                                                            Day of

Number                                        Findings                                                    Occurrence

 

MALES  

 

3301                                             Rales (moist)                                             CR1-1

                                                     Hypoactivity                                             CR-2

                                                     Irregular respiration                                  CR-3, 5-11

                                                     Gasping                                                    0 (2 hrs)-1

                                                     Ano-genital staining                                  2

                                                     Ocular discharge (black, left eye)             3-8

                                                     Active and healthy                                    12-14

 

3302                                             Rales (moist)                                            CR-3

                                                     Hypoactivity                                             CR-4

                                                     Irregular respiration                                  CR-13

                                                     Gasping                                                    0 (2 hrs)-1, 8-11

                                                     Nasal discharge (red)                                1-2

                                                     Facial staining (black)                               3-5

                                                     Active and healthy                                    14

 

3303                                             Rales (moist)                                            CR-1

                                                     Irregular respiration                                  CR-4

                                                     Hypoactivity                                             0 (1 hr)-1

                                                     Active and healthy                                    5-14

 

3304                                             Rales (moist), irregular respiration            CR-6

                                                     Hypoactivity                                             0 (1 hr)-6

                                                     Ano-genital staining                                  1-3

                                                     Dead                                                         7

 

3305                                             Hypoactivity                                             CR-0 (2 hrs)

                                                     Irregular respiration                                  CR-2

                                                     Active and healthy                                    3-14

 

FEMALES  

 

3306                                             Irregular respiration                                  CR-6

                                                     Hypoactivity                                             0 (1 hr)-3

                                                     Rales (moist)                                             1-4

                                                     Ano-genital staining                                  2-8

                                                     Active and healthy                                    9-14

 

3307                                             Irregular respiration                                  CR-4

                                                     Hypoactivity                                             0 (1 hr)-2

                                                     Rales (moist)                                             1-4

                                                     Ano-genital staining                                  2

                                                     Dead                                                         5

 

3308                                             Irregular respiration, hypoactivity             CR-1

                                                     Rales (moist)                                             1

                                                     Dead                                                         2

 

3309                                             Hypoactivity                                            CR-1, 6 (am)

                                                     Irregular respiration                                  CR-3, 5-6 (am)

                                                     Rales (moist)                                            1

                                                     Ano-genital staining                                  1-2

                                                     Active and healthy                                    4

                                                     Dead                                                         6 (pm)

 

3310                                             Hypoactivity                                             CR-0 (2 hrs)

                                                     Irregular respiration                                  CR-2

                                                     Active and healthy                                    3-14

1CR - removal from the exposure tube

 

TABLE 5: INDIVIDUAL NECROPSY OBSERVATIONS (5.10 mg/L)

Animal

Number                                        Tissue                                                 Findings

 

MALES

 

3301-3303, 3305                         All tissues and organs                          No gross abnormalities

 

3304                                             Lungs                                                 Extremely red

                                                     Intestines                                            Slightly distended

 

FEMALES

 

3306, 3310                                   All tissues and organs                          No gross abnormalities

 

3307                                             Lungs                                                 Slightly red

                                                     Stomach, intestines                             Slightly distended

 

3308                                             Lungs                                                 Extremely red

                                                     Liver                                                  Mottled

                                                     Stomach, intestines                             Slightly distended

 

3309                                             Lungs                                                 Slightly red

                                                     Liver                                                  Mottled

Applicant's summary and conclusion

Interpretation of results:
other: Irritating to the respiratory tract according to Regulation(EC) No. 1272/2008
Conclusions:
CLP: STOT Single Exp. 3, H335