Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 - 29 Mar 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
2 dose levels only

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
adopted 07 Sep 2009
Deviations:
yes
Remarks:
only 2 dose groups instead of 3
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Version / remarks:
EC No 440/2008, part B., May 2008, ammended by Comission Regulation (EU) No. 260/2014 (24 January 2014)
Deviations:
yes
Remarks:
only 2 dose groups instead of 3
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Version / remarks:
adopted Aug 1998
GLP compliance:
yes
Test type:
traditional method
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han), Outbred, SPF-Quality
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10-13 weeks
- Weight at study initiation: 316 – 386 g (males) and 209 – 251 g (females)
- Housing: 5 animals of the same sex and same dosing group were housed together in polycarbonate cages (Makrolon MIV type; height 18 cm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J. Rettenmaier & Söhne GmbH + CO KG, Rosenberg, Germany). Animals were separated during designated procedures/activities.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
> 3.4 - < 4.3 µm
Remark on MMAD/GSD:
The Mass Median Aerodynamic Diameter (MMAD) ± Geometric Standard Deviation (GSD) was 3.4 ± 2.0 µm and 3.6 ± 1.9 µm at 1 mg/L air and 4.3 ± 1.7 µm and 3.7 ± 1.8 µm at 5 mg/L air, respectively. Agglomeration of aerosol particles at this high concentration might have resulted in the MMAD values to fall outside the recommended range of 1 - 4 µm. The MMAD just exceeded this range and there was no evidence for item deposition in the upper airways. Since it is generally known that good distribution throughout the lung requires particles with an aerodynamic diameter between 1 and 5 μm, it can be assumed that sufficient deposition in the lower respiratory tract occurred during the exposure.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Polycarbonate restraining tubes, which were connected to the exposure chamber. The chamber consists of animal sections with eight animal ports each. Each animal port has its own test atmosphere inlet and exhaust outlet. 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. 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 test item were made of stainless steel, glass, rubber or plastic.
- Rate of air: 1 L/min
- System of generating aerosols: An aerosol was generated by nebulization of the test item using a Collison nebulizer. The primary aerosol was let through a cyclone, allowing large particles to settle, and diluted with pressurized air before it entered the exposure chamber. The mean total airflows were 39 and 19 L/min, at 1 and 5 mg/L, respectively.
- Method of particle size determination: The particle size distribution was characterized twice during each exposure period. The samples were drawn with a flow of 2 L/min. From the test atmosphere through a tube mounted in one of the free animal ports of the exposure chamber. The samples were collected with an 8 stage Marple personal cascade impactor containing fiber glass filters and a fiber glass back-up filter. Amounts of test item collected were measured gravimetrically. Subsequently the Mass Median Aerodynamic Diameter (MMAD) and the Geometric Standard Deviation (gsd) were determined based on OECD guidance document No 39.
- Treatment of exhaust air: From the exposure chamber the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood.
- Temperature and humidity in air chamber: 18.7 – 21.6 °C, 5.6 – 6.8%

TEST ATMOSPHERE
- Brief description of analytical method used: Sample volumes were measured by means of a dry gas meter. The collected amount of test item in the air sample was measured gravimetrically.
- Samples taken from breathing zone: yes. Samples were drawn from the test atmosphere through a tube mounted in one of the free animal ports of the exposure chamber.

Analytical verification of test atmosphere concentrations:
yes
Remarks:
gravimetric
Duration of exposure:
4 h
Concentrations:
1.1 ± 0.04 and 5.2 ± 0.2 mg/L air (analytical concentrations)
3.0 and 7.9 mg/L air (nominal concentrations)
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: Animals were checked for mortality, behavioral signs of distress and effects on respiration at least three times during exposure. Post exposure observations were performed at periodic intervals on the day of exposure (at least two times) and once daily thereafter. Animals were weighed individually on Day 1 (pre exposure), 2, 4 and 8 and 15.
- Necropsy of survivors performed: Yes, all animals were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded.

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 1 - < 5 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
1 mg/L: 0/5 males and 1/5 females died approximately two hours after exposure.
5 mg/L: 5/5 males and 5/5 females died or were sacrificed in moribund condition during exposure or within one hour after exposure.
Clinical signs:
other: 1 mg/L: Slow breathing was seen in 1/5 males during exposure. Lethargy, hunched posture, labored respiration and/or piloerection were noted in 5/5 males and 5/5 females after exposure, fully reversible within 24 h. In addition, clonic spasms, muscle twitc
Body weight:
The mean body weight gain shown by the surviving animals at 1 mg/mL over the study period was within the range expected for rats of this strain and age used in this type of study.
Gross pathology:
1 mg/L: Necropsy revealed no abnormalities
5 mg/L: Mucous or gelatinous contents of the stomach in 5/5 males and 5/5 females, dark red foci or dark red discoloration of the thymus in 2/5 males and 2/5 females and dark red discoloration of the mandibular lymph nodes (both sides) in 0/5 males and 2/5 females were noted.

Any other information on results incl. tables

Table 1: Aerodynamic Particle Size Distribution in the Test Atmosphere

Concentration: 1 mg/mL air Sampling Time 1 Sampling Time 2
Stage Cut point
(µm)
Mass sampled
(mg)
Relative mass
(%)
Cumulative mass
(% of total sampled)
Mass sampled
(mg)
Relative mass
(%)
Cumulative mass
(% of total sampled)
1 21 0.06 0.73 99.27 0.06 0.63 99.37
2 15 0.02 0.24 99.03 0.04 0.42 98.95
3 10 0.19 2.3 96.73 0.31 3.25 95.71
4 6 0.94 11.38 85.35 1.25 13.09 82.62
5 3.5 3.32 40.19 45.16 4.02 42.09 40.52
6 2 3.31 40.07 5.08 3.56 37.28 3.25
7 0.9 0.26 3.15 1.94 0.21 2.2 1.05
8 0.5 0.02 0.24 1.69 0.02 0.21 0.84
Back up 0.25 0.14 1.69 0 0.08 0.84 0
MMAD1(mm): 3.4 3.6  
gsd2:     2     1.9  
Concentration: 5 mg/mL air Sampling Time 1 Sampling Time 2
Stage Cut point
(µm)
Mass sampled
(mg)
Relative mass
(%)
Cumulative mass
(% of total sampled)
Mass sampled
(mg)
Relative mass
(%)
Cumulative mass
(% of total sampled)
1 21 0.06 0.74 99.26 0.07 0.67 99.33
2 15 0.01 0.12 99.13 0 0 99.33
3 10 0.4 4.94 94.19 0.45 4.3 95.03
4 6 1.36 16.81 77.38 1.67 15.95 79.08
5 3.5 3.02 37.33 40.05 3.77 36.01 43.08
6 2 2.94 36.34 3.71 3.9 37.25 5.83
7 0.9 0.3 3.71 0 0.52 4.97 0.86
8 0.5 0 0 0 0 0 0.86
Back up 0.25 0 0 0 0.09 0.86 0
MMAD 1(mm): 4.3 3.7  
gsd 2:     1.7     1.8  
1 Mass Median Aerodynamic Diameter; 2 Geometric Standard Deviation      

Applicant's summary and conclusion

Interpretation of results:
other: Acute Inhal. 4, H332 according to Regulation (EC) No. 1272/2008
Conclusions:
CLP: Acute Inhal. 4, H332 according to Regulation (EC) No. 1272/2008