Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-10-15 to 2009-11-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
, adopted 2009-09-07
Deviations:
no
Guideline:
other: additional examinations performed: clinical signs, gross pathology, histopathological examination of nose, larynx, trachea and lungs (five levels) 24 hours and 14 days after cessation of exposure
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium hexahydroxoantimonate
EC Number:
251-735-0
EC Name:
Sodium hexahydroxoantimonate
Cas Number:
33908-66-6
Molecular formula:
NaSb(OH)6
IUPAC Name:
sodium stibanylidyne hexahydroxide
Test material form:
not specified

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: males: 52 days; females: 66 days
- Weight at study initiation (main study): males: 224 - 238 g; females: 214 - 230 g
- Weight at study initiation (satellite group): males: 217-242 g; females: 228 - 233 g
- Fasting period before study: Feeding was discontinued approx. 16 hours before exposure; only tap water was then available ad libitum.
- Housing: Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages.
- Diet (ad libitum): Commercial diet, ssniff® R/M-H V1534 served as food (ssniff Spezialdiäten GmbH, 59494 Soest, Germany). During the 14-day observation period the animals are kept by sex in groups of 2 - 3 animals in MAKROLON cages (type III plus).
- Water (ad libitum): Drinking water
- Acclimation period: At least 5 days; Animals were acclimatised to the test apparatus for approx. 1 hour on 2 days prior to testing. The restraining tubes did not impose undue physical, thermal or immobilization stress on the animals.

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C±3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus, exposure chamber volume and method of holding animals in test chamber : The study was carried out using a dynamic inhalation apparatus (RHEMA-LABORTECHNIK, 65719 Hofheim/Taunus, Germany) (air changes/h (≥ 12 times)) with a nose-only exposure of the animals according to KIMMERLE & TEPPER. The apparatus consists of a cylindrical exposure chamber (volume 40 L) which is able to hold 10 animals in pyrex tubes at the edge of the chamber in a radial position.
Exposition started by locating the animals’ noses into the exposure chamber after equilibration of the chamber concentration for at least 15 minutes.
Before initiating the study with the animals, a pre-test was carried out with the exposure system in order to verify that under the experimental settings chosen, the limit concentration of 5 mg/L air could be achieved by gravimetric analysis.
- System of generating particulates/aerosols: The dust of the test material was generated with a rotating brush dust generator (RBG 1000, PALAS GmbH Partikel und Lasermesstechnik,76229 Karlsruhe, Germany). The generator was fed with compressed air (5.0 bar) from a compressor (ALUP Kompressorenfabrik, 73257 Köngen, Germany)(air was taken from the surrounding atmosphere of the laboratory room and filtered using an in-line disposable gas filter). At the bottom of the exposure chamber, the air was sucked off at a lower flow rate than it was created by the dust generator in order to produce a homogenous distribution and a positive pressure in the exposure chamber (inflow 900 L/h, outflow 800 L/h).

The tests with the main study animals and the recovery animals were conducted in the same inhalation chamber but on different days. Between the exposure times the chamber was cleaned carefully.

- Method of particle size determination: An analysis of the particulate size distribution was carried out twice during the exposure period using a cascade impactor according to MAY. The dust from the exposure chamber was drawn through the cascade impactor for 5 minutes at a constant flow rate of 5 L/min. The slides were removed from the impactor and weighed on an analytical balance (SARTORIUS, type 1601 004, precision 0.1 mg). Delta of slides’ weight were determined. The correct functioning of the dynamic separation of particles was controlled microscopically during spot-checks.
The mass median aerodynamic diameter (MMAD) was estimated by means of non-linear regression analysis. The 32 μm particle size range and the filter (particle size range < 0.5 μm) were not included in the determination of the MMAD in order not to give undue weight to these values. The Geometric Standard Deviation (GSD) of the MMAD was calculated from the quotient of the 84.1%- and the 50%-mass fractions, both obtained from the above mentioned non-linear regression analysis.
- Temperature, humidity, pressure in air chamber: A manometer and an air-flow meter ( ROTA Yokogawa GmbH & Co. KG, 79664 Wehr/Baden, Germany) were used to control the constant supply of compressed air and the exhaust, respectively. Flow rates were checked hourly and corrected if necessary.
The oxygen content in the inhalation chamber was 21%. It was determined at the beginning and at the end of the exposure with a DRÄGER Oxygen-analysis test set (DRÄGER Tube Oxygen 67 28 081). Carbon dioxide concentration did not exceed 1%.
Temperature (21.2°C ± 0.4°C (main study) or 21.5°C ± 0.2°C (satellite group)) and humidity (58.1% ± 0.7% (main study) or 58.8% ± 0.4 (satellite group)) were measured once every hour with a climate control monitor (testo 175-HZ data logger).

TEST ATMOSPHERE
- Brief description of analytical method used: The actual dust concentration in the inhalation chamber was measured gravimetrically with an air sample filter (Minisart SM 17598 0.45 μm) and pump (Vacuubrand, MZ 2C (Membrane Pump,Vacuubrand GmbH + Co. KG, 97877 Wertheim/Main, Germany)) controlled by a rotameter. Dust samples were taken once every hour during the
exposure. For that purpose, a probe was placed close to the animals' noses and air was drawn through the air sample filter at a constant flow of air of 5 L/min for 1 minute. The filters were weighed before and after sampling (accuracy 0.1 mg). The correct loading of the filter was checked by the airflow via the rotameter and by a positive weight increase of the filter after the sampling period of 1 minute.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): main study: 4.731 µm (GSD: 2.85); satellite group: 4.077 µm (GSD: 2.60)
No further infromation on the inhalation exposure was stated.

Analytical verification of test atmosphere concentrations:
yes
Remarks:
see above ("details on inhalation exposure")
Duration of exposure:
4 h
Concentrations:
Actual concentration main study: 5.40 +/- 0.07 mg/L
Actual concentration satellite group: 5.19 +/- 0.22 mg/L
Nominal concentration main study: 5.40 mg/L
Nominal concentration satellite group: 5.20 mg/L
No. of animals per sex per dose:
Main study: 3 males / 3 females
Satellite group: 3 males / 3 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Careful clinical examinations were made at least twice daily until all symptoms subsided, thereafter each working day. Observations on mortality were made at least once daily (in the morning starting on test day 2). Individual weights of animals were determined once during the acclimatisation period, before and after the exposure on test day 1, on test days 2, 3, 8 and 15.
- Necropsy of survivors performed: Yes; Necropsy of all main study and satellite animals (3 + 3 males and 3+3 females) was carried out and all gross pathological changes were recorded:
1. Satellite animals: necropsy at 24 hours after cessation of exposure, as this is likely to be the time at which any signs of respiratory irritation would have manifested;
2. Main study animals: necropsy at the end of the 14-day observation period.
All main study and satellite animals were subjected to the same level of histopathological examination upon necropsy at the end of the respective observation period. During histopathology, attention was paid to alterations that might be indicative of respiratory irritation, such as hyperaemia, oedema, minimal inflammation, thickened mucous layer.
The following organs of all animals were fixed in 10% (nose, i.e. head without brain, eyes and lower jaw) or 7% (other organs) buffered formalin for histopathological examination:
1.Nose (5 levels of the nasal turbinates):
The tip and Level 1 of the nose were taken from a cut just anterior to the incisor teeth. With tip removed, Level 2 was taken approximately 2 mm posterior to free tip of the incisor teeth. Level 3 was cut through the incisive papilla. Level 4 was cut through the middle of the second palatal ridge, which is located just anterior to the molar teeth. Level 5 was cut through the middle of the molar teeth. All sections were embedded face down to yield a section from the anterior section, except the nose tip was embedded posterior surface down.
2. Larynx
3. Trachea
4. Lungs (five levels)
Paraffin sections were prepared of all above mentioned organs and stained with haematoxylin-eosin.
- Other examinations performed: Cageside observations included, but were not limited to: changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, as well as somatomotor activity and behaviour pattern.
Particular attention was directed to observation of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The animals were also observed for possible indications of respiratory irritation such as dyspnoea, rhinitis etc.. Changes in weight were calculated and recorded when survival exceeded one day.

Assessment of respiratory tract irritation effects:
The Assessment of respiratory tract irritation effects were conducted according to the criteria set forth in the OECD proposal document ENV/JM/HCL(2004)9/REV:
1. There are currently no validated animal tests that deal specifically with respiratory tract irritation. However, useful information may be obtained from single and repeated inhalation toxicity tests. For example, animal studies may provide useful information in terms of toxicity (dyspnoea, rhinitis etc) and histopathology (e.g. hyperaemia, oedema, minimal inflammation, and thickened mucous layer) which are reversible and may be reflective of the characteristic clinical symptoms described above. Such animal studies can be used as part of weight of evidence evaluation.
2. The special classification would occur only when more severe organ/systemic effects including the respiratory system were not observed.
No further infromation on the study design was stated.
Statistics:
Since no mortality occurred, the calculation of an LC50 was not required.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.4 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: There were no signs of respiratory irritation, based on an absence of test item-related clinical signs and histopathological effects in the nose, larynx and trachea 24 hours and 14 days after exposure.
Mortality:
No mortality occurred in the main or satelite group.
Clinical signs:
other: A 4-hour inhalation exposure to Sodium hexahydroxoantimonate at a concentration of 5.40 mg/L air revealed slight ataxia, slight to moderate tremor, slight dyspnoea and nose-bleeding in all 3 male and 3 female rats. Rats were free of symptoms after 3 hours
Body weight:
All animals in the main study gained the expected body weight throughout the study period.
Gross pathology:
No pathological changes were detected in the main or satellite group at necropsy.
Other findings:
- Histopathology: A 4-hour inhalation exposure to Sodium hexahydroxoantimonate at a concentration of 5.40±0.07 mg/L air (14-day sacrifice) or 5.19±0.22 mg/mL air (24-hour sacrifice) did not reveal any test material-related histopathological changes in the lungs and nose.
The following non test material-related observations were made in regards to the nose (five levels):
The nasal cavity of level 1 and 2 showed a normal respiratory epithelium with cilia and a normal olfactory epithelium without inflammatory reactions – that is the respiratory epithelium contained three major cell types, the basal cells above the basement membrane, the ciliated epithelial cells and the secretory goblet cells. The number of goblet cells varied between the animals of the main study and the satellite animals, but there was no difference between both groups.
Only a minimal to mild lympho-histiocytic infiltration and lymphocytic follicles were noted in the subepithelial area of the normal respiratory epithelium. The normal olfactory epithelium with 5 to 7 nuclear layers showed normal basal cells, olfactory sensory cells and sustentacular cells. There were no degenerative changes recorded.
The nasoturbinates of the nose in level 5 showed only normal olfactory epithelium.
The following observations were made in regards to the lungs (five levels):
No relevant pulmonary changes were observed in the 5 localisations of the lung neither in the rats sacrificed 14 days after exposure nor the rats sacrificed 24 hours after exposure.
Minimal to mild foamy macrophages (alveolar histiocytosis) were observed in 1 male rat in the left lobe of the lung and in 1 female rat in all 5 lungs localisations.


Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the present test conditions, the LC50 value for rats following inhalation of Sodium hexahydroxoantimonate for 4 hours was determined as follows (gravimetric concentration):
LC50: > 5.40 Sodium hexahydroxoantimonate/L air
Based on the results of the histopathological and macroscopic investigations, Sodium hexahydroxoantimonate does not require classification for respiratory irritation.
According to the EC-Commission directive of September 1st, 1993 on the approximation of the laws, regulations and administrative provision relating to the classification, packaging and labelling of dangerous substances (67/548/EC and its subsequent amendments) and the results obtained under the present test conditions Sodium hexahydroxoantimonate does not require classification for neither acute inhalation toxicity nor respiratory irritation.
Also, according to the EC Regulation 1272/2008 and subsequent regulations, the test material is not classified for acute inhalation toxicity.