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Toxicological information

Specific investigations: other studies

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Administrative data

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012-02-09 to 2012-03-19
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:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class (ATC) Method) (2009-09-07)
Deviations:
no
Principles of method if other than guideline:
The study includes a satellite group of test animals, which were sacrificed and subject to detailed histopathology of the respiratory tract shortly after exposure. The main study group animals were subject to the same detailed histopathology of the respiratory tract, following sacrifice after the 14-day post-exposure observation period.
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-11-12
Type of method:
in vivo
Endpoint addressed:
respiratory irritation

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: powder (particle size of original material: D50 = 1.9 µm; mass median aerodynamic diameter as determined in the inhalation chamber during study: 2.3 µm)
Details on test material:
- Name of test material (as cited in study report): silver powder
- Molecular formula (if other than submission substance): Ag
- Molecular weight (if other than submission substance): 107.87
- Substance type: metal powder
- Physical state: solid
- Analytical purity: 99.2%
- Batch No.: PMC 2
- Stability under test conditions: stable
- Storage condition of test material: room temperature, closed container
- Other: Volume based particle size distribution of the sample: D10=0.8µm, D50=1.9µm, D90=11.2µm. This does not represent the characteristics of the actual test atmosphere (which was analysed during the study) but of the test item as such.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals 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: approx. 8 weeks; females: approx. 9 weeks
- Weight at study initiation: males: 241 - 257 g; females: 223 - 238 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. During the 14-day observation period, the animals are kept by sex in groups of 2 - 3 animals in MAKROLON cages (type III plus).
- Diet: commercial diet, ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: at least 5 adaption 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

Administration / exposure

Route of administration:
inhalation
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the study was carried out using a dynamic inhalation apparatus (RHENA-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.

- 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).
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 exhaust air was drawn through gas wash-bottles.

- Method of particle size determination: an analysis of the particle size distribution was carried out twice during the exposure period using a cascade impactor according to MAY (MAY, K. R. Aerosol impaction jets, J.Aerosol Sci. U6U, 403 (1975), RESEARCH ENGINEERS Ltd., London N1 5RD, UK).
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). Deltas of slides’ weight were determined.
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.
In addition, a sample of approx. 10 g test material was taken from the exposure chamber to determine the median physical particle size with a CILAS 715 by My-Tec, 91325 Adelsdorf, Germany. This determination was non-GLP.

- Temperature, humidity, oxygen content, carbon dioxide content: 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 (23.0°C ± 0.1°C (main study) or 22.0°C ± 0.2°C (satellite group)) and humidity (62.2% ± 0.1% (main study) or 61.4% ± 0.1% (satellite group)) were measured once every hour with a climate control monitor (testo 175-HZ data logger).

Exposition started by locating the animals 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.

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.

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). Individual chamber concentration samples did not deviate from the mean chamber concentration.
The inhalation chamber was equilibrated for at least 15 minutes (t95 approximately 8 minutes).
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- MMAD / GSD:
Main study group: 2.270 μm ± 3.03
Satellite group: 2.269 μm ± 3.11
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
please refer to "details on exposure" above
Duration of treatment / exposure:
4 hours
Frequency of treatment:
once
Post exposure period:
Duration of observation period following administration: 24 hours (satellite group) and 14 days (main study)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5.16 ±0.01 mg/L air (main group)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
13.89 mg/L air (main group)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
5.15 ±0.01 mg/L air (satellite group)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
13.89 mg/L air (satellite group)
Basis:
nominal conc.
No. of animals per sex per dose:
Main study group: 3 males / 3 females
Satellite group: 3 males / 3 females
Control animals:
no

Examinations

Examinations:
- Duration of observation period following administration: 24 hours (satellite group) and 14 days (main study)
- 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) to minimize loss of animals to the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals.
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..
Individual weights of animals were determined once during the acclimatisation period, before and after the exposure on test day 1, on test days 3, 8 and 15. Changes in weight were calculated and recorded when survival exceeded one day. At the end of the test, all animals were weighed and sacrificed.
- 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:
- 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;
- Main study animals: necropsy at the end of the 14-day observation period, also to assess whether any respiratory tract irritation persists or abates.
-Histopathology:
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) Nasal cavity, nasopharynx and paranasal sinus:
Organs: nasal cavity, nasopharynx, paranasal sinus
Localisations: posterior part of upper incisors, incisive papilla, second palatine crest, first molar teeth
Direction: transverse
Remarks: embedded with the rostral faces down, decalcified
The tip and Level 1 of the nose were taken from a cut just anterior to the incisor teeth. With the tip romoved, Level 2 was taken approximately 2 mm posterior to free the 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; whoch 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:
Organ: larynx
Localisations: base of epiglottis, ventral pouch, cricoid cartilage
Direction: transverse
3) Trachea (one section, including the bifurcation, longitudinal horizontal):
Organ: trachea
Localisations: including the bifurcation
Direction: longitudinal horizontal
Remarks: embedded in toto; careful microtome sectioning until recommended cutting level was obtained.
4) Lung:
Localisations: left lobe, right caudal lobe, right cranial lobe, right middle lobe, accessory lobe
Direction: Section 1, 2: longitudinal horizontal; Section 3, 5: transverse; Section 4: longitudinal vertical
Remarks: instillation obligatory, longitudinal horizontal section comprising the lobar bronchus and its mainbranches.
Sample size(s) adapted to the size of the cassette(s); preferentially, the diaphragmatic margin was trimmed off.
Alternative procedure: right and left loves (separate blocks) embedded ventral suface down.
Paraffin sections were prepared of all above mentioned organs and stained with haematoxylin-eosin.
The histopathology was conducted in consideration of the suggestions made in the OECD Guidance Document on Histopathology for Inhalation Toxcity Studies, Supporting TG 41 (Subacute Inhalation Toxcity: 28-day Study) and TG 413 (Subchronic Inhalation Toxicity: 90-day Study). OECD Series on Testing and Assessment No. 125, Document No. ENV/JM/MONO (2010) 16, June 1, 2010.
Positive control:
not applicable

Results and discussion

Details on results:
LC50 (male and female rats, 4 hours) > 5.16 mg/L air
- No animal died prematurely.
- Under the present test conditions, a 4-hour inhalation exposure to Silver Powder Batch PMC 2 at a concentration of 5.16 mg/L air revealed slightly reduced muscle tone on test day 1 immediately after the end of exposure until 30 minutes post exposure, slight ataxia until 60 minutes post exposure and slight dyspnoea (reduced frequency of respiration with increased volume) until 3 hours post exposure in all 3 of 3 male and 3 of 3 female animals, respectively. However, this effect is considered to be an overall clinical sign of general toxicity common to inert dust exposure, but not necessarily test item-related. Since the detailed histopathology of the respiratory tract revealed no pathologically noteworthy findings, Silver Powder Batch PMC 2 is not considered to be irritating to the respiratory tract.
- No influence in body weight gain was observed.
- Macroscopic changes in the nasal cavity and lungs: marbled lungs were observed in all animals of the main study (14-day sacrifice) and in all satellite animals (24-hour sacrifice).
- Histopathology:
1) Test item-related histopathological changes
- Male and female animals of the main study: all 5 lung localisations analysed microscopically from the rats of this group showed a minimal to mild increase of macrophages with large cytoplasm in the alveolar lumen of the lungs. In several animals a focal minimal to mild peribronchitis and minimal pneumonic foci with neutrophilic granulocytes were observed in the alveolar lumen. These findings were not noted in the satellite animals.
2) Non-test item-related histopathological changes
Male and female animals of the main study and the satellite group:
- Observations made for the nose (five levels): the nasal cavity of level 1 showed a normal squamous epithelium and a normal respiratory epithelium with goblet cells. A mild congestion was noted in all animals. The levels 2 and 3 showed similar normal morphological characteristics.
The normal respiratory epithelium partially with cilia contained three major cell types, the basal cells above the basement membrane, the ciliated epithelial cells and the secretory goblet cells.
A normal olfactory epithelium with 5 to 7 nuclear layers, normal basal cells, olfactory sensory cells and sustentacular cells was noted in the male and female animals.
The levels 4 and 5 of the nose showed a normal olfactory epithelium.
In some animals minimal to mild lympho-histiocytic infiltrations or lymphocytic follicles in the subepithelial region of the respiratory epithelium (naso-pharynx region) were observed as normal findings.
- Observations made for the lungs (five levels): the 5 localisations of the lungs in the satellite animals (24-hour sacrifice) showed a normal structure without inflammatory reactions.
The epithelial cells of the larynx and trachea in the main study and the satellite groups showed a normal structure without inflammatory reactions.

Applicant's summary and conclusion

Conclusions:
LC50 (male and female rats, 4 hours) > 5.16 mg/L air
Based on the results of the histopathological and macroscopic investigations, Silver Powder Batch PMC 2 does not require classification for respiratory irritation.
According to the criteria specified by Directive 67/548/EEC and subsequent regulations, Silver Powder Batch PMC 2 does not require classification either for acute inhalation toxicity or for respiratory irritation.
According to the EC Regulation No. 1272/2008 and subsequent regulations, Silver Powder Batch PMC 2 does not require classification for acute inhalation toxicity or specific target organ toxicity - single exposure.