Registration Dossier

Administrative data

Description of key information

Reliable experimental studies in animals indicate a low acute toxicity of elemental silver (including nanoforms), following exposure via the oral, dermal or inhalation route. No mortality or any relevant clinical signs of acute toxicity were observed and the following effect levels were established for silver as follows: LD50oral > 5000 mg/kg, LD50dermal > 2000 mg/kg and LC50inhalation > 5.16 mg/L.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-08-09 to 1993-09-01
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, however no information on the purity of test item is given.
Qualifier:
according to guideline
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Manston, Kent, U.K.
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 154 - 175 g (males) and 140 - 155 g (females)
- Fasting period before study: yes, an overnight fast immediately before dosing and for approximately 2 hours after dosing
- Housing: in groups of five by sex in solid floor polypropylene cages with sawdust bedding
- Diet: ad libitum (Rat and Mouse Expanded Diet No. 1, Special Diet Servoces Limited)
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 23
- Humidity (%): 48 - 57
- Air changes (per hr): 15
- Photoperiod: 12 hours dark/light cycle
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 200 mg/mL

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg

DOSAGE PREPARATION:
- Rationale for the selection of the starting dose: a range-finding study with a fmale and a male rat receiving 2000 mg/kg of the test substance
Doses:
2000 mg/kg body weight
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: for 14 days
- Frequency of observations and weighing: The animals were observed for deaths or signgs of toxicity 0.5, 1, 2 and 4 hours after dosing and subsequently once daily. Individual body weights were recorded prior to dosing on Day 0 and on Days 7 and 14.
- Necropsy of survivors performed: yes
Statistics:
no data
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
There were no deaths observed.
Clinical signs:
There were no clinical signs of toxicity during the study.
Body weight:
All animals showed expected gain in body weight during the study.
Gross pathology:
No abnormalities were noted at necropsy.
Other findings:
No other findings were observed.
Interpretation of results:
practically nontoxic
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute oral median lethal dose (LD50) of the test material, silver powder CAP 9, in the Sprangue-Dawley strain rat was found to be greater than 2000 mg/kg body weight. No symbol and risk phrase are required.
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Publication, not a full study report, GLP. However, the study has apparently been conducted in accordance with OECD 423 and relevant experimental details and results are reported.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
2001-12-17
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Koatec Co. Ltd. (Pyeongtaek, Korea)
- Age at study initiation: 8-week-old
- Weight at study initiation: 300 mg/kg: 159.03 ± 3.84 (1st step) and 164.43 ± 3.02 (2nd step); 2000 mg/kg: 153.00 ± 3.74 (1st step) and 159.40 ± 5.47 (2nd step)
- Housing: three animals were housed per cage, plus during the administration and observation periods, no more than three animals were housed in a wire cage.
- Diet (ad libitum): commercial rodent chow (TEKLAD CERTIFIED GLOBAL 18% PROTEIN RODENT DIET, 2918C Harlan Co. Ltd., Indianapolis, IN, USA
- Water (ad libitum): tap water sterilised by ultra-violet irradiation
- Quarantine and acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 3°C
- Relative humidity: 55 ± 15%
- Air changes: 10 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: 1% citric acid
Details on oral exposure:
MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg

DOSAGE PREPARATION:
The AgNPs were dispersed in 1% citric acid.

CLASS METHOD
- Rationale for the selection of the starting dose: due to the absence of any toxicity data on the test substance, the dose level for the first step was 300 mg/kg (dosing volume were 10 mL/kg).
Doses:
300 and 2000 mg/kg
No. of animals per sex per dose:
6 rats (3 rats per step)
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days (daily)
- Frequency of observations and weighing: the mortality, clinical signs, body weights and necropsy findings were observed. The body weights were recorded on day 1, 3, 7 and 24.
- Necropsy of survivors performed: yes
Statistics:
The statistical analyses were performed using SPSS 12.1, Chicago, IL, USA and the data expressed as the mean ± SD. A chi-square test and one-way analysis of variance (ANOVA) were applied to test all the data. A value of p < 0.05 indicated statistical significance.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 other: mg/kg
Based on:
test mat.
Mortality:
300 and 2000 mg/kg: no deaths occurred
Clinical signs:
300 and 2000 mg/kg: normal appearance
Body weight:
300 and 2000 mg/kg: there were no significant differences in the body weights during 14 days after administration.
Gross pathology:
300 and 2000 mg/kg: no abnormal gross findings at necropsy.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LD50 (female rats) > 2000 mg/kg
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not classified as acute toxic via the oral route.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as acute toxic via the oral route.
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Publication, not a full study report, non-GLP. However, the study has apparently been conducted in accordance with OECD guideline 425 and relevant experimental details and results are reported.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)
Version / remarks:
adopted 2001-12-17
Deviations:
no
GLP compliance:
not specified
Test type:
up-and-down procedure
Limit test:
yes
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: National Laboratory Animal Centre, Mahidol University
- Age at study initiation: 10 - 12 weeks old
- Weight at study initiation: 28 - 35 g
- Diet (ad libitum): pelleted food
- Water (ad libitum): filtered tap water

ENVIRONMENTAL CONDITIONS - the animals were housed in an animal facility under negative pressure.
- Temperature: 24 ± 1°C
- Relative humidity: 55 ± 10%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
DOSAGE PREPARATION:
Preparation of AgNPs: A 0.094 M aqueous solution of silver nitrate (AgNO3; Merck) was prepared with soluble starch (Merck) as a stabilizer. An aqueous solution of 0.07 M sodium borohydride (NaBH4; Merck) with the soluble starch solution as a solvent was sequentially prepared as reducing agent. By mixing both solutions, the AgNO3 solution was added dropwise to the NaBH4 solution under vigorous stirring.
The purification of the AgNPs was precipitated using the centrifugation. Then, the purified AgNPs were washed three times with DI water and adjusted to the same volume before dilution. The percentage purity of the AgNPs was measured from free Ag ion concentration in the original AgNPs solution (10,000 ppm) using the macro- and microelectrode (Ngeontaea, W. et al., 2008)*. The results showed that the Ag ion concentration was contented at 3.77 ± 0.2 and 3.61 ± 0.2 ppm with the macro- and microelectrode, respectively. The AgNPs solutions were diluted with distilled water to obtain the concentration of AgNPs prior to use.
The particle morphology of AgNPs was observed using JEOL JEM-2010 analytical transmission electron microscope. The AgNps had a spherical configuration which had a primary particle diameter of 10 - 20 nm. The plasmon extinction of AgNPs was measured by Ocean Optics Portable UV-Visible spectrometer (USB 4000-UV-VIS detector). The maximum of extinction spectrum (λmax) of AgNPs was at 395 nm with a narrow full width at half height (FWHH) about 45 nm. This result indicated that the size distribution of AgNPs was narrow.

*Reference:
Ngeontaea, W., Janrungroatsakula, W., Morakot, N., Aeungmaitrepiroma, W. and Tuntulania, T. 2008. New silver selective electrode fabricated from benzothiazole calyx [4] arene: Speciation analysis of silver nanoparticles. Sens. Actuators. 134: 377 -385.
Doses:
5000 mg/kg
No. of animals per sex per dose:
9 male and 9 female mice
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 14 days)
- Frequency of observations and weighing: the animals were observed for toxic symptoms continuously for the first 3 hours after dosing. Finally, the number of survivors was noted after 24 hours and these animals were then maintained for 14 days further with observations made daily.
- Other examinations performed: at 1, 7 and 14 days after gavage, six mice in each group were sacrificed. Whole blood was collected for routine clinical pathology and blood chemical parameters including serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum creatinine, triglyceride, cholesterol and total protein. Various organs such as lung, hilar lymph node, heart, liver and kidney were collected in 10% buffered neutral formalin for routine histopathological evaluations.
Statistics:
Data were analysed using analysis of variance (ANOVA; Tukey' multiple comparison method). Values of P<0.05 were considered as the level statistical significance. Statistical analysis was performed using the SPSS statistical software for Windows, version 12.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Mortality:
No deaths were recorded during the 14 day observation period.
Clinical signs:
The animals did not show any significant changes in the general appearance during the observation period.
Males of the treatment group showed bite wounds in scrotal areas and tails.
Body weight:
There were no significant differences in the percentage of weight gain between the control and treatment groups of both male and female mice given 5000 mg/kg of the colloidal AgNPs orally.
Percentage of body weight gain AgNPs in male mice decreased on the first day .
Gross pathology:
There was no significant lesion found during the observation time.
Other findings:
- Haematology and clinical chemistry: the haematological analysis showed no significant changes of RBC, Hb, Ht, MCV, MCH, MCHC, platelets and WBC in the male and female treatment groups compared to the control groups. The leukocyte differential count showed no significant difference between the control and treated groups. There were no significant differences in any of the biochemical parameters examined in either the control or treated group of the male and female mice.
- Histopathology: histopathological examination of various organs in the control and treated animals showed no remarkable lesions that could be attributed to the effect of oral exposure of AgNPs at all observation times. Accumulation of free aggregated AgNPs was found on the mucosal areas of the gastrointestinal tracts of the treated mice after 24 hours post exposure. However, no evidence of penetration or infiltration of AgNPs was observed in the accumulated areas.

Test animal source:

National Laboratory Animal Centre, Mahidol University

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LD50 (male and female mice) > 5000 mg/kg bw
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not classified as acute toxic via the oral route.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as acute toxic via the oral route.
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Version / remarks:
adopted 2001-12-17
Deviations:
yes
Remarks:
number of tested animals higher as required by the guideline
GLP compliance:
yes
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orient Bio (Seongnam, Korea)
- Housing: housed in temperature- and light-controlled animal facilities
- Diet (ad libitum): rodent diet (Teklad Certified Irradiated Global 18 % Protein Rodent Diet; HArlan Laboratories, Madison, WI, USA)
- Water (ad libitum): tap water
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
no data
Doses:
2061 mg/kg bw
No. of animals per sex per dose:
5 males / 5 females
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: clinical observations and mortality checks were conducted once per hour for 6 hours after dosing and once daily thereafter for 14 days.
Body weights were measured on the day of treatment and on test days 1, 7, and 14.
- Necropsy of survivors performed: yes
At the end of the study, all of the animals were anestheized with isoflurane, and whole blood was collected from anesthetized animals via the posterior vena cava.
Statistics:
All of the data were expressed as means ± SD. Data were analysed using the Student's t-test.
Preliminary study:
no data
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 061 mg/kg bw
Based on:
test mat.
Mortality:
No mortalities were observed in the treatment and control groups.
Clinical signs:
no data
Body weight:
No treatment-related changes in body weights
Gross pathology:
Necropsy findings also revealed no treatment-related efects.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LD50 (male/female rats) > 2061 mg/kg bw
According to the Directive 67/548/EEC and its subsequent amendments, the test substance is not acutely toxic via the oral route.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not acutely toxic via the oral route.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Acute toxicity: via inhalation route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
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. QA statement)
Remarks:
signed 2009-11-12
Test type:
acute toxic class method
Limit test:
yes
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: 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
Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation 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 test atmosphere concentrations:
yes
Remarks:
please refer to "details on inhalation exposure" above
Duration of exposure:
4 h
Concentrations:
Actual concentration:
Main study group: 5.16 ±0.01 mg/L air
Satellite group: 5.15 ±0.01 mg/L air
Nominal concentration:
Main study group: 13.89 mg/L air
Satellite group: 13.89 mg/L air
No. of animals per sex per dose:
Main study group: 3 males / 3 females
Satellite group: 3 males / 3 females
Control animals:
no
Details on study design:
- 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.
Statistics:
Since no animal died prematurely, the calculation of an LC50 was not required.
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.16 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No animal died prematurely.
Clinical signs:
other: 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, slig
Body weight:
No influence in body weight gain was observed.
Gross pathology:
- 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).
Other findings:
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.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
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.
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Publication, based on results form a OECD (403, 1995) study in accordance with GLP. Relevant experimental details and results are reported.
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
adopted 1981-05-12
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS - Specific-pathogen free Sprague Dawley rats (Slc:SD; originally derived from the Charles River SD in 1968)
- Source: Orient Bio (Seongnam, Korea)
- Age at study initiation: 7 weeks
- Weight at study initiation: approximately 218 g (males) and 153 g (females)
- Housing: during the acclimation and experimental periods, the rats were housed in five mesh cages (five rats per cage) in a room with controlled temperature and humidity. During the exposure period, the animals were housed in individual wire cages.
- Diet (ad libitum): a rodent diet (Harlan Teklab, Plaster International Co., Seoul
- Water (ad libitum): filtered water
- Acclimation period: 1 week before starting the experiments

ENVIRONMENTAL CONDITIONS
- Temperature: 23°C ± 2°C
- Humidity: 55% ± 7%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus and exposure chamber volume: a whole-body-type exposure chamber (1.3 m^3, Dusturbo, Seoul)

- System of generating particulates/aerosols: the silver nanoparticles were generated as described in previous reports (Ji et al., 2007a; 2007b; Jung et al., 2006; Sung et al., 2009)*. The generation consisted of a small ceramic heater connected to an AC power supply (AC 95 V) and housed within a quartz tube case. The heater dimensions were 50 X 5 X 1.5 mm^3, and a surface temperature of about 1500°C within a local heating area of 5 X 10 mm^2 was achieved within about 10 sec (Jung et al., 2006)*. The source material (about 160 mg, Daedeok Science, Daejeon) was positioned at the highest temperature point. The quartz tube case was 70 mm in diameter and 140 mm long. Clean (dry and filtered) air was used as the carrier gas, and the gas flow maintained at 54.0 L/min (Re 1/4 572, laminar flow regime) using a mass flow controller (MFC, AERA, FC7810CD-4V, Japan; Ji et al., 2007a,b)*. This generator has already been shown to generate nanoparticles from 5 to 95 nm in diameter that do not agglomerate in air. Plus, an X-ray diffraction analysis using an X-ray diffractometer utilising CuK2 radiation showed that the particles generated are metallic silver, not silver oxides (Jung et al., 2006)*. In the current study, the system produced different concentrations of nanoparticles (high, middle, and low) in three separate chambers.

- Method of particle size and test atmosphere determination: in each chamber, the nanoparticle distribution with respect to size was measured directly in real-time using a differential mobility analysing system (DMAS); combining a differential mobility analyser (Short type-DMA, 4220, HCT Co., Ltd, Korea, range 2 - 150 nm) and condensation particle counter (CPC, 4312, HCT Co., Ltd, Korea, 0 - 10^8/cm^3 detection range). The nanoparticles were measured using sheath air at 5 L/min and polydispersed aerosol air at 1 L/min for the DMA and CPC, respectively. The particle concentration in the fresh-air control chamber was measured using a particle sensor (4123, HCT Co., Ltd, Korea) that consisted of two channels; 300 - 1000 nm and over 1000 nm.

TEST ATMOSPHERE (if not tabulated)
(Please also refer to Table 1 in the field "Any other information on materials and methods incl. tables" below)

*References:
- Ji, J.H., Jung, J.H., Yu, I.J., and Kim, S.S. (2007a) Long-term stability characteristics of metal nanoparticle generator using small ceramic heater for inhalation toxicity studies. Inhalation Toxicology 19(9): 745 - 751.
- Ji, J.H., Jung, J.H., Kim, S.S., Yoon, J.U., Park, J.D., Choi, B.S., et al. (2007b) A twenty eight-days inhalation toxicity study of silver nanoparticles in Sprague-Dawley rats. inhalation Toxicology 19(10): 857 - 871.
- Jung, J.H., Oh, H.C., Noh, H.S., Ji, J.H., and Kim, S.S. (2006) metal nanoparticle generation usng a small-sized ceramic heater with a local heating area. Journal of Aerosol Science 37: 1662 - 1670.
- Sung, J.H., Ji, J.H., PArk, J.D., Yoon, J.U., Kim, D.S., Jeon, K.S. et al. (2009) Subchronic inhalation toxicity of silver nanoparticles. Toxicological Science 108(2): 452 - 461.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
please refer to "Details on inhalation exposure" above.
Duration of exposure:
4 h
Concentrations:
Actual concentrations:
- low-dose group: 75.84 +/- 3.25 µg/m^3
- middle-dose group: 134.62 +/- 5.99 µg/m^3
- high-dose group: 750.00 +/- 35.62 µg/m^3

Note by the authors: "While more than 3 10^6 particles/cm3 (ca. 730 µg/m^3) could be achievable, this would generate a lot of sub-micron-sized agglomerated/ aggregated particles, which are less than desirable conditions for studying the effects of nanosized particles.

(Please also refer to Table 1 in the field "Any other information on materials and methods incl. tables" below)
No. of animals per sex per dose:
5 males / 5 females
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: daily on weekdays for any evidence of exposure-related effects including: respiratory, dermal, behavioural, nasal, or genitourinary changes suggestive of irritation.
- Body weights: at purchase, at the time of grouping, and 1, 3, 7, and 14 days after the 4-hour inhalation exposure and before necropsy.
- Other examinations performed: Lung function tests were conducted on four rats from each dose group. The lung function of the exposed rats was evaluated at purchase and 1, 3, 7, and 14 days after the 4-hour inhalation exposure using a ventilated bias flow whole-body plethysmograph (WBP; SFT3816, Buxco Electronics, Sharon, Connecticut, USA), consisting of a reference chamber and animal chamber interconnected by a pressure transducer (MAX1320, Buxco Electronics). The parameters for the pulmonary function test included the tidal volume (TV, mL), minute volume (MV, mL/min), respiratory frequency (BPM, breath/min), inspiration time (Ti, sec), expiration time (Te, sec), peak inspiration flow (PIF, mL/sec), and peak expiration flow (PEF, mL/s). After being exposed to the silver nanoparticles for 4 hours, the rats were placed in the animal chamber, left for 40 min to stabilize, and the plethysmography initiated by measuring the selected parameter values for 5 min (Sung et al., 2008)*.

* Reference:
- Sung JH, Ji JH, Yun JU, Kim DS, Song MY, Jeong J, et al. (2008) Lung function changes in Sprague-Dawley rats after prolonged inhalation exposure to silver nanoparticles. Inhalation Toxicology 20(6): 567–574.
Statistics:
All the results are expressed as the means ± standard error (SE). An analysis of variance (ANOVA) test and Duncan’s multiple range tests were used to compare the body weights and parameters from the lung function test obtained for the three dose groups with those obtained for the control rats.
The level of significance was set at p < 0.05 and p < 0.01.
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 750 other: µg/m^3
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: 750 µg/m³ was the maximum attainable concentration that was possible without masking the effects assessment of nano-particles by generation of non-nano-sized agglomerates.
Mortality:
No mortality was observed related to the test substance.
Clinical signs:
other: No toxic signs were observed related to the test substance.
Body weight:
There were no significant changes in the body weights of the male and female rats.
Gross pathology:
No significant gross effects were observed.
Other findings:
- Food consumption: no significant differences were observed in food consumption between the exposed rats and the control group.
- Lung function test: although some statistically significant differences were found in the lung function parameters, such as the tidal volume and minute volume, among the dose groups, no significant dose-dependent changes were found in the lung function parameters for the male and female rats exposed to the silver nanoparticles
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LC50 (male and female rats; 4 h) > 750 µg/m^3 ( or 3.1 x 10^6 particles/cm^3)
The following statement was given by the authors of the publication:
"While more than 3 x 10^6 particles/cm^3 could be achievable, this would generate a lot of sub-micron-sized agglomerated/aggregated particles, which are less than desirable conditions for studying the effects of nanosized particles. Plus, exposure to more than a million silver nanoparticles/cm^3 is unlikely in a living environment or from silver nanoparticle-containing consumer products. Thus, the highest concentration in the current exposure assessment study would not be aniticpated in a real silver nanoparticle manufacturing workplace (mansucript in submission)."
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Publication, not a full study report, GLP. However, the study has apparently been conducted in accordance with OECD guideline 402 and relevant experimental details and results are reported.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
adopted 1987-02-24
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Koatec Co. Ltd. (Pyeongtaek, Korea)
- Age at study initiation: 8-week-old
- Weight at study initiation: 0 mg/kg: 252.93 ± 4.07 (males) and 228.69 ± 3.70 (females); 2000 mg/kg: 256.82 ± 5.99 (males) and 228.36 ± 8.40 (females)
- Housing: three animals were housed per cage, plus during the administration and observation periods, no more than three animals were housed in a wire cage.
- Diet (ad libitum): commercial rodent chow (TEKLAD CERTIFIED GLOBAL 18% PROTEIN RODENT DIET, 2918C Harlan Co. Ltd., Indianapolis, IN, USA
- Water (ad libitum): tap water sterilised by ultra-violet irradiation
- Quarantine and acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 3°C
- Relative humidity: 55 ± 15%
- Air changes: 10 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
not specified
Vehicle:
other: 1% citric acid
Details on dermal exposure:
An electric clipper was used to expose the skin, then the test substance was applied and covered with a dressing. The dressing was removed after 24 hours.
Duration of exposure:
24 hours
Doses:
0 and 2000 mg/kg
No. of animals per sex per dose:
5 males / 5 females
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 15 days
- Frequency of observations and weighing: the animals were observed twice a day over 15 days for any irritation and toxicity. The mortality, clinical signs, body weights and necropsy findings were observed and the results compared with those for the vehicle control group. The body weights were recorded on day 1, 3, 7 and 24.
- Necropsy of survivors performed: yes
Statistics:
The statistical analyses were performed using SPSS 12.1, Chicago, IL, USA and the data expressed as the mean ± SD. A chi-square test and one-way analysis of variance (ANOVA) were applied to test all the data. A value of p < 0.05 indicated statistical significance.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 other: mg/kg
Based on:
test mat.
Mortality:
The Ag-NPs did not induce any abnormal signs in this study.
Clinical signs:
The Ag-NPs did not induce any death in this study.
Body weight:
All the animals, including the negative control group and test groups, lost weight on day 1, which was assumed to be stress related.
Gross pathology:
There were no abnormal gross findings for any of the treated groups at necropsy.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LD50 > 2000 mg/kg
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not classified as acute toxic via the dermal route.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as acute toxic via the dermal route.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Acute oral toxicity:

Four reliable studies are available regarding the acute oral toxicity of metallic silver. In a study according to OECD TG 401, conducted in 1993 by Allen, silver metal powder (particle size described as < 40 µm) was used as the test item. No deaths or other signs of acute toxicity were observed in this limit test in rats. The LD50 was established as >2000 mg/kg.

Kim et al. (2012) published results of a study conducted according to OECD TG 423 using silver nanoparticles (average particle size: 10 nm). Doses of 300 and 2000 mg/kg were applied to three rats each. No deaths or other signs of acute toxicity were observed in this study. The LD50 was established as >2000 mg/kg.

In 2011, Maneewattanapinyo published a study using material characterised as “colloidal silver nanoparticles“, consisting of 99.96% elemental silver and less than 0.04% ionic silver. The study was conducted according to OECD TG 425 as a limit test, administering a dose of 5000 mg/kg to male and female mice. No deaths or other signs of acute toxicity were observed in this study. The LD50 was established as >5000 mg/kg.

Yun et al. (2015) published results of a study conducted according to OECD TG 420 using citrate-capped silver nanoparticles. A dose of 2061 mg/kg was applied to five male and female rats. No deaths or other signs of acute toxicity were observed in this study. The LD50 was established as >2061 mg/kg.

Similar acute toxicity studies with other silver substances are included in this dossier for metallic silver for comparative reasons.

 

Acute inhalation toxicity:

Two reliable acute inhalation toxicity studies with elemental silver are available. In a study according to OECD TG 436 by Haferkorn (2012), silver metal powder was used and inhaled by rats for 4 hours at an exposure concentration of 5.16 mg/L. The mass median aerodynamic diameter of inhaled silver particles as determined in the inhalation chamber during the study was MMAD = 2.3 µm. No deaths were observed. Clinical signs, such as slight ataxia and reduced breathing frequency were restricted to the first few hours post exposure and are considered to be general signs in response to inert dust exposure, but not necessarily test item-related. The LC50 (4h) was established at > 5.16 mg/L (>5.16 g/m³). The study included a satellite group of animals for the assessment of respiratory irritation potential (see respective section in this dossier).

Sung et al. (2011) reported a 4-hour acute inhalation toxicity study in rats with silver nanoparticles (acc. to OECD TG 403). Groups of ten rats (5m+5f) were exposed to three different exposure concentrations of ca. 76, ca. 135 and ca. 750 µg/m³ (highest attainable concentration). The median particle size of particles in the inhalation chamber was 18 -20 nm. No mortalities were observed and no other signs of acute toxicity were observed. Some influence of the exposure on lung function parameters, such as tidal volume and minute volume were reported, but these were not dose-dependent. As in the study by Haferkorn, these effects can be considered as generic responses to the inhalation of inert dusts, but are not considered to be related to the test substance silver as such. The LC50 is established in this study as > 750 µg/m³ (highest concentration tested).

A large number of mechanistic studies were identified, in which silver nanoparticles were intratracheally instilled in rats and mice. The major endpoints investigated in such studies are primarily (1) time course and dose-response intensity of pulmonary inflammation via cytokine measurements, differential cell count in BALF, (2) airway and lung parenchymal cell proliferation and (3) histopathological evaluation of lung tissue. Whereas such information might be appropriate to assess the direct effects of a substance on the pulmonary region, it is not considered relevant for hazard and risk assessment purposes. The introduction of the toxicant via instillation techniques is nonphysiologic, involving invasive delivery, usually at a dose and/or dose rate substantially greater than that which would have occurred during inhalation. In addition, the distribution of an instilled material within the respiratory tract will likely differ from the distribution of an inhaled material.The influence of the instilled vehicle in which the test material is suspended or dissolved may have an impact on the distribution of the test substance within the lung, produce effects itself or, if it alters the physicochemical nature of the test material, may alter the effects of the material on the lungs (Driscoll, K.E. et al. 2000; Osier, M. and Oberdörster, G., 1997). The references usingintratrachealinstillation are given below in a tabulated summaryfor information only.

Reference

Information on study design

Silva et al. (2015)

Species:

Male Sprague Dawley rats

# of animals:

6 rats/dose/time point

Dose:

0, 0.1, 0.5, or 1.0 mg/kg bw

Route of administration:

intratracheal

Sacrifice:

1, 7, and 21 days after administration

Examinations

BAL cell analysis, Lung histopatholgy, Airway cytotoxicity,

ICP-MS analysis (lung, heart, spleen, kidney, liver)

Arai et al. (2015)

Species:

Male ICR mice

# of animals:

3 mice (experiment 1) or 8 mice (experiment 2)

Dose:

10 µg Ag/mouse (target dose); 7.5 µg Ag/mouse (actual dose)

Route of administration:

intratracheal

Sacrifice:

4 and 24 hours after administration

Examinations

BALF (interleukin conc., silver conc., # of cells), Silver conc. in lung, liver, kidneys, spleen and urine

Gosens et al. (2015)

Species:

Female C57BL/6NTac inbred mice

# of animals:

3 mice/group

Dose:

0, 1, 4, 8, 16, 32, 64 and 128 µg/mouse

Route of administration:

intratracheal

Sacrifice:

24 hours after administration

Examinations

BALF, (cytokines, LDH, ALP, albumin, total protein, cell count, cell differential count), Haematology (total number of cells, cell differential count, haemoglobin content), Liver analysis (glutathione content, silver content)

Seiffert et al. (2015)

Species:

Sprague Dawley rats

# of animals:

5 – 6 rats

Dose:

0.1 mg/kg bw

Route of administration:

intratracheal

Sacrifice:

1, 7, and 21 days after administration

Examinations

Lung mechanics (resistance, dynamic compliance, airway responsiveness), Bronchoalveolar lavagage (differential cell count,, total protein, KC, eosinophilic cationic protein, IL-13, IgE, IL-5, CCL11, and bronchoalveolar lavage malondialdehyde), Lung histology.

Kaewamatawong et al. (2013)

Species:

Male ICR mice

# of animals:

3 mice/dose/time point

Dose:

0, 10, 100, 1000 and 10000 ppm

Route of administration:

intratracheal

Sacrifice:

1, 3, 7 and 15 days after administration

Examinations

Histopathology, Immunohistochemistry, Distribution/accumulation AgNPs

References:

Osier, M and Oberdörster, G. 1997. Intratracheal Inhalation vs Intratracheal Instillation: Differences in Particle Effects. Fundamental and applied Toxicology 40, 220-227

 

Driscoll KE, Costa DL, Hatch G, Henderson R, Oberdörster G, Salem H, Schlesinger RB. 2000. Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations. Toxicol Sci., 55(1):24-35

 

Acute dermal toxicity:

Two acute dermal toxicity studies with elemental silver are available, both using nanoforms of silver as the test items. Kim et al. (2012) published results of an acute dermal toxicity study in accordance with OECD TG 402, using „nano-sized colloidal silver“ (average particle size = 10 nm). The limit dose of 2000 mg/kgbw applied to the skin of rats (5m+5f) did not produce deaths or any signs of acute toxicity. The LD50 (dermal) is established as >2000 mg/kg.

Maneewattanapinyo (2011) reported on a study conducted similarly to OECD TG 434. The test item was characterised as „colloidal silver nanoparticles“, consisting of 99.96% elemental silver and less than 0.04% ionic silver. Doses of ca. 0.17 and 348 mg Ag/kgbwwere applied to the skin of guinea pigs. No mortality and no clinical signs indicating toxicity were observed. Gross pathology also revealed no signs of toxiciy. In this study, the LD50 (dermal) for elemental silver was established as > 348 mg/kg. Because of some reporting deficiencies and because the highest dose in this study is below the limit dose for such tests, this study is considered merely as supporting data.

 

Acute toxicity other routes:

The references contained in the summary entry for the acute toxicity via non-physiological routes of application are of limited value for risk assessment purposes. The references do not fulfil the criteria for quality, reliability and adequacy of experimental data for the fulfilment of data requirements under REACH and hazard assessment purposes (ECHA guidance R4 in conjunction with regulation (EC) 1907/2006, Annexes VII-X). The information contained therein were included for information purposes only and the deficiencies of the studies are listed below. Generally, the intravenous and intraperitoneal administration is not relevant for the hazard assessment of industrial chemicals.These routes are not considered physiologically relevant because, although it is occasionally used for dosing of chemotherapeutic drugs, humans are not exposed to environmental chemicals via the peritoneum or intravenously.After oral administration a chemical is absorbed by the digestive system and then carried through the portal vein into the liver before it reaches the rest of the body. After i.p. or i.v. administration a portion of the administered chemical will be carried through the hepatic portal system to the liver. Thus, both activation and detoxification of chemicals is less effective when using these non-physiological routes. As a result, only a small amount of active chemical emerges from the liver to the rest of the circulatory system, and many chemicals are significantly less toxic by the oral route than by i.p. or i.v. injection (Wang et al, 2015). Secondly, the i.p. or i.v. administration of a chemical may cause serious bolus effects, due to an escalated increase of the internal dose, whereas the oral route shows a delayed and slow increase of the internal dose, leading to milder and more physiological responses. In summary, there are many reasons to conclude that the i.p. or i.v. route is inappropriate because it limits first-pass metabolism and normal liver detoxification processes, does not necessarily enhance systemic exposures or detection of adverse effects, and can lead to abnormal localised effects.

 

In the studies by Sarhan et al., 2014 and Ansari et al., 2015, rats were given doses of 2 and 5 g/kg bw respectively by intraperitoneal injection. Despite the identical study design in both references, a discrepancy was observed in the overall findings. The animals exposed to 5 g/kg bw showed no signs of toxicity, whereas less than half of the dose showed signs of liver and kidney toxicity. Due to the irrelevant route of exposure and overall poor reporting and experimental quality, the references were not considered further for hazard and risk assessment purposes.

In a study by Xue et al., 2012 mice were intravenously exposed to doses of 7.5, 30 and 120 mg/kg silver nanoparticles.

Justification for classification or non-classification

Reliable experimental studies in animals indicate a low acute toxicity of elemental silver (including nanoforms), following exposure via the oral, dermal or inhalation route. No mortality or any relevant clinical signs of acute toxicity were observed and the following effect levels were established for silver as follows: LD50oral > 5000 mg/kg, LD50dermal > 2000 mg/kg and LC50inhalation > 5.16 mg/L. In consequence, classification for acute toxicity is not required.