Silver

Endpoint summary

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

Reference

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:

guideline study with acceptable restrictions

Remarks:

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:

other: There were no clinical signs of toxicity 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 conclusion

Endpoint conclusion:

no adverse effect observed

Acute toxicity: via inhalation route

Link to relevant study records

Reference

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 conclusion

Endpoint conclusion:

no adverse effect observed

Acute toxicity: via dermal route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: dermal

Type of information:

other: Route-to-route extrapolation from inhalation study

Adequacy of study:

weight of evidence

Study period:

2012-02-09 to 2012-03-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

cf. section 'Cross-reference' - Weight of evidence approach described in 'Silver metal (massive and powder): Weight of Evidence' (document attached in IUCLID section 13 - "CSR Annex 11 - Weight of Evidence Justification for Silver metal - human health endpoints).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Version / remarks:

2009-09-07

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

other: acute toxic class method

Limit test:

yes

Sex:

male/female

Dose descriptor:

other: LC50

Effect level:

> 5.16 other: mg/L air

Based on:

test mat.

Reference 2

Endpoint:

acute toxicity: dermal

Type of information:

other: Route-to-route extrapolation from oral study

Adequacy of study:

weight of evidence

Study period:

1993-08-09 to 1993-09-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no information on the purity of test item is given.

Justification for type of information:

cf. section 'Cross-reference' - Weight of evidence approach described in 'Silver metal (massive and powder): Weight of Evidence' (document attached in IUCLID section 13 - "CSR Annex 11 - Weight of Evidence Justification for Silver metal - human health endpoints").

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Reference 3

Endpoint:

acute toxicity: dermal

Type of information:

other: Dermal absorption study for route-to-route extrapolation

Adequacy of study:

weight of evidence

Study period:

27 June 2006-August 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

cf. section 'Cross-reference' - Weight of evidence approach described in 'Silver metal (massive and powder): Weight of Evidence' (document attached in IUCLID section 13 - "CSR Annex 11 - Weight of Evidence Justification for Silver metal - human health endpoints).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

13 April 2004

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Test Guidelines for in vitro assessment of dermal absorption and percutaneous penetration of cosmetic ingredients, Diembeck et al, 1999, Food and Chemical Toxicology, 37:191-205

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Opinion of the Scientific Committee on Cosmetic Products and Non-Food Products intended for Consumers on "Basic Criteria for the in vitro Assessment of Dermal Absorption of Cosmetic Ingredients", SCCNFP/0750/03 final

Version / remarks:

20 October 2003

Deviations:

not specified

GLP compliance:

yes

Dose descriptor:

other: absorption

Effect level:

0 other: mg cm-2 h-1

Based on:

test mat.

Remarks:

0.5% silver in formulation

Remarks on result:

other: assessed as 'bioavailability', driven by amount of test item in dermis (and residual epidermis)

Dose descriptor:

other: absorption

Effect level:

ca. 0 other: mg cm-2 h-1

Based on:

test mat.

Remarks:

1.5% silver in formulation

Remarks on result:

other: assessed as 'bioavailability', driven by amount of test item in dermis (and residual epidermis)

Executive summary:

The dermal absorption/percutaneous penetration of silver was determined in vitro (Bornatowicz, 2006). Silver was tested as micro-sized powder in a hydrophilic formulation at 0.5 and 1.5% Ag. The test substances was assessed via determination of Ag using ICP-MS.

Three integrity checked dermatomed skin preparations of one young pig were used in each experiment. Skins were inserted in static penetration cells (Franz-cells) with an application area of 1.0 cm².

The test substance formations were applied topically to the horny layer of the skin in nominal quantities of 20 mg/cm². A non-occlusive exposure under temperature controlled conditions was performed and formulations were left on the skin for 24h.

24h after application, the stratum corneum was removed by repeated stripping (absorbed test substance). The remaining skin was taken to determine absorbed test substance. Penetration was calculated via the mass of test substance in the receptor fluid. The amount of bioavailable test substance is defined as sum of absorbed + penetrated test substance.

Most of the test substance is wiped off at the end of exposure (60-67%). Tape stripping removed a large amount of test substance from the upper layers of the skin with the amount of silver decreasing with later strippings suggesting a low level of Ag in the deeper layers of the stratum 5%, absorption 1.38-2%). A very low amount of silver penetrates the skin (0.0007-0.0014%), with the amount of test item in the receptor fluid being below the Limit of Quantification for both formulations. The bioavailability of silver was calculated at 1.38-2.0%, corresponding to 1.52-6.54 µg/cm².

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Introduction:

An in-vivo comparative toxicokinetic study, via oral route, was performed using a rodent model (according to OECD TG 417 and GLP compliant; Melvin et al., 2021 and Charlton et al., 2021). The test items included two ionic silver salts (silver nitrate and silver acetate), a well-characterized nanosilver reference material (15 nm AgNP) and a powder-form of silver metal (size ~0.3 μm, representing a conservative silver metal powder). Comparative toxicokinetics data were obtained after both single and 28-days repeated dose administration, including the measurements of Ag levels in blood and in tissues.

The key findings were:

• silver metal was substantially less absorbed than soluble silver salts and nanosilver. Based on matched dose assessments, the extent of systemic exposure was about 10 to 30-fold lower in the case of silver metal versus reference ionic silver salts.


• silver metal was considerably less distributed in tissues and organs than silver salts (ionic silver compounds). This links to predictions that silver metal (massive and powder) represents a correspondingly lower health hazard, i.e., is less likely to cause toxicity effects.

It is generally accepted that systemic toxicity of simple silver salts substances is driven by the silver ion (Ag+) as the primary species relevant for tissue exposure, and hence hazard assessment. Thus, a low bioavailability of silver metal (massive and powder), leading to a low internal concentration of silver ions (as toxicophore) leads to a lack of biological interaction and hence an absence of adverse outcome in comparison with high bioavailable silver salts. Therefore, it is assumed that silver metal represents a lower health hazard than the more bioavailable forms of silver at comparable nominal Ag levels.
Therefore, following the new in-vivo TK study findings, a direct Read-Across of mammalian toxicity datasets from simple silver salts and nanosilver to silver metal (massive and powder) is considered not appropriate.

Alternatively, a Weight of Evidence (WoE) approach considering:

1. the available mammalian toxicity data of simple silver salts and nanosilver and


2. the demonstrated difference in bioavailability of simple silver salts and nanosilver vs. silver metal (massive/powder)
   is justified to complete the REACH data requirements for Ag metal (massive/powder) and to avoid any new animal testing.

 

The approach and justification for the applied human health hazard assessment is detailed in the weight of evidence justification document attached to the silver IUCLID file in section 13. 

 

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 toxicity – dermal route

There is no study available to evaluate the dermal acute toxicity of silver metal (massive and powder). However, acute toxicity studies of silver metal (massive/powder) via oral and inhalation route of exposure are available. These studies are used as source of information and a weight of evidence (WoE) approach is built based on the route-to-route extrapolation including a correction factor for dermal penetration/absorption.

The WoE-approach for acute dermal toxicity of silver metal (powder/massive) is built based on acute toxicity studies via oral and inhalation route for silver massive (powder) and nanosilver (cfr data in table below)
Note that:

• based on the outcome of the comparative in-vivo toxicokinetics data, the data of nanosilver cannot be directly read-across to silver metal powder because of the demonstrated difference in bioavailability.


• it is proven that dermal penetration is dependent on the dissolution of the substance with the dissolution being proportionate to surface area (higher for nano particles12).
  Since it is acknowledged that the silver ion is the relevant toxicophore for systemic toxicity, the outcome of studies performed with nanosilver can be considered as worst-case to assess the potential effects of silver metal (massive/powder).

In the below table, the quality of the individual studies is summarised via assessment of their relevance and reliability (assessed as Klimisch-score (‘K’)).

There are several lines of evidence taken into account to fill the acute toxicity endpoint:

1. An oral acute toxicity study with silver metal powder shows no effect up to 2000 mg/kg bw and the inhalation acute toxicity study shows no effect up to 5.16 mg/L air.


2. The bioavailability of silver metal powder via the oral route was estimated between 0.3 to 0.5% and the inhalation bioavailability was calculated around 1%. As the dermal absorption of silver metal is orders of magnitude lower (0.0014%), it is reasonable to assume that the acute dermal toxicity of silver metal (massive/powder) at comparable dosing level is lower than the acute toxicity via oral and inhalation routes.


3. Furthermore, acute oral toxicity and acute inhalation toxicity studies with nanosilver do not show a hazard up to classification threshold concentrations and are taken as supporting evidence for the non-hazard conclusion for silver metal (massive/powder) via dermal route, knowing that the bioavailability of nanosilver is at least equal but most likely higher than that of silver metal (massive/powder), as demonstrated experimentally by e.g. in TDp testing and in vivo TK testing.

In conclusion, it is considered justified to use:

• The acute toxicity studies (oral and inhalation route of exposure) performed with silver powder and applying a route-to-route extrapolation, and


• the studies performed nanosilver which are considered as a worst-case test item compared to massive/powder form to complete the acute dermal toxicity endpoint of silver metal (massive/powder).

This Weight of Evidence is considered strong and reliable, and demonstrates that silver metal (massive/powder) does not trigger adverse effects via single exposure by dermal route up to threshold exposure level.

Please see section 13 for further details in the development of the Weight of Evidence Approach for silver metal.

 

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.

 

1 Pang C, Brunelli A, Zhu C, Hristozov D, Liu Y, Semenzin E, Wang W, Tao W, Liang J, Marcomini A, Chen C, Zhao B (2016) Demonstrating approaches to chemically modify the surface of Ag nanoparticles in order to influence their cytotoxicity and biodistribution after single dose acute intravenous administration. 

2 Patchin ES, Anderson DS, Silva RM, Uyeminami DL, Scott GM, Guo T, Van Winkle LS, Pinkerton KE (2016) Sizedependent deposition, translocation, and microglial activation of inhaled silver nanoparticles in the rodent nose and brain. Environ Health Perspect. 124: 1870-1875. 

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.