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Basic toxicokinetics

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Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-2009
Reliability:
other: Standard reliability rating not applicable: Non standard test (in-vitro bioaccessibility). Methodology and results are very well documented.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Objective of study:
other: bioaccessibility
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Solubility of test item in simulated human fluids.
GLP compliance:
no

Test material

Constituent 1
Reference substance name:
silver (metal), disilver oxide, silver nitrate
IUPAC Name:
silver (metal), disilver oxide, silver nitrate
Details on test material:
Ag-1:
- Name of test material (as cited in study report): silver metal powder
- CAS No.: 7440-22-4
- Molecular formula (if other than submission substance): Ag
- Molecular weight (if other than submission substance): 107.87g/mol
- Substance Type: metallic
- Physical state: powder
- Analytical purity: 99.2% (stated by the producer)
- Purity test date:
- Batch No.: Batch PMC 2
- Expiration date of the batch: March 2009
- Storage condition of test material: closed container, cool, dry, ventilated area
- Other:
Water solubility: considered "insoluble"
Relative density: 10.5 (MSDS provided by the producer)
Tapped density: 3.20g/cm³ (stated by the producer)
Surface area: 3.0m²/g (stated by the producer)
Particle size: volume based particle size distribution (provided by the producer)
D10: 0.8 μm
D50: 1.9 μm
D90: 11.2 μm

Ag-2:
- Name of test material (as cited in study report): silver metal powder
- CAS No.: 7440-22-4
- Molecular formula (if other than submission substance): Ag
- Molecular weight (if other than submission substance): 107.87 g/mol
- Substance type: metallic
- Physical state: powder (very fine, nanometer range)
- Analytical purity: min. 98% (stated by producer)
- Batch No.: Batch PMC 3
- Stability: chemical modification is not anticipated during storage, the powder particles may agglomerate
- Storage condition of test material: closed container, cool, dry, ventilated area
- Other:
Water solubility: considered “insoluble”
Relative density: 10.5 (MSDS provided by the producer)
Tapped density: 2.7 g/cm3 (stated by the producer)
Surface area: 11.04 m2/g (stated by the producer)
Particle size: volume based particle size distribution (provided by the producer)
D10: 20 nm
D50: 35 nm
D90: 60 nm

Ag2O:
- Name of test material (as cited in study report): disilver oxide powder
- CAS No: 20667-12-3
- Molecular formula (if other than submission substance): Ag2O
- Molecular weight (if other than submission substance): 231.74 g/mol
- Physical state: solid, light coloured
- Analytical purity: 93.7 % (stated by the producer), corresponds to > 99.9 % Ag2O
- Storage condition: in tightly closed container in cool, well ventilated area, separate from acids, alkalis, reducing agents and combustibles
- Batch No.: Batch PMC 1
- Expiration date of the batch: until March 2009
- Other:
Water solubility: “insoluble in hot or cold water”
Relative density: 7.14 g/cm3 (20°C) (stated by the producer)
Tapped density: 1.32 g/cm3 (stated by the producer)
Surface area: 0.32 m2/g (stated by the producer)
Particle size: volume based particle size distribution (provided by the producer)
D10: 2.2 μm
D50: 3.9 μm

AgNO3
- Name of test material (as cited in study report): silver nitrate
- CAS No: 7761-88-8
- Molecular formula (if other than submission substance): AgNO3
- Molecular weight (if other than submission substance): 169.87 g/mol
- Physical state: white crystalline
- Analytical purity: min 99.9 % AgNO3 (stated by the producer)
- Storage conditions: tightly closed container, avoid light effect and humidity; product has a corrosive effect on aluminium or steel
- Batch No.: Batch PMC 1
- Expiration date of the batch: no expiration date if product is kept on original package
-Other:
Water solubility: approx. 2.16 kg/L (20°C) (stated by the producer)
Relative density: approx. 4.35 g/cm3 (stated by the producer)
Surface area: 0.15 m2/g (stated by the producer)
Particle size: volume based particle size distribution (provided by the producer)
D10: 231 μm
D50: 367 μm
D90: 486 μm

Test animals

Species:
other: in vitro (simulated human body fluids)
Details on test animals or test system and environmental conditions:
The silver metal, silver oxide and silver nitrate particles were exposed to five different test media separately at a pH range from about 4.5 to 7.4. The test media were:
• Gamble’s solution (GMB, pH 7.4) which mimics interstitial fluid within the deep lung under normal health conditions (Stopford et al, 2004).
• Phosphate-buffered saline (PBS, pH 7.4), is a standard physiological solution that mimics the ion strength of human blood serum. It is widely used in the research (e.g. Norlin et al, 2002) and medical health care community (e.g. Hanawa et al, 2004; Okazaki and Gotoh, 2004) as a reference test solution for comparison of data under simulated physiological conditions.
• Artificial sweat (ASW, pH 6.5) simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body when sweating. The fluid is recommended in the available standard for testing of nickel release from nickel containing products (EN1811, 1998).
• Artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions (Stopford et al, 2004).
• Artificial gastric fluid (GST, pH 1.5) mimics the very harsh digestion milieu of high acidity in the stomach. (ASTM D5517)

The test media were selected to simulate relevant inhalation scenarios (as far as practical) where silver metal, silver oxide or silver nitrate particles may enter the human body through inhalation and, subsequently by ingestion of inhaled particles that are translocated to the gastro-intestinal tract. It should be stressed though, that the different test media only simulate physiological conditions to a limited extent. The complexity and function of the real body fluids are difficult to simulate. However, in vitro results in such synthetic biological media can, in a simple way, provide information that could be relevant for a real situation.

The test solutions were prepared using ultra-pure water and chemicals of analytical grades.
The pH of ALF and GMB was adjusted using 50% NaOH and 25% HCl respectively.
The pH of PBS was adjusted with 50% NaOH.
For ASW, the pH was adjusted using 1% dilute ammonia solution

Administration / exposure

Route of administration:
other: in vitro (simulated human body fluids)
Details on study design:
Experimental Procedure
Triplicate powder samples were prepared for exposure in the different test media, each for two time periods. In addition, one blank sample (without addition of any test material) containing only the test solution was incubated together with the triplicates for each time period. 5 ± 0.5 mg of the test material was weighed using a Mettler AT20 balance with readability of 2 μg, and placed in a TPX Nalge® jar. 50 mL of the test solution was then added to the TPX Nalge® jar containing the powder sample, before incubated at dark conditions in a Stuart shaker incubator S180 regulated at 37 ± 0.5°C. The solution was gently shaken (bi-linearly) with an intensity of 25 cycles per minute fpr 2 and 24 hours, respectively
Details on dosing and sampling:
The particle loading of 0.1 g/L (corresponding to 5 mg of the test material per 50 mL volume of solution) was selected since it is experimentally feasible even when low concentrations of released metal are expected.

The time periods for the exposure of the silver metal, silver oxide and silver nitrate particles, 2 hour and 24 hours respectively, were selected to have some relevance to the inhalation/ingestion scenario and to enable comparison with other metal ion release/dissolution data generated for these time periods. The approximate time for the gastric phase of digestion is about 2 hours, and therefore this exposure time period was considered relevant for testing in artificial gastric fluid (Hamel et al, 1998). The 24 hour exposure was selected as a standard time duration that is relatively easy to compare with existing metal ion release/dissolution data as well as toxicity data for further evaluation of the bioaccessibility of released silver.

After the testing period, the samples were allowed to cool down to ambient room temperature before the final pH of the test solution was measured. The test medium was then separated from the powder particles by centrifugation at 3000 rpm for 10 minutes, resulting in a visually clear supernatant with remaining particles in the bottom of the centrifuging tube. In some cases fine particles were floating on the solution surface after centrifugation. These particles were removed using acid cleaned pasteur pipettes. The supernatant solution was decanted into a LDPE storage flask and acidified to pH <2 with 65% pure HNO3 prior to solution analysis (a standard procedure for metal analysis).

Results and discussion

Main ADME resultsopen allclose all
Type:
other: Bioavailability based on dissolution in artificial biological fluids
Results:
The dissolved concentrations of silver in various artificial physiological media were very similar and seem independent of material type (silver metal, disilver oxide, silver nitrate).
Type:
other: Bioavailability based on dissolution in artificial biological fluids
Results:
It may be hypothesised that the complex ionic environment and the likely formation of poorly soluble silver chloride – an ubiquitous ion in physiology - leads to very similar equilibrium concentrations of dissolved silver, independent of the origin.

Any other information on results incl. tables

Particle characterization:

Ag-1

The silver metal powder, shows a broad particle size distribution that contains a fraction of smaller sized particles ( <0.1 μm in diameter). The silver particles show a fairly rough surface structure that contributes to the relatively large specific surface area, 3.1 m²/g, measured by BET analysis.

The Ag test material shows large variations in composition between different areas investigated. Areas with silver peaks (Ag 3d5/2) with a binding energy of 368.3 eV (associated with silver oxides) were observed as well as areas with the silver peak significantly shifter to higher binding energies of 369.2-370.7 eV (Ag 3d5/2). This shift is associated to metallic silver. The results imply particles with a thin surface oxide layer, or particles of varying surface coverage and thickness of the surface oxide. In addition, significant amounts of strongly oxidized carbon (carbon with single, and/or double oxygen bonding, carboxyl groups etc) of varying binding energies were detected on these particles. Their definite assignment is hazardous at this stage although it is clear that the origin is not from atmospheric contamination.

Ag-2

Ag-2, has a completely different appearance and surface morphology, just like “fine gravel” as seen from SEM images. Ag-2 particles show a broad distribution in size, including a large fraction of smaller sized particles and a large specific surface area of 7.9 m^2/g. Nano-sized silver particles demonstrate a high tendency to agglomerate, as depicted from particle size measurements in liquid medium (PBS).

The Ag-2 test material shows large variations in composition between different areas investigated. Areas with silver peaks (Ag 3d5/2) with a binding energy of 368.3 eV (most probably associated with silver oxides) were observed as well as areas with the silver peak significantly shifted to higher binding energies of 369.9-370.1 eV (Ag 3d5/2). This shifted peak is associated to metallic silver. The results imply particles with a thin surface oxide layer or particles of varying surface coverage and thickness of the surface oxide. In addition, significant amounts of strongly oxidized carbon of varying binding energies (carbon with single, and/or double oxygen bonding, carboxyl groups etc) were detected on these particles. Their definite assignment is hazardous at this stage although it is clear that the origin is not from atmospheric contamination.

Ag2O:

Silver oxide particles, Ag2O, seem to consist of larger agglomerates of finer, spherical silver oxide particles. The particles are all of similar size range and if the particles consist of agglomerated finer oxide particles, they are very stable since they do not fall apart when agitated during the size distribution analysis (this is seen from the agreement of measured particle size distribution). Silver nitrate particles are large, white salt crystal particles with a smooth surface morphology that looks like “wet ice bergs” when studied by SEM. The silver nitrate particles are large in size, which is also reflected by the small specific surface area, 0.03 m²/g, measured by BET analysis.

The Ag2O test material shows a silver peak (Ag 3d5/2) with a binding energy of 368.2 eV, associated with silver oxides. Both AgO and Ag2O show closely overlapping binding energies (367.9-368.2) why no unambiguous phase identification can be made. The oxygen component (O 1s) consists of three main peaks, located at 529.7, 531.8 and 533.6 eV, respectively. These peaks are associated with silver oxide, adsorbed hydroxyl groups, and/or bulk hydroxides, respectively Carbon (C 1s) is present on the surface as typical adventitious carbon due to atmospheric contamination.

AgNO3:

The AgNO3 test material, Figure 3, shows a silver peak (Ag 3d5/2) with a binding energy of 368.6, slightly higher compared to peaks usually associated with silver oxides (368.2 eV) and in addition, peaks of nitrogen (N 1s) and oxygen (O 1s) associated with nitrate. Carbon (C 1s) is present on the surface as typical adventitious carbon due to atmospheric contamination.

[XPS:

All test materials reveal adventitious carbon on the surface (285.0 eV) and small amount of oxidized carbon. A surface contamination layer of carbon is always observed to different extent due to the surface history, and its source is usually atmospheric and not possible to avoid.]

Bioaccessibility data- silver release:

The concentrations of silver that were released from the different particles were all over very similar and seem independent of material type (pure metal, oxide, nitrate). Since the concentrations measured after 2 hours and 24 hours of exposure were practically constant, it is concluded that the release/dissolution of silver takes place relatively fast and at these test conditions equilibrium is obtained during the time period prior to the 2 hour exposure and sampling. It may be speculated that silver dichloro complexes may have dominated the dissolution process. However no chemical speciation measurements were made to confirm this hypothesis. The released silver concentrations from all test materials were highest in phosphate buffered saline (PBS), having a neutral pH but the highest chloride content of the media investigated. Released silver concentrations were lowest in artificial gastric fluid (GST) with the most acidic pH and the lowest concentration of chlorides. The chloride concentration of the different test media is highest in PBS (5.35 g/L) > GMB (3.97 g/L) > ASW (3.05 g/L) > ALF (2.02 g/L) > GST (0.97g/L).

Table: Total concentration of released silver [μg/L] in the different test media

 

Test Material

GST  

Ag conc.

μg/L

ALF 

Ag conc.

μg/L

ASW 

Ag conc.

μg/L

GMB 

Ag conc.

μg/L

PBS 

Ag conc.

μg/L

Ag-1 2h

36.7±6.8

131.3±1.2

215.0±1.0

282.7±6.7

349.0±7.5

Ag-1 24h

35.3±1.2

123.0±1.0

224.0±59.8

270.0±3.5

352.3±6.5

Ag2O 2h

36.0±1.0

129.3±1.2

190.0±7.8

237.3±41.3

281.7±6.4
Ag2O 24h 36.0±1.0 123.0±0.0 184.7±2.1 264.3±1.5 338.0±2.6
AgNO32h 36.0±1.0 127.0±1.0 190.0±1.0 272.0±5.2 355.7±11.6
AgNO3 24h 34.0±1.0 120.0±1.0 186.0±2.6 259.3±1.5 347.3±2.1
Ag-2 2h  42.0 ±2.6 127.7 ± 1.2   209.3 ±14.6  278.3 ±8.1  280.7 ±92.2
Ag-2 24h   42.0 ±1.0  120.3 ±0.6 184.0 ± 1.0   260.3 ±1.5  340.0 ±1.0

Results presented as release rates of silver per unit surface area and hour of exposure (μg/cm²/hour), are shown in the table below. These rates are calculated from the released silver concentration considering the solution volume, measured BET area and sample weight, and the exposure time period. Error bars indicate the standard deviation of release rates from triplicate samples. The silver metal, silver oxide and silver nitrate particles all show higher average release rates of silver per surface area after 2 hours exposure and lower release rates after 24 hours of exposure. In agreement to the concentration findings, the highest release rates are observed when the test materials are exposed in PBS. Since there was no kinetic behaviour observed from the measured silver concentrations, the decreasing release rates are purely an effect of the normalisation by time. The release rate of silver decreased according to the following sequence for all samples investigated after 2 hours of exposure: PBS > GMB > ASW > ALF >> GST

Table: Release rates of silver [μg/cm²/hour] in the different test media.

Test Material

GST

Ag rate

μg/cm²/h

ALF 

Ag rate

μg/cm²/h

ASW 

Ag rate

μg/cm²/h

GMB 

Ag rate

μg/cm²/h

PBS 

Ag rate

μg/cm²/h

Ag-1 2h

0.070±0.012

0.25±0.01

0.41±0.00

0.51±0.01

0.67±0.03

Ag-1 24h

0.006±0.000

0.02±0.00

0.03±0.01

0.04±0.00

0.06±0.00

Ag2O 2h

0.068±0.001

0.24±0.01 0.35±0.02 0.43±0.07 0.53±0.02
Ag2O 24h 0.006±0.000 0.02±0.00 0.03±0.00 0.04±0.00 0.05±0.00 
AgNO3 2h 0.064±0.003  0.22±0.00 0.35±0.01 0.49±0.02 0.61±0.02
AgNO3 24h 0.005±0.000 0.02±0.00 0.03±0.00 0.04±0.00 0.05±0.00 
Ag-2 2h 0.078 ±0.005 0.24 ±0.01 0.39 ±0.03 0.52 ±0.02 0.54 ±0.18
Ag-2 24h 0.007±0.000 0.02±0.00 0.03±0.00 0.04±0.00 0.06±0.00

Another comparison of release data is enabled by normalizing the released amount of silver by the amount of particles loaded for a given time period (μg/μg). This quotient can function as an indicative measure of the percentage of the pure metal particles that has been released (dissolved) into solution. The results indicate that less than 0.5% of the silver metal particles have been dissolved during the 24 hours in all test media. From a bioaccessibility perspective, only a small fraction of silver is hence released regardless of the nature of the test material investigated. The results are presented as the maximum percentage determined from the triplicate samples investigated to avoid underestimating the amount of dissolved silver. This conclusion could also easily be drawn based on the results of silver concentration.

Table: Amount of released silver per amount of particles loaded [μg/μg] in the test media.

 Test Material GST Ag ratio μg/μg ALF Ag ratio μg/μg  ASW Ag ratio μg/μg GMB Ag ratio μg/μg PBS Ag ratioμg/μg
 Ag-1 2h  0.0004±0.0001  0.0014±0.0000  0.0022±0.0000  0.0028±0.0000  0.0037±0.0002
 Ag-1 24h  0.0004±0.0000  0.0013±0.0000  0.0022±0.0006  0.0027±0.0001  0.0036±0.0001
 Ag2O 2h  0.0004±0.0000  0.0013±0.0000  0.0019±0.0001  0.0024±0.0004  0.0029±0.0001
 Ag2O 24h  0.0004±0.0000  0.0013±0.0000  0.0019±0.0000  0.0026±0.0000  0.0036±0.0001
 AgNO3 2h  0.0003±0.0000  0.0012±0.0000  0.0019±0.0000  0.0026±0.0001  0.0033±0.0001
AgNO3 24h   0.0003±0.0000  0.0011±0.0000  0.0018±0.0000  0.0026±0.0001  0.0034±0.0001
Ag-2 2h  0.0004 ±0.0000  0.0013 ±0.0001  0.0021 ±0.0002 0.0028 ±0.0001  0.0030 ±0.0010
Ag-2 24h  0.0004 ±0.0000  0.0012 ±0.0000  0.0019 ±0.0000  0.0027 ±0.0000  0.0036 ±0.0000

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: see conclusion and summary
The dissolved concentrations of silver in various artificial physiological media were very similar and seem independent of material type (silver metal, disilver oxide, silver nitrate).It may be hypothesised that the complex ionic environment and the likely formation of poorly soluble silver chloride leads to very similar equilibrium concentrations of dissolved silver, independent of the originating substance. Chloride ion is ubiquitous in physiological systems.
Executive summary:

The concentrations of silver that were released from the different particles were all over very similar and seem independent of material type (pure metal, oxide, nitrate). The concentrations measured after 2 hours and 24 hours of exposure were practically constant. The released silver concentrations from all test materials were highest in phosphate buffered saline (PBS), having a neutral pH but the highest chloride content of the media investigated. Released silver concentrations were lowest in artificial gastric fluid (GST) with the most acidic pH and the lowest concentration of chlorides. The silver metal, silver oxide and silver nitrate particles all show higher average release rates of silver per surface area (μg/cm²/hour) after 2 hours exposure and lower release rates after 24 hours of exposure. In agreement to the concentration findings, the highest release rates are observed when the test materials are exposed in PBS. Since there was no kinetic behaviour observed from the measured silver concentrations, the decreasing release rates are purely an effect of the normalisation by time. The release rate of silver decreased according to the following sequence for all samples investigated after 2 hours of exposure: PBS > GMB > ASW > ALF >> GST.

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