Registration Dossier

Administrative data

Link to relevant study record(s)

Description of key information

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
1
Absorption rate - dermal (%):
1
Absorption rate - inhalation (%):
1

Additional information

Introduction:

Toxicokinetic information on “silver” is required for the assessment of the relative contribution of the possible routes of entry into the human body (inhalation, skin, ingestion), and for a comparison of relative bioavailability of different silver substances. ATSDR (1990) previously summarised the available information in a comprehensive review, from which relevant paragraphs have been directly extracted below. The summarised findings are considered to apply to all silver substances in general, but distinguish between different bioavailabilities where relevant (reference: ATSDR (1990): Toxicological profile for silver. ATSDR - Agency for Toxic Substances and Disease Registry). Further references mentioned in text directly cited from ATSDR can be found in the ATSDR report.

The earlier information extracted from the ATSDR review is supplemented by more recent data on (i) in-vitro bioaccessibility on metallic silver, disilver oxide and silver nitrate (unpublished), and (ii) published in-vitro and in-vivo investigations relating to the bioaccessibility/bioavailability of silver, in particular from silver nanomaterials. A highly relevant side-by-side comparison of the toxicokinetics of a soluble silver substance (silver acetate) vs. silver nanoparticles of different sizes has recently been conducted by US FDA (Boudreau, 2012), which is however not yet published in full; preliminary results are already considered in this dossier.

The water solubility of chemical substances is widely used as a first tier for screening purposes when assessing bioavailability. An overview of water solubilities for several silver substances is presented in the table below:

 

Water solubility of the substance (typically at 20 °C)

silver nitrate, AgNO3

up to ca. 2 kg/L (handbooks data range from 710 g/L to 2150 g/L)

silver acetate, AgCH3COOH

ca. 10 g/L

silver sulfate, Ag2SO4

8.2 g/L

silver carbonate, Ag2CO3

63 mg/L

silver chloride, AgCl

1.9 mg/L

silver bromide, AgBr

140 µg/L

silver iodide, AgI

30 µg/L

disilver oxide, Ag2O

considered practically insoluble (handbook data range from 1.6 mg/L at 20°C to 46 mg/L at 25°C)

silver sulfide, Ag2S

considered practically insoluble

silver (metal), Ag

considered practically insoluble (very slow dissolution depending on particle size/surface area and medium; see chapter 1.3 of the CSR)

 

Comparative in-vitro bioaccessibility of Ag, Ag2O and AgNO3(unpublished, Midander and Wallinder, 2009)

Metallic silver (two powder samples: D50=1.9 µm and D50=35 nm), disilver oxide and silver nitrate have been subject to in-vitro bioaccessibility testing in five different artificial physiological media. Phosphate-buffered saline (PBS, pH 7.4), is a standard physiological solution that mimics the ion strength of human blood serum, artificial sweat (ASW, pH 6.5), Gamble’s solution (GMB, pH 7.4) which mimics interstitial fluid within the deep lung under normal health conditions , artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions and artificial gastric fluid (GST, pH 1.5). The test items were put into these solutions at a loading of 100 mg/L and incubated in the dark at 37 °C for 2 h and 24 h, respectively. Subsequently, undissolved particles were removed by filtration, and the filtrate was analysed for dissolved silver.

The dissolved concentrations of silver in all five artificial physiological media were very similar and apparently independent of the silver substance tested (i.e., metal, oxide and nitrate), despite their vastly differing water solubility. It is hypothesised that the complex ionic environment and the formation of poorly soluble silver chloride precipitates leads to very similar equilibrium concentrations of dissolved silver, independent of the originating substance. Chloride ions are ubiquitous in physiological systems, so that the formation of silver chloride particles can be assumed to be a limiting factor for systemic bioavailability of silver following exposure via the dermal, oral or inhalation route.

 

Other, published in-vitro bioaccessibility data

Several authors have conducted studies relating to the bioaccessibility of silver in physiological media, including investigations on the solubility/bioaccessibility of nano forms of silver (see for example the studies by Mwilu et al. (2013), Ma et al. (2012), Levard et al. (2013) and Zhang, et al. (2013), all summarised in tabular format in this CSR). As expected, the dissolution behaviour of metallic silver, including nanoforms, depends strongly on the type of material tested, and particle size distribution, aggregation or agglomeration and the presence of coatings have been shown to influence dissolution behaviour. As a general conclusion, the presence of chloride ions and formation of poorly soluble silver chloride or silver chloride complexes can reasonably be expected to limit the concentration of free silver ions in physiological media.

 

Specific investigations on the chemical transformation of nanosilver in biological environments (Liu et al., 2012)

Liu et al. (2012) studied the dissolution and chemical transformation of nanosilver in biological environments and draw the following conclusions from their studies: Silver nanoparticles undergo a set of biochemical transformations, incl. accelerated oxidative dissolution in gastric acid, thiol binding & exchange, photoreduction of thiol- or protein-bound silver to secondary zerovalent silver nanoparticles. Also, silver nanoparticles undergo rapid reactions between silver surfaces and reduced selenium species. Selenide is observed to rapidly exchange with sulfide in preformed Ag2S solid phases. The combined results allow proposing a conceptual model for silver nanoparticle transformation pathways in the human body: argyrial silver deposits are secondary particles formed by partial dissolution in the gastrointestinal tract followed by ion uptake, systemic circulation as organo-Ag complexes, and immobilization as zerovalent silver nanoparticles by photoreduction in light-affected skin regions. The secondary silver particles then undergo detoxifying transformations to sulfides and partly further to selenides or Se/S mixed phases through exchange reactions. The formation of secondary nano-sized particles in biological environments implies that silver nanoparticles are not only a product of industrial nanotechnology but can also also been present in the human body following exposure to more traditional chemical forms of silver. The research presented above supports the hypothesis that nanosilver particles are not absorbed to any relevant extent and/or further distributed within the body as intact particles, but rather in ionic form after dissolution. Depending on the chemical environment, secondary particles may be formed via transformation into poorly soluble forms such as metallic silver, chloride, sulfide or -selenide.

In a study comparing tissue distribution after oral dosing with Ag-NPs and AgNO3, the authors concluded that tissue levels were generally much higher in oral dosing with AgNO3, and also concluded that (i) these tissue levels were highly correlated with the dissolved (ionic) fraction of the AgNP suspension, and (ii) silver nanoparticles were detected not only in AgNP-treated rats, but also in those dosed with AgNO3, obviously demonstrating formation of these from ionic silver in vivo (van der Zande et al., 2012).

Below, available key information on absorption, distribution, metabolism and excretion of “silver” as relevant for human health risk assessment of silver is presented.

 

Absorption

Oral absorption

ATSDR, 1990: “Based on medical case studies and experimental evidence in humans, many silver compounds, including silver salts and silver-protein colloids, are known to be absorbed by humans across mucous membranes in the mouth and nasal passages, and following ingestion. The absorption of silver acetate following ingestion of a 0.08 mg/kg/day dose of silver acetate containing radiolabelled silver (110mAg) was studied in a single female by in vivo neutron activation analysis and whole body counting: approximately 21% of the dose was retained in the body at 1 week (East et al. 1980; MacIntyre et al. 1978). ”

However, the reliability of this method is not documented and several assumptions in the publication by East et al. leading to this estimate render this a likely overestimate. In a well-documented comparative investigation assessing the bioavailability of 110msilver nitrate in mice, rats, monkeys and dogs via oral, intravenous and intraperitoneal administration, only about 1% or less of an oral dose was absorbed with the exception of dogs (<10%) (Reference: Furchner et al. 1968: Comparative metabolism of radionuclides in mammals-IV. Retention of silver-110m in the mouse, rat, monkey, and dog, Health Physics 15:505-514).

A study comparing tissue distribution levels in rats after dosing with AgNPs and AgNO3 showed that silver tissue levels were much higher in liver after AgNO3-dosing than with AgNPs, and also demonstrated that the tissue levels were highly correlated to the dissolved (ionic) fraction of the AgNP suspension; this suggests that mainly Ag+ passes the intestine upon oral intake (van der Zande et al., 2012).

More conclusive data may become available from a study currently being conducted in the US (Boudreau, 2012) when the results are published.

 

 

Dermal absorption

 

Several published sources report information on percutaneous absorption of silver, with varying degrees of reliability and relevance for risk assessment.

 

Some older information exists which can however be used as supportive information. Since this data is often only available from secondary sources, dedicated endpoint records have not been prepared in the technical dossier on the studies mentioned in this paragraph. Therefore, for technical reason, these references do not appear in the automatically generated reference list in the CSR. In a well-documented study with guinea pigs less than 1% of the applied dose of silver nitrate was absorbed through the skin. However, this study has major methodical deficiencies, and is therefore considered only as supporting data (Wahlberg et al., 1965: Percutaneous toxicity of metal compounds. A comparative investigation in guinea pigs. Arch. Environ. Health 11, 201-204). A review article exists which contains references to earlier published investigations, in which an in-vivo percutaneous absorption study in guinea pigs with110Ag as tracer is described. Despite that this study also has considerable methodological shortcomings compared to current standards, the authors likewise conclude on a dermal absorption rate of <1% (Skog & Wahlberg, 1964: A comparative investigation of the percutaneous absorption of metal compounds in the guinea pig by means of the radioactive isotopes: 51Cr, 58Co, 65Zn, 110mAg, 115mCd, 203Hg. J. Invest. Derm. 43, 187-192, 1964. Cited in Hostynek, 1993: Metals and the skin. Crit. Rev. Toxicol. 171-235). Other similarly outdated data relate to either non-standard test systems or absorption through wounded or burnt skin, and is therefore not considered relevant for the assessment of percutaneous absorption through intact skin, as required for risk assessment purposes. For example, one case report study of 11 human volunteers on absorption of silver from the nasal septum after cauterisation for nose bleeds suggests a significant increase of silver blood concentrations 3 hours after administration. This study is not considered as particularly relevant for human health risk assessment because of the involved skin injury (Nguyen et al., 1999: Argyremia in septal cauterization with silver nitrate.J. Otolaryng. 28, 211-216).

 

Most recently, increased interest in the safety assessment of silver nanomaterials has produced a number of relevant publications on these materials:

 

Larese et al. (2009) compared the percutaneous absorption of polyvinylpirrolidone-coated silver nanoparticles in-vitro through intact and damaged human skin. Whereas the publication has some reporting deficiencies, the percutaneous absorption rate (percentage of the applied dose absorbed during 24h of exposure) can be calculated: the exposure concentration is given as 70 µg Ag/cm2, and the median penetration rates (over 24h) are 0.46 ng/cm2and 2.32 ng/cm2for intact and damaged skin, respectively. Thus, the percentage of the applied dose absorbed during 24h of exposure is 0.00066 % for intact skin and 0.0033 % for damaged skin.

Samberg et al. (2010) studied dermal absorption and irritation due to silver nanoparticles in-vivo in pigs. By microscopic investigations silver nanoparticles were localized only in the superficial layers (50nm particles) and on the top layer (20nm particles) of the stratum corneum and did not appear to penetrate into the deeper dermis. This may be considered as supportive of the assumption that silver does not penetrate through human skin to any relevant extent.

 

Brandt (2012) compared the percutaneous absorption of silver from two antimicrobial topical creams in mice in-vivo. No absorption rates are reported, but the study concludes that percutaneous absorption of silver from an antimicrobial cream containing nanoscale silver was much lower than from a cream containing (soluble) silver sulfadiazine. Analysis of inner organs and blood of mice treated with the commercial cream containing 0.1% nanoscale silver revealed extremely low percutaneous absorption rates, resulting in barely detectable silver ion concentrations with values not differing significantly from those of the untreated group.

 

Moiemem et al. (2010) studied the systemic absorption of silver in six patients with large scale burn wounds (median of 46.1% of the total body surface affected). Patients were treated with a commercial wound dressing containing “nanocrystalline” silver. Silver levels in blood serum were analysed over time. A significant increase of silver in blood was observed, and levels decreased following the end of treatment. None of the patients had any symptoms or signs suggesting argyria. The authors conclude that elevated silver levels in blood were similar to those reported following the use of silver sulfadiazine.

In an earlier study by the same group of researchers (Vlachou, 2007) similar investigations were carried out with patients with smaller scale burn wounds (median of 12% of the total body surface affected).A significant increase of silver in blood was observed during treatment, and levels decreased to baseline levels following the end of treatment. No haematological or biochemical indicators of toxicity associated with the silver absorption were observed. The overall finding is not considered relevant for human health risk assessment of industrial chemicals, in view of the involvement of damaged/wounded skin.

 

George et al. (2013) studied the absorption of silver from a nanocrystalline silver wound dressing when applied for 4-6 days to intact human skin of 16 healthy patients. The analysis of absorption/penetration was conducted by microscopy (optical and SEM) and XRD analysis of skin samples, as well as by silver analysis in blood serum. Although, according to the authors silver nanoparticles may penetrate into intact human skin in vivo beyond the stratum corneum as deep as the reticular dermis, the absorbed silver species appear to precipitate in clusters across the epidermis. However, despite this silver deposition in the dermis, silver nanoparticles did not reach systemic circulation.

Trop et al. (2006) observed argyria-like symptons and increased blood silver levels in a clinical case report on treatment of burn wounds with a silver-coated wound dressing. This indicates some absorption of silver through damaged/burned skin, but the absorption rate was not quantified. Therefore, and since uptake through damaged skin is not relevant for the risk assessment under REACH, this study is not considered further.

 

 

As supportive information, reference is made to a study not directly related to silver as such: Campbell (2012) assessed the disposition of inert polystyrene nanoparticles in mammalian skin using confocal microscopy. These polystyrene nanoparticles when applied in aqueous suspension could only infiltrate the stratum disjunctum, i.e. skin layers in the final stages of desquamation. This minimal “uptake” was independent of contact time size of nanoparticles tested. Overall, these results demonstrate objectively and semi-quantitatively that (inert) nanoparticles of a wide size range cannot penetrate beyond superficial skin layers of the barrier and are unlikely to reach the viable cells of the epidermis or beyond.

 

Overall, the available data for soluble silver substances as well as silver nanomaterials indicate dermal absorption rates well below 1% of the applied dose. Data obtained with silver nanomaterial preparations (which can be assumed to also contain a certain amount of dissolved, ionic silver) indicate some penetration into the stratum corneum, but detailed follow-up investigations have shown that this bound material does not become systemically available, but instead is lost via desquamation.

 

These conclusions above are consistent with the methodology proposed in HERAG guidance for metals (HERAG fact sheet - assessment of occupational dermal exposure and dermal absorption for metals and inorganic metal compounds; EBRC Consulting GmbH / Hannover /Germany; August 2007). The following default dermal absorption factors (reflective of full-shift exposure, i.e. 8 hours) are therefore considered adequately conservative both for (soluble) silver substances as well as silver nanomaterials, i.e. 1.0% from exposure to liquid/wet media, and 0.1% from exposure to dry (dust).

 

Inhalation absorption

Model calculations of inhalation absorption based on laboratory dustiness tests with representative materials

Experimental investigations have been conducted on six samples of different silver compounds: silver metal (3 different sizes), disilver oxide (2 different batches) and one representative sample of silver nitrate (crystalline powder). The samples were subject to mechanical agitation in a rotating drum apparatus and the mass fraction of the material that becomes airborne was determined (“total dustiness”). In addition, the particle size distribution of the airborne dusts was determined with a cascade impactor. Then, the MPPD model was used, to estimate the fractional deposition of such dusts in three regions of the human respiratory tract: (i) extrathoracial fraction (head), (ii) tracheo-bronchial fraction (TB) and (iii) the pulmonary fraction (EBRC, 2010: Investigations on dustiness and particle size of airborne dusts of six silver compound samples. Unpublished report for the Precious Metals Consortium, EBRC Consulting GmbH, Hannover, Germany). The results are presented in tabular form below.

MPPD model results: Fractional deposition (%) in different regions of the respiratory tract. Data on physical particle size of the original test materials and on total dustiness is also given:

 

 

Silver powder Batch PMC1

Silver powder Batch PMC2

Silver powder Batch PMC3

Disilver oxide Batch PMC1

Disilver oxide Batch PMC2

Silver nitrate Batch PMC1

D50

ca. 30 µm

ca. 2 µm

ca. 35 nm

ca. 4 µm

ca. 7-18 µm

ca. 370 µm

Total dustiness

155 mg/g

91 mg/g

248 mg/g

149 mg/g

126 mg/g

0.83 mg/g

Deposition (total)

45.4 %

51.2 %

53.3 %

46.1 %

49.1 %

37 %

Deposition (head), fH

45.3 %

50.1 %

52.4 %

45.3 %

47.9 %

36.8 %

Deposition (TB), fTB

0.1 %

0.4 %

0.3 %

0.3 %

0.5 %

0.1 %

Deposition (pulmonary)

fPU

0.0 %

0.7 %

0.6 %

0.6 %

0.8 %

0.1 %

Based on the fractional deposition, an inhalation absorption factor can be calculated under the following assumption: The material deposited in the head and tracheobronchial regions would be translocated to the gastrointestinal tract without any relevant dissolution, where it would be subject to an assigned default gastrointestinal uptake at a ratio of 1% (see above). The material that is deposited in the pulmonary region may conservatively be assumed by default to be absorbed to 100%. This absorption value is chosen in the absence of relevant scientific data regarding alveolar absorption although knowing that this is a conservative choice specifically for poorly soluble substances. Formula: (fH + fTB) * absoral + fPU * abspul = inhalation absorption factor. Example calculation for batch PMC3: (52.4% + 0.3%) * 1 % + 0.6 % * 100% = 1.13 %

Estimated inhalation absorption factors, assuming 1% absorption in the gastrointestinal tract (considered applicable to soluble silver substances; for the absorption of metallic silver this is considered a conservative worst-case) of material initially deposited in the head or tracheobronchial region and 100% absorption of material deposited in the pulmonary region:

 

Silver powder Batch PMC1

Silver powder Batch PMC2

Silver powder Batch PMC3

Disilver oxide Batch PMC1

Disilver oxide Batch PMC2

Silver nitrate Batch PMC1

Estimated inhalation absorption factor

0.45 %

1.21 %

1.13 %

1.06 %

1.28 %

0.47 %

Given the inherently conservative assumption of 100% absorption for pulmonary deposition and 1% oral absorption for the poorly soluble substances, it is considered adequate to take a value of 1% forward for inhalation absorption of silver substances.

Distribution

ATSDR, 1990: "The distribution of silver to various body tissues depends upon the route and quantity of silver administered and its chemical form. An oral dose of silver, following absorption, undergoes a first pass effect through the liver resulting in excretion into the bile, thereby reducing systemic distribution to body tissues (Furchner et al. 1968). The subsequent distribution of the remaining silver is similar to the distribution of silver absorbed following exposure by the inhalation and dermal routes and following intramuscular or intravenous injection. Silver distributes widely in the rat following ingestion of silver chloride (in the presence of sodium thiosulfate) and silver nitrate in drinking water (at 88.9 mg silver/kg/day for silver nitrate) (Olcott 1948); The amount of silver in the various tissues was not measured, although qualitative descriptions of the degree of pigmentation were made. High concentrations were observed in the tissues of the reticuloendothelial system in the liver, spleen, bone marrow, lymph nodes, skin, and kidney. Silver was also distributed to other tissues including the tongue, teeth, salivary glands, thyroid, parathyroid, heart, pancreas, gastrointestinal tract, adrenal glands, and brain. Within these tissues advanced accumulation of silver particles was found in the basement membrane of the glomeruli, the walls of blood vessels between the kidney tubules, the portal vein and other parts of the liver, the choroid plexus of the brain, the choroid layer of the eye, and in the thyroid gland (Olcott 1948; Moffat and Creasey 1972; Walker 1971)".

Tissue distribution studies involving i.v. administration of soluble radiotracer (110m) silver nitrate indicate that liver is a likely target organ for silver: 2 hours p.a., the highest tissue concentrations were in liver (12.4% of dose), with substantially lower levels in other organs (spleen 1.2% and kidneys 1.1%, for example (Gregus & Klaassen, 1986).

Some information on tissue distribution following exposure of experimental animals to nanosilver is available from recent toxicological studies by Sung et al. (2009) and Kim et al. (2010). Tables with the corresponding data are contained in the respective study summaries in the technical dossier and only the brief summary is presented here.

In a subchronic inhalation toxicity study conducted with nanosilver (Sung et al. 2009) rats were exposed to 49, 133 and 515 µg/m³ for 6h per day, 5 days per week for 13 weeks. At the end of the study, the authors measured silver concentrations in liver, kidneys, olfactory bulb, brain and lung tissues and report the following finding: “Silver concentration in lung tissue from groups exposed to silver nanoparticles for 90 days were a statistically significant (p < 0.01) and increased with dose. There was also a clear dose-dependent increase in the silver concentration in the blood, and dose-dependent increase in the liver silver concentration for both genders. Silver concentration in the olfactory bulb was higher than in brain, and increased in a dose dependent manner in both the male and female rats (p < 0.01). Interestingly, silver concentrations in the kidneys showed a gender difference, with the female kidneys containing two to three times more silver accumulation than in male kidneys”.

 

In a subchronic oral toxicity study with nanosilver (Kim et al. 2010), four groups of rats received 0, 30, 125 and 500 mg/kg/day of silver via gavage for 13 weeks and silver levels in selected tissues were measured: Results for testes, liver, kidney, brain and lung are reported in the publication and the authors summarise their findings as follows: “There was a statistically significant (P < 0.01) dose dependent increase in the silver concentration of all the tissue samples from the groups exposed to silver nanoparticles in this study. In addition, a two-fold higher accumulation of silver in the kidneys of female rats when compared with the male rats occurred across all the dose groups indicating a marked gender-dependent distribution”.

Further data are anticipated to originate from toxicokinetic and toxicity studies currently being conducted in the US (Boudreau, 2012).

 

Metabolism

Silver is not subject to any metabolism in its true sense regardless of its original chemical speciation, with one exception which relates to the formation of argyria particles through reaction of ionic silver to sulfide/selenide particles (postulated mechanism: Liu et al. 2012; van der Zande et al., 2012 – for details, see section above).

 

Excretion

ATSDR, 1990: "Following oral exposure to silver acetate in humans, silver is eliminated primarily in the faeces, with only minor amounts eliminated in the urine (East et al. 1980). The rate of excretion is most rapid within the first week after a single oral exposure (East et al. 1980). Whole-body retention studies in mice and monkeys following oral dosing with radiolabelled silver nitrate indicate that silver excretion in these species follows a biexponential profile with biological half-lives of 0.1 and 1.6 days in mice and 0.3 and 3 days in monkeys. In similarly exposed rats and dogs, silver excretion followed a triexponential profile with biological half-lives of 0.1, 0.7, and 5.9 days in rats and 0.1, 7.6, and 33.8 days in dogs (Furchner et al. 1968). Data for whole body clearance of silver at two days after exposure for these four species are presented in Table 2-5 (Furchner et al. 1968). Transit time through the gut may explain some of these interspecies differences in silver excretion. Transit time is approximately 8 hours in mice and rats, and approximately 24 hours in dogs and monkeys (Furchner et al. 1968). Animals excrete from 90% to 99% of an administered oral dose of silver in the feces within 2 to 4 days of dosing (Furchner et al. 1968; Jones and Bailey 1974; Scott and Hamilton 1950). Excretion in the faeces is decreased and deposition in tissues, such as the pancreas, gastrointestinal tract, and thyroid, is increased when saturation of the elimination pathway in the liver occurs as a result of chronic or high level acute exposure to silver (see Table 2-4) (Constable et al. 1967; Olcott 1948; Scott and Hamilton 1950). "

In studies with bile-duct cannulated rats, the total average silver excretion of an i. v. -dose in the first 4 days p. a. was 72.3% of dose and almost exclusively via faeces (72.0%), whereas only 0.3% were excreted via urine. This underlines the high relevance of biliary excretion in the case of silver, being rapid and extensive in rats, with 45% of the dose appearing in bile already in the first 2 hours p. a. (Gregus and Klaassen, 1986).