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REACH

Silver

EC number: 231-131-3 | CAS number: 7440-22-4

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Uses advised against

Toxicological information

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Link to relevant study record(s)

Reference

Endpoint:: basic toxicokinetics in vivo
Type of information:: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: key study
Justification for type of information:: Substance considered to fall within the scope of the read-across (document attached in IUCLID section 13).
Reason / purpose for cross-reference:: read-across source
Preliminary studies:: Comprehensive enabling work was done to support the main study. The AgNP and AgMP test items were characterised to confirm suitability of the test items and to confirm stability in an appropriate aqueous-based vehicle. For the AgNP suspension, the ionic Ag was ~5% of the total Ag concentration.
Type:: absorption
Results:: Based on matched dose assessments, nanosilver (AgNP) exhibits a systemic exposure profile between sub-micron Ag metal powder (‘AgMP’) and a reference ionic Ag salt.
Type:: distribution
Results:: Levels of Ag distributed into tissues and organs after AgNP exposure are between those after AgMP exposure and an those after exposure to an ionic Ag compound
Details on absorption:: Comparisons via normalised administered doses (i.e. Ag equivalent dose) can be made between different Ag test items. Based on these matched dose assessments, it can be concluded that oral dosing of AgMP leads to appreciably lower Ag amplitude of systemic exposure in comparison to ionic Ag forms, while AgNP exhibits an intermediate systemic exposure profile.
Details on distribution in tissues:: - Levels of Ag distributed into tissues and organs are considerably lower in the case of AgMP than an ionic Ag compound. AgNP exhibits an intermediate TK profile with achieved tissue levels more closely resembling those of the ionic Ag test items rather than those evident for Ag metal powder (AgMP).
- The study shows that the testis and brain are minor sites of distribution. In contrast, the ovary has been shown to be an important site of distribution. However, ancillary investigations strongly suggest silver is present in the ovary as chemically stable silver sulphide/selenide complexes with very low local bioavailability.
: Key result
Test no.:: #1
Toxicokinetic parameters:: AUC: 686
Remarks:: Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 3.6 mg/kg bw/d
: Key result
Test no.:: #1
Toxicokinetic parameters:: AUC: 1080
Remarks:: Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 3.6 mg/kg bw/d
: Key result
Test no.:: #2
Toxicokinetic parameters:: AUC: 186
Remarks:: Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 36 mg/kg bw/d
: Key result
Test no.:: #2
Toxicokinetic parameters:: AUC: 276
Remarks:: Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 36 mg/kg bw/d
: Key result
Test no.:: #3
Toxicokinetic parameters:: AUC: 35.3
Remarks:: Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 360 mg/kg bw/d
: Key result
Test no.:: #3
Toxicokinetic parameters:: AUC: 67.3
Remarks:: Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 360 mg/kg bw/d
Conclusions:: This in-vivo toxicokinetic study (in rats) by oral gavage comparing silver metal powder, nanosilver, silver nitrate and silver acetate shows a lower absorption/distribution and lower systemic exposure to tissues and organs for silver metal powder than for the other silver substances. The amount of Ag+ in blood and tissues is much higher for silver acetate/silver nitrate than for silver metal powder at comparable Ag dosing levels. Although all silver-containing substances have the ability to release Ag+, the extent of released silver ions is significantly different between substances. Therefore, a direct read-across of mammalian toxicity datasets for simple ionic silver salts like silver acetate or silver nitrate and nanosilver to silver metal powder/massive is not supported (from a scientifically valid toxicokinetic perspective).
Executive summary:: This in-vivo toxicokinetic study is an oral route investigation using adult CD Sprague Dawley rats. The design conformed to OECD TG 417 and was conducted in compliance to GLP.cThe study was designedcas a comparative toxicokinetic assessment of the test items Silver Acetate (AgAc), Silver Nitratec(AgNO3), Micron-sized Silver (AgMP) and Nanoparticulate Silver (AgNP). This included assessment of

bioavailability following single oral doses relative to single intravenous doses and systemic exposure following single and repeat oral doses. Groups of 4 male and 4 female CD Sprague Dawley rats were administered the test items, either as a single intravenous dose, a single oral dose or repeated daily oral doses for 4 weeks.

The systemic exposure (28 days) results show a difference in the extent of absorption and systemic circulation for the different Ag test items. Based on a matched dose assessment, the extent of systemic exposure was about 10 to 30-fold lower in the case of silver metal powder versus silver acetate (as reference ionic silver compound), while AgNP exhibits an intermediate systemic exposure profile.

The silver tissue levels after repeated dose administration are considerably lower in the case of silver metal powder (AgMP) than for silver acetate, while AgNP exhibits an intermediate TK profile with achieved tissue levels more closely resembling those of the ionic Ag test items rather than those evident for Ag metal powder (AgMP).

In conclusion, it is assumed that the systemic toxicity of silver substances is driven by the release potential of Ag ionic species as the primary moiety for systemic circulation and tissue exposure, and hence hazard assessment. The comparative TK study is a high quality GLP investigation and the first which permits a direct comparison of systemic circulation and tissue distribution of Ag ionic species between soluble Ag salts (AgNO₃and Ag acetate), AgNP and AgMP. This new evidence demonstrates that oral intake of AgMP results in markedly lower absorption, distribution and systemic tissue/organ exposure to silver than more bioavailable forms like AgAc. As a generic observation, the following trend is being observed: AgAc > AgNO₃>> AgNP >>> AgMP.

The study findings show that the direct read-across of mammalian toxicity data with soluble silver salts (like silver acetate and silver nitrate) and nanosilver to silver metal (powder and massive) is not justified (based on their individual toxicokinetic profiles).

Description of key information

The oral absorption of silver was determined in an in vivo TK study (GLP and OECD417 compliant). The study demonstrated that the oral availability of silver metal (as powder) was significantly lower than nanosilver and -especially- soluble silver salts.

The inhalation absorption of silver was determined experimentally using modelling of laboratory dustiness testing.

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:

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:

the available mammalian toxicity data of simple silver salts and nanosilver and
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 for Silver Metal and the read-across justification documents attached to the silver IUCLID file in section 13. The various human health endpoints of the silver REACH dossiers have been updated on an individual basis to reflect the approach that is described there.

The key toxicokinetic information from the in-vivo comparative toxicokinetic study have been supplemented by publishedin-vitroandin-vivoinvestigations relating to the bioaccessibility/bioavailability of silver, in particular from silver nanomaterials.

Note thatin vivoTK data have been generated since in vitro bioaccessibility testing (‘bio elution testing’) is not applicable to assess the bioaccessibility of silver substances because of the strong binding of Ag ions with chloride resulting in an immediate and excessive AgCl precipitate.

Based on theoretical electrochemical considerations (Batchelor-McAuley et al., 2014), one can assume that under physiological (near-neutral) pH, the oxygen reduction on the surface of AgNPs leads to the formation of hydrogen peroxide, which is of relevance for the dissolution of silver nanoparticles in aqueous oxygenated systems: the relatively slow decomposition of H2O2 on silver and the dependency of the mass-transport away from the particle means that for sufficiently small particles (≤ 1μm) the dissolution process will be driven by the two-electron, two-proton reduction of oxygen. This value of the 1μm is obtained through consideration of the relative rate of the mass-transport of hydrogen peroxide from an isolated particle compared to its rate of decomposition (1.3x10-2 cm s-1). Above this size limit, the dissolution reaction will be similar to that of silver metal powder and massive, where the formation of water will become more pronounced. The presence of Ag+ complex species such as chlorides or thiols may however make dissolution more thermodynamically favourable.

Comparative in-vivo toxicokinetic study of silver metal powder, nanosilver, silver nitrate and silver acetate

A comparative in-vivo toxicokinetic study has been conducted by the European Precious Metals Federation (EPMF) and is reported in Melvin et al. (2021) and Charlton et al. (2021). It is an oral route investigation using adult CD Sprague Dawley rats. The design conformed to the OECD TG 417, and was conducted in compliance to Good Laboratory Practices (GLP). Four test items were used: two ionic Ag salts (silver acetate (AgAc) and silver nitrate (AgNO3); both > 99.5% pure); a well-characterised and small-size Ag nanoparticles reference material (AgNP; surfactant stabilized aqueous dispersion with 10% Ag; d50= 15nm); and a sub-micron sized silver powder form (AgMP; uncoated; >99% pure; d50~0.3 µm; crystalline powder of highly uniform spheroidal shape). The silver metal powder (d50 ~0.3 µm) tested in the study is the finest representative powder on the EU market and considered a worst-case type of silver metal powder in terms of expected bioavailability.

It is noted that a technical failure during the conduct of the Ag in tissue analysis in the initial study necessitated a repeat of one part of the programme relating to the micron-sized silver powder (AgMP), nanosilver (AgNP) and silver nitrate test items. As a result, the findings for single dose investigations on all test items together with the repeat dose phase outcomes for silver acetate are covered in Melvin et al. (2021); with all other repeat dose phase findings being addressed in Charlton et al. (2021). An official statement released by the originating contract laboratory confirms that both parts of the overall programme were performed to an exactly equivalent design and under matched experimental conditions. Hence, it is scientifically justifiable to compare findings obtained for the different forms of silver across the two study segments.

Given that absolute bioavailability (F-values) had only been reported in the literature for a few silver compounds and nanosilver substances, a conventional single dose bioavailability assessment was attempted for all the test items (Melvin et al. 2021). However, as mentioned in that report, anomalous results were obtained in the case of the silver powder (AgMP) relating to the blood concentration profiles which were evident following intravenous bolus administration. The derived F-value was clearly incompatible with the observed AUC data via the oral route after a single dose. Furthermore, separately performed mass-balance calculations indicated that very rapid clearance of AgMP had occurred from the blood compartment. An explanation for the artefactually elevated F-value results for AgMP has not yet been fully elucidated. However, it is established in the scientific literature that certain micron-size particulates can be very rapidly sequestered by the reticuloendothelial system when administered via the i.v. route. We cannot exclude the possibility that the F-value derived for the suspension particulate AgNP test item may also have been subject to some artefactual distortion. In alignment with the direction taken by the CRO investigator, comparative TK assessment of the various Ag test items should instead be based on their respective p.o. dose-normalised AUC group mean values (as an indicator of relative systemic exposure). Further insights can also be obtained by comparison of AUC profiles obtained after repeated oral dosing.

Achieved systemic exposure (Ag in blood) following repeated (28 days) dosing: Comparison of the AUC(0-t) of all the respective Ag test items clearly shows the TK difference between AgMP and the other test items. The AgMP reference selected for investigation had a median particle size of ca. 0.3 μm (identified as the smallest representative silver powder form put on the market by EPMF members) and can be considered a worst-case type of silver powder in terms of expected bioavailability. Based on a matched dose assessment, the extent of systemic exposure was about 10 to 30-fold lower in the case of silver metal powder versus silver acetate (as reference ionic silver compound). Silver nanoparticles exhibit an intermediate systemic exposure profile.

Differentials in Ag in tissue concentrations following repeated dosing:When predicting the potential for toxicity for forms of Ag with variable bioavailability, it is reasonable to also consider differentials in key tissue/target organ exposure levels. As an example, and adopting the most conservative approach, such a comparison may be based on the attained tissue Ag concentrations arising from the highest administered/tolerated dose of Ag in respect of a soluble silver compound versus that for a Ag powder form. In the case of the latter, this represents the tissue exposure attained at a Limit Dose of 1000 mg Ag/kg bw/d administered via the oral route for a period of 4 weeks. Such a data analysis shows that for the majority of tissues, exposure to the soluble silver compound leads to tissue Ag levels which are 2 to 3 orders of magnitude greater than those evident for the silver powder. In confirmation of the markedly different TK characteristics of soluble silver compounds, similar differentials are apparent when comparing Ag in tissue levels attained at the maximally administered doses of silver nitrate versus AgMP. The AgNP exhibits an intermediate toxicokinetic profile with achieved tissue levels more closely to those of the ionic Ag test items than to AgMP.

In conclusion, it is assumed that the systemic toxicity of silver substances is driven by the release potential of Ag ionic species as the primary moiety for systemic circulation and tissue exposure, and hence hazard assessment. The comparative TK study is a high quality GLP investigation and the first which permits a direct comparison of systemic circulation and tissue distribution of Ag ionic species between soluble Ag salts (AgNO3 and Ag acetate), AgNP and AgMP. This new evidence demonstrates that oral intake of AgMP results in markedly lower absorption, distribution and systemic tissue/organ exposure to silver than more bioavailable forms like AgAc. As a generic observation, the following trend is being observed: AgAc > AgNO3 >> AgNP >>> AgMP.

The study findings show that the direct read-across of mammalian toxicity data with soluble silver salts (like silver acetate and silver nitrate) and nanosilver to silver metal (powder and massive) is not justified (based on their individual toxicokinetic profiles).

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.

Maurer et al. (2014) analysed the dissolution behaviour of silver metal NPs (10 and 50nm) by tangential flow filtration, in water was well as in simulated physiological fluids such as cell culture media, artificial lysosomal fluid and artificial alveolar fluid (acc. to Stopford et al. 2003). The study aimed at separating dissolved silver ions from Ag-NPs. Whereas centrifugation-based separation techniques may lead to NP agglomeration and very small NPs are retained in the supernatant even after ultracentrifugation, in this case the recirculating tangential flow filtration system retained large particles within the continuous flow path, while allowing ions to pass through molecular filters, providing a reproducible NP separation from dissolved ionic silver. The ionic dissolution of Ag-NPs was dependent on exposure time, chemical composition and temperature of the exposure solution. Ionic dissolution was found to be greatest in alveolar fluid for both sizes of Ag-NPs.

Braydich-Stolle et al. (2014) characterised the impact of artificial interstitial, alveolar, and lysosomal fluid on the physical properties of Ag NPs and their rate of ionic release. The test items studied were (i) hydrocarbon-coated (Ag-HC) (25nm), and (ii) polysaccharide-coated (Ag-PS) (25nm). After dispersion in artificial fluids, the Ag-HC and Ag-PS NPs exhibited significant alterations in morphology, aggregation patterns, and particle reactivity: water-dispersed Ag-HC and Ag-PS had a primary size of 27 and 236.3 nm, respectively, with predominantly spherical morphology. In lysosomal fluid, extensive aggregation was observed for both coatings with a concurrent loss of spherical morphology (Ag-HC), with primary and aggregate particle sizes of 120 and 290 nm, respectively. For Ag-PS, the corresponding primary and aggregate diameters were 50 and 160nm. In alveolar fluid, both Ag-HC and Ag-PS also showed significant aggregation: individual and aggregate particles sizes were 68 and 204 nm for Ag-HC, and 53 and 341 for Ag-PS, respectively.

Loza et al. (2014) studied the formation of Ag-Cl NPs by adding silver nitrate to different simulated biological media at concentrations between 0.01g/L-0.1g/L (stated as being the range of the cytotoxicity of silver), with stirring at room temperature for 7d under sterile conditions without exclusion of light. The precipitated silver particulates were isolated by ultracentrifugation, re-dispersed in pure water, again subjected to ultracentrifugation and then analysed by X-ray powder diffraction, SEM and energy-dispersive X-ray spectroscopy. The initially present silver ions are bound as silver chloride due to the presence of chloride. Only in the absence of chloride, glucose was able to reduce Ag+ to Ag0. The authors concluded that the predominant silver species in biological media is dispersed nanoscopic silver chloride, surrounded by a protein corona which prevents the growth of the crystals and leads to colloidal stabilisation.

In another paper, Loza et al. (2014) describes the dissolution/precipitation behaviour of silver ions in physiological media further: whereas the equilibrium concentration of free ionic silver is determined by the solubility product of AgCl (1.7x10-10mol2/L2), ionic silver can also form complexes with organic compounds and thereby be removed from the equilibrium. However, the concentration of ionic silver is typically low in biological experiments (between 1 and 100 ppm) thereby possibly explaining why precipitation is typically not observed, since the resulting particles are very small. Conversely, AgNPs release silver ions when oxygen or hydrogen peroxide are present. In the presence of chloride ions, precipitation of silver chloride nanoparticles occurs.

Walczak, A. P. et al. (2013) performed an in vitro study on silver metal NPs (60nm) in simulated human digestion fluids (saliva, gastric and intestinal medium, with and without addition of proteins). After gastric digestion in the presence of proteins, the number of particles dropped significantly, however they increased to original values after the intestinal digestion. SEM-EDX revealed that the reduction in the number of particles was caused by their clustering; these clusters being composed of AgNPs and chlorine. During intestinal digestion, these clusters disintegrated back into single 60 nm AgNPs. In comparative experiments, intestinal digestion of AgNO3 solution in the presence of proteins also resulted in the formation of silver nanoparticles (20–30 nm) composed of silver, sulphur and chlorine.

Specific investigations on the chemical transformation of nanosilver in biological environments

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).

The dissolution of silver metal NPs (60nm, 5% w/w suspension in ethylene glycol) and binding of released Ag+ ions in cellulo was studied in primary murine macrophages, exposed to a total dose of 5 μg/mL AgNPs either as single exposure (acute mode) for 6h or 24h, or as 1.25 μg/mL AgNPs per day for 4 days (chronic mode). Dissolution rates were dependant on the exposure scenario: chronic exposure lead to a higher Ag+ release than acute exposure; Ag-S bond lengths were 2.41 ± 0.03A and 2.38 ±0.01A in acute and chronic exposure respectively, compatible with diagonal AgS2 coordination. Glutathione was identified as the most likely putative ligand for Ag+. Internalised AgNPs in macrophages were characterised by TEM as electron-dense deposits in the cytoplasm.

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 AgNO₃showed 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).

Furthermore, in a comparativein-vivotoxicokinetic study of silver metal powder (AgMP), nanosilver (AgNP), silver nitrate and silver acetate, comparisons via normalised administered doses (i.e. Ag equivalent dose) can be made between different Ag test items (Melvin et al. 2021 and Charlton et al. 2021). Based on these matched dose assessments, it can be concluded that oral dosing of AgMP leads to appreciably lower Ag amplitude of systemic exposure in comparison to ionic Ag forms, while AgNP exhibits an intermediate systemic exposure profile. Levels of Ag distributed into tissues and organs are considerably lower in the case of AgMP than an ionic Ag compound. AgNP exhibits an intermediate TK profile with achieved tissue levels more closely resembling those of the ionic Ag test items rather than those evident for silver metal powder (AgMP).

Dermal absorption

The dermal absorption/percutaneous penetration of silver was determined in vitro (Bornatowicz, 2006). 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.

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), f_H	45.3 %	50.1 %	52.4 %	45.3 %	47.9 %	36.8 %
Deposition (TB), f_TB	0.1 %	0.4 %	0.3 %	0.3 %	0.5 %	0.1 %
Deposition (pulmonary) f_PU	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: (f_H+ f_TB) * abs_oral+ f_PU* abs_pul= 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) ".

A comparativein-vivotoxicokinetic study of silver metal powder, nanosilver, silver nitrate and silver acetate shows that levels of Ag distributed into tissues and organs are considerably lower in the case of AgMP than an ionic Ag compound (Melvin et al. 2021, Charlton et al. 2021). For the majority of tissues, exposure to the soluble silver compound leads to tissue Ag levels which are 2 to 3 orders of magnitude greater than those evident for the silver powder. The AgNP exhibits an intermediate toxicokinetic profile with achieved tissue levels more closely to those of the ionic Ag test items than to AgMP.

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”.

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).

Interactions of silver with other metals

Silver is well-known for its antimicrobial activity. It is therefore not surprising that animal experimental studies with high oral doses of silver have caused a disruption of the intestinal microflora involving a massive shift in composition of the intestinal microbiota resulting in severe gastroenteritis, and thus contributing to a disruption of homeostasis in the intestinal tract (Boudreau, 2012 and Williams et al. 2014).

Secondly, dietary administration of a very high doses of silver may cause disruption of maternal copper homeostasis resulting in copper deficiency by modifying copper-ceruloplasmin binding as a “secondary non-specific mechanism”. For this reason, a discussion of the relevance of ceruloplasmin and the influence of administration with either silver and/or copper is provided.

Disruption of intestinal microbiota by high oral dosing with inorganic silver substances

High oral dosing with inorganic silver substances severely impairs intestinal microbiota, leading to gastrointestinal disruption which in turn additionally interferes with the uptake of essential micronutrients, as demonstrated in the following information:

Preliminary results of an experimentally completed subchronic (90-day) study in rats conducted at National Center for Toxicological Research (NCTR) within the National Toxicology Program have shown that oral administration of silver acetate at doses of 200 and 400 mg/kg bw/d resulted in significant decreases in body weight gain and an increased morbidity at 400 mg/kg bw/d caused by severe gastroenteritis. No overt dose-response pathologies were observed for silver nanoparticles (Boudreau, 2012).

As published by Williams et al. (2014), it was investigated how the endogeneous microbial community (microbiota) of the rat intestine was affected during treatment with silver acetate and silver nanoparticles (AgNP). The focus of the analyses was directed to the two predominant bacterial phyla Firmicutes and Bacteroidetes constituting more than 90% of the microbiota in the intestine. Three genera out of them (Bacteroides, Lactobacillus and Bifidobacterium) and also the family Enterobacteria that includes, along with many harmless symbionts, many of the more familiar Gram-negative pathogens were examined by real-time PCR analyses. In addition, intestinal mucosa-associated immune responses were measured at the level of mucosal protective layer (MUC2 and MUC3), pathogen-associated molecular patterns (Toll like receptors 2 and 4; and NOD2), regulatory molecules (FoxP3 and GRP43) and inflammatory cytokines (interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß). Silver acetate (AgAc) was administered at doses of 100, 200 and 400 mg/kg bw/d by oral gavage to male and female Sprague-Dawley rats (10 rats/sex/group) twice daily, 7d/week, for 13 weeks. The selected doses of AgAc provided approximately 64, 128 and 192 mg silver/kg bw/d on a mass basis. Effects of silver acetate were compared to those of AgNP with discrete sizes (10, 75 and 110nm) gavaged at doses of 9, 18 and 36 mg/kg bw/d under the same time conditions. Culture-based analysis of ileal mucosa-associated bacterial populations showed a size- and dose-dependent antimicrobial effect of AgNP. The pattern of results indicated greater antimicrobial activity (measured as CFU) with decreasing nanoparticle size. The dose effect (gavaged with the same size AgNP) was significant for 110nm AgNP in males, whereas in females 75nm and 110nm AgNP showed dose-dependent responses. The antimicrobial activity of rats gavaged with 100 mg/kg bw/d AgAc (equal to 64 mg Ag/kg bw/d) was comparable to that given lower dose of 10nm AgNP (9 mg Ag/mg bw/d). All male rats (10/10) and eight female rats gavaged with 400 mg/kg bw AgAc were moribund. Most of the animals dosed with 200 mg/kg bw had severe gastroenteritis. Taken together, chronic exposure of rats to AgNP and AgAc apparently resulted in the killing of some bacterial populations in the rat ileum. Therefore, DNA-based microbiota analysis was used to determine how the major bacterial phyla were affected during silver treatment. The examination indicated that administration of low dose AgAc (100 mg/kg bw/d) altered the ratio of Firmicutes and Bacteroidetes in the ileal mucosa of both male and female rats from about 90:10 (controls) to 65:35 indicating a shift in the bacterial subpopulations representing the two phyla which could be due to changes in the subpopulations of any of the members of these two phyla. Relative expression of genes of some beneficial bacterial genera, e.g., Bacteroides, Lactobacillus and Bifidobacterium genera, in both male and female rats showed decreased proportions of the Lactobacillus population. The expression of Bifidobacterium genus was completely lacking in the AgAc-treated male rats. The reduction of Gram-positive bacteria (belonging to Lactobacillus and Bifidobacterium genus) caused by low doses of AgAc and AgNP was thought to be due to prolonged interaction of silver ions with charged molecules in the multilayer cell wall of Gram-positive bacteria. There was a highly significant increase in the expression of Enterobacteria family-specific genes including many of the more familiar Gram-negative pathogens, such as Salmonella, Escherichia coli, Yersinia, Klebsiella and Shigella, both in male and female AgAc-treated rats. The lower expression of symbiotic population of gut microbiome, as well as, an increase in the expression of bacterial genes representing Enterobacteria family may be a contributory factor for the gastrointestinal distress observed in AgAc gavaged rats. Exposure to AgNP prompted size- and dose-dependent changes to ileal-mucosal microbial populations as well as of intestinal gene expression and induced an apparent shift in the gut microbiota towards greater proportions of Gram-negative bacteria. DNA-based analyses revealed that exposure to 10nm AgNP and low-dose silver acetate caused a decrease in populations of beneficial Firmicutes phyla, along with a decrease in the Lactobacillus genus. The analysis of host gene expression in the ileum demonstrated that smaller sizes and lower doses of AgNP exposure prompted the decreased expression of important immune-modulatory genes, including MUC3 (mucosal protective layer), TLR2 and TLR4 (involved in microbial recognition), GPR43 and FOXP3 (involved in T-cell regulation). Gender-specific effects of AgNP exposure were more prominent for the gut-associated immune responses. Although exposure to AgNP seemed to generally cause down-regulation of the examined genes, the AgAc and vehicle control animals seemed to show little change or a general up-regulation of gene expression. This seems to indicate that the gene expression responses observed in this study may be the result of nanoparticles-specific impacts and not simply the effect of silver ions. In any case, exposure to AgNP may lead to changes in the gut-associated immune response.

Overall conclusions (disruption of intestinal microflora): the available data indicate that oral exposure of rats to silver acetate (64 mg Ag+/kg bw/d, lowest dose in the 90-day NTP study) causes alterations at the phylum-level in the ileum mucosa-associated microbiota and of the overall homeostasis of the intestinal tract. Oral exposure to Ag nanoparticles as well (lowest dose 9 mg AgNP/kg bw/d) prompted size- and dose-dependent changes to ileal-mucosal microbial populations, as well as, intestinal gene expression and induced an apparent shift in the gut microbiota towards greater proportions of Gram-negative Enterobacteria that may be the cause for the severe gastroenteritis. In addition, exposure to AgNP modulated the gut-associated immune response and the overall homeostasis of the intestinal tract. Taken together, these changes in rat microbiome caused by exposure to silver ions or silver nanoparticles indicate that ingestion of silver affect the gastrointestinal tract function adversely.

Influence of silver salts and silver powder on copper status in rodents

Pribyl et al. (1989) had shown that feeding rats a silver nitrate-supplemented diet (60 mg AgNO3/kg bw/d, equivalent to 38 mg of silver/kg bw/d) for more than 7 days decreased the ceruloplasmin-dependent oxidase activity in the blood to about 20%. Further treatment for up to 16 days led to a complete loss of oxidase activity in blood and resulted in the formation of a structurally modified ceruloplasmin as indicated by substitution of copper atoms for four silver atoms and loss of the characteristic absorption band at 610 nm.

More recently, Shavlovski et al. (1995) administered sparingly soluble silver chloride (50 mg/animal, corresponding to about 250 mg/kg bw/d which is equal to 188 mg of silver/kg bw/d) in diet to 5 inbred albino female rats during days 7-15 of gestation. The AgCl treatment during the period of organogenesis did not affect the development of embryos. Pre-implantation and post-implantation losses, the number and the body weight of fetuses corresponded to those of the controls (20 animals). No external abnormalities appeared on day 20 of gestation. A group of 20 rats was also administered the same high amount of AgCl throughout the entire period of gestation (gd 1-20). This treatment resulted in a considerably increased incidence of post-implantation losses of 36.0% compared to that of 9.6% of the control group, whereas the pre-implantation losses were not affected. The number of live fetuses and the fetal weight were decreased by 81% and 22%, respectively. Five out of 145 embryos (3.4%) showed visible abnormalities. The number of embryos having visceral damages was considerably higher in this trial group. The incidences of hydronephrosis (31%) and cryptorchidism (35%) increased considerably compared to controls (5.3% and 1.3%, respectively). In addition, the incidence of haemorrhages increased. Moreover, all of the totally little number of newborns died within 24h of birth. AgCl administration throughout the entire gestational period resulted in a reduced copper content of about 65% in maternal tissues (liver, heart, and kidney, cf. Table 3 in the original paper) and the absence of copper in placenta, embryonic tissues and maternal serum which correlated with the disappearance of oxidase activity from circulation (data not shown). Simultaneous i.p. injection of purified human ceruloplasmin into pregnant rats treated with AgCl especially during the second half of gestation caused a considerable reduction of adverse effects in dams and in the embryonic development including lower mortality of newborns. On the other hand, simultaneous introduction of the copper chelator penicillamine during the whole term increased the post-implantation loss to totally 79% thus providing additional substance to the argument of an involvement of copper deficiency to the toxicity caused by AgCl. Based on these findings, the authors concluded on an embryo-/fetotoxic effect of AgCl which is caused by the ability of silver to interfere with copper metabolism, in particular by altering the copper-transporting function of ceruloplasmin and, consequently, resulting in a copper deficiency in the developing tissues of embryos.

Conclusions: whereas the mechanistic information in silver-induced modification of copper ceruloplasmin-binding is of value, the limitations of the study by Shavlovski et al. to serve as substitute for a prenatal developmental toxicity study (OECD TG 414) need to be recognised (i.e., lack of data relating to potential maternal toxicity such as body weight change, body weight gain, food intake, water consumption and clinical signs; actual dietary intake not reported; maternal effects were only mentioned in an the abstract, no data on litter size and individual fetal body weights presented; only one dose tested). Thus, an interpretation of effects in connection with litter size and fetal weight data, the establishment of a dose-response relationship and finally the derivation of NOAELs for both maternal toxicity and embryo-fetal toxicity is not possible. However, the reported loss of the copper-dependent oxidase activity and the lack of copper in the maternal blood serum demonstrate the appearance of possibly adverse effects in dams. In addition, the copper content in maternal tissues (liver, heart, and kidney) was reduced to 65% indicating a distinct copper deficiency. Moreover, copper was not detectable in the placenta. Taking together the results from both studies, treatment of rats with doses of about 38 mg of silver/kg bw (AgNO3) or estimated 188 mg of silver/kg bw (AgCl) for 16–20 days resulted obviously in formation of a structurally modified ceruloplasmin in which 4 copper atoms were replaced by silver atoms. The consequence of this is an impairment of the copper transport function of ceruloplasmin and the deactivation of its copper-dependent oxidase activity in circulation.

A comprehensive assessment of the effects of repeated administration of silver salts on the copper status in rats and mice merits consideration of several other published investigations:

(i) Changes of copper concentrations in rat tissues after silver administration as well as in the status of serum ceruloplasmin have been shown by Hirasawa et al. (1994). Fischer F344 rats (6 males per group) received i.p. about 1 mg of silver/kg bw as silver nitrate for 6 successive days. The administered silver deposited in all the tissues examined, particularly in the pancreas, followed in that order in spleen = liver > kidney > lung > heart. Hepatic and pancreatic copper concentrations were significantly increased after silver administration, whereas renal and serum copper concentrations were significantly decreased. These results suggest that silver causes a re-distribution of tissue copper in the rat body. Serum copper concentration and ceruloplasmin oxidase activity were decreased to 60% and 25% of the control values, respectively. Immunoblotting of serum ceruloplasmin of silver-treated groups showed only a slight decrease in its concentration in serum suggesting that most of the ceruloplasmin was still present but as an oxidase inactive form. Simultaneous i.p. injection of about 3 mg of zinc (as zinc sulfate) for 6 days did not reverse the silver-provoked decreases in serum copper concentration as well as serum ceruloplasmin oxidase activity levels. No consistent changes in iron concentration were observed in any of the tissues examined after silver and/or zinc administration. In conclusion, the results indicate that silver administration to rats caused a disturbance of copper metabolism and of ceruloplasmin metabolism, but zinc did not protect against such changes. Hirasawa et al. (1997) purified an enzymatically inactive ceruloplasmin from the serum of Fischer F344 rats treated i.p. with 1 mg of silver/kg bw as silver nitrate for 6 days. Silver treatment resulted in decreases of copper concentration and ceruloplasmin oxidase activity in serum to 60% and 10% of the control values, respectively, and a marked accumulation of silver in serum. The metal contents of the Ag-modified ceruloplasmin were estimated as about 0.8 atoms of silver and 4.2 atoms of copper per molecule compared to 5.9 atoms of copper for ceruloplasmin from control rats. The Ag-modified ceruloplasmin showed neither a blue colour (characteristic for native protein, cf. Hellman & Gitlin, 2002) nor an EPR signal as characteristics of normal holo-ceruloplasmin. Thus, silver binding to native holo-ceruloplasmin resulted in a loss of one or two of the copper atoms essential for the oxidase activity.

(ii) Change of copper status in blood serum (copper concentration, ceruloplasmin oxidase activity, and ceruloplasmin protein content) of adult Wister rats and C57B1 mice was caused by feeding AgCl-containing diet (50 mg/kg bw/d, equal to 37.6 mg of silver/kg bw/d) for 4 weeks (Ilyechova et al., 2011). In rats, no ceruloplasmin oxidase activity was detectable in serum at that time. Simultaneously, copper concentration in rat blood serum was decreased by 90%, while in mice the copper concentration decreased only by 60%, but serum oxidase activity disappeared after one week on Ag-diet. The ceruloplasmin content in blood sera of Ag-treated animals remained unchanged as evidenced by immunoblotting. Ceruloplasmin existed as two protein forms with different electrophoretic mobility. One of them corresponded by electrophoretic mobility to the holo-ceruloplasmin, while the other one with lower mobility was considered the apo-ceruloplasmin. Ag-containing ceruloplasmin purified from blood serum of Ag-treated rats contained a molar ratio [Ag]:[Cu] of 4.5:1 and was shown to exist in a considerably misfolded tertiary/secondary structure of the protein as shown by CD spectra and calorimetric measurements. Reverse transcription PCR analysis indicated the same levels of ceruloplasmin mRNA in liver of both control and Ag-mice. The same results were reported for Ag-treated rats. Thus, the activity of the ceruloplasmin gene was not affected neither in mice nor rats by Ag-feeding. Recovery of the copper status in rats and mice, fed with Ag-diet for 4 weeks (no oxidase activity in serum), was tested after single i.p. or p.o. injection of CuSO4 (2.6 mg Cu/kg bw) under continuing the Ag-feeding. The Cu2+ injection caused a significant increase of the oxidase activity in the serum of both species starting after 20 min already and recovering to control levels after 4h which was accompanied by rapid insertion of copper into newly synthesized ceruloplasmin in liver. No significant differences between i.p. or p.o. modes of copper injection were observed. The recovered copper status persisted for 3 days under continuing Ag-diet. Direct addition of CuSO4 to oxidase inactive serum of Ag-treated animals did not lead to recovery of oxidase activity. Thus, the restored oxidase activity is not the result of an exchange of silver in the existing Ag-modified ceruloplasmin by copper but rather the result of de novo synthesis of holo-ceruloplasmin due to the availability of Cu2+ ions which compensate for the copper lack provoked by silver treatment of animals. Taken together, the results of that study indicate that dietary silver treatment of rats and mice (37.6 mg of silver/kg bw/d) for 4 weeks caused disappearance of the ceruloplasmin-dependent oxidase activity and reduction in copper content in serum indicating serum copper deficiency. However, ceruloplasmin gene expression in liver was not affected. Single injection of CuSO4 (2.6 mg Cu/kg bw i.p. or p.o.) to Ag-treated animals resulted in recovery of the oxidase activity of serum within 4 h in both species presumably due to de novo synthesis of copper-containing ceruloplasmin.

(iii) The influence of short (4w) and prolonged (6m) dietary exposure to silver chloride (corresponding to 50 mg/kg bw/d) on copper metabolism was studied: one group of adult rats received a Ag-diet for one month (Ag-A1), and another group received a Ag-diet for 6 months from birth (Ag-N6). In Ag-A1 rats the Ag-diet caused a dramatic decrease of copper status indexes that was manifested as ceruloplasmin-associated copper deficiency. In Ag-N6 rats, copper status indexes decreased only 2-fold as compared to control rats. In rats of both groups, silver entered the bloodstream and accumulated in the liver. Silver was incorporated into ceruloplasmin (Cp), but not SOD1. In the liver, a prolonged Ag-diet caused a decrease of the expression level of genes, associated with copper metabolism. Comparative spectrophotometric analysis of partially purified Cp fractions has shown that Cp from Ag-N6 rats was closer to holo-Cp by specific enzymatic activities and tertiary structure than Cp from Ag-A1 rats. One of the Cp isoforms is of hepatic origin, and the other is of extrahepatic origin; the latter is characterized by a faster rate of secretion than hepatic Cp. The authors hypothesised that that the disturbance of holo-Cp formation in the liver is compensated by induction of extrahepatic Cp synthesis (Ilyechova et al., 2014).

(iv) In pregnant Sprague-Dawley rats administered intravenously with radioactive 67Cu, the infused copper was either bound to ceruloplasmin or to albumin and transcuprein (constituting the exchangeable plasma copper pool), since the radioactivity in ceruloplasmin entered the placenta and the foetus much more rapidly than that from the exchangeable copper pool. The inhibition of the ceruloplasmin biosynthesis by cycloheximide reduced the appearance of radiotracer in plasma ceruloplasmin and was accompanied by a reduction in the 67Cu levels in placenta and the foetuses, further corroborating the importance of maternal ceruloplasmin for the foetus (Lee, 1993). In a comprehensive review, Gambling et al. (2011) concluded from the above that there is substantial evidence that offspring born to copper-deficient dams suffered from developmental abnormalities due to an impairment of intracellular copper-containing enzymes (cytochrome c oxidase, lysyl oxidase) and showed a significantly reduced 24h survival time. Further, the extent to which copper deficiency affects pregnancy outcome is very much dependent on the degree of copper limitation: severe copper deficiency can lead to reproductive failure, early embryonic death and gross structural malformations in the fetus, while moderate or mild copper deficiency has little effect on either number of live births and neonatal weight.

While above studies clearly show that administration of silver salts result in decreased copper content, this is not the case for silver powder. In thein-vivocomparative toxicokinetic study (Melvin et al. 2021), the copper status of rats exposed to micron-sized silver (AgMP) has been evaluated and compared to results for an ionic Ag reference (AgNO3). For AgMP, there was no indication of any depletion in circulating Cu levels after 28 days treatment via the oral route, even with treatment corresponding to a limit dose (1000 mg/kg bw/d). This equates to a Ag equivalent dose level which was 12 times higher than that employed as the AgNO3 high dose comparator. In contrast, a depression in circulating Cu levels was apparent from a quite low exposure in the case of the ionic reference compound (being evident from 35 mg Ag/kg bw/d).

Other studies on copper status in mice

In another study, C57B1 mice (5 animals per group) received in diet 50 mg AgCl/kg bw/d (equal to 37.6 mg of silver/kg bw/d) for one, two or three weeks to evaluate the rate of development of blood serum copper deficiency (Zatulovskiy et al., 2012). After silver chloride treatment for 3 weeks one group was given a standard diet for 14 days to study the effect of silver removal on serum copper status which was characterized by ceruloplasmin oxidase activity, ceruloplasmin protein concentration and copper content. Serum oxidase activity was not detected after one week of Ag-diet and remained undetectable during the entire period of Ag-diet. After the 3rd day following silver removal from the diet, the ceruloplasmin oxidase activity was progressively restored. Serum copper concentrations changed in accordance with oxidase activity, i.e., they decreased to about 30% after three weeks and increased to the normal value following Ag-free diet. The ceruloplasmin concentration in serum of Ag-mice treated for 3 weeks was comparable to that of control mice. Immunoblotting showed that ceruloplasmin was present as two protein forms with different electrophoretic mobility. One of them corresponded by electrophoretic mobility to the holo-ceruloplasmin, while the other one had a lower mobility. Neither of them, however, displayed oxidase activity. The Ag-treatment did not change the expression of some genes involved in copper transport (Ctr1 and ATP7B) and of intracellular copper enzymes (superoxide dismutase 1 (SOD1) and cytochrome c oxidase (COX)) as determined by semi-quantitative reverse transcription PCR of total RNA fraction isolated from livers of control and Ag-mice treated for 3 weeks. Similar results were obtained at the protein level, i.e., Ag-diet did not alter the protein level of Ctr1, ATP7B, SOD1, and COX (Cox4i1 subunit). The intracellular distribution and activity of SOD1 were also unchanged. As well, the rates of oxygen consumption by mitochondria isolated from liver of Ag-treated mice and control mice were identical. Together these data indicate that dietary silver treatment for three weeks does not affect the copper homeostasis in liver. The copper status in serum was clearly restored in Ag-treated mice within three days after the removal of silver from the diet.

In summary, the results indicate that silver administration to mice (37.6 mg of silver/kg bw/d) for three weeks caused a reduction in the ceruloplasmin-dependent oxidase activity and copper content in serum indicating a serum copper deficiency. However, no changes in ceruloplasmin gene expression in liver occurred. In addition, the SOD1 and COX activity were not decreased. Also Ctr1 and ATP7B gene expression and level of these proteins were not affected. Apparently, copper homeostasis in liver cells was not disturbed.

Overall summary and conclusions, interaction of silver with other metals

The alterations in serum copper level and ceruloplasmin oxidase activity in rats after i.p. administration of 1 mg Ag/kg bw for 6 days (Hirasawa et al. 1994, 1997) are qualitatively similar to those reported by the group of Shavlovski (Pribyl et al., 1989, Shavlovski et al., 1995) and recently by Ilyechova et al. (2011; 2014). However, there are also very distinct quantitative differences:

Treatment of rats with 1 mg Ag/kg bw for 6 days (as AgNO3) resulted in decreased copper content and oxidase activity in serum to 60% and 10%, respectively, and formation of a modified ceruloplasmin containing about 0.8 Ag atoms and 4.2 Cu atoms in contrast to 5.9 Cu atoms in native holo-ceruloplasmin from control rats (Hirasawa et al., 1994).

However, feeding rats a higher amount of 38 mg Ag/kg bw/d for 16 days (as AgNO3) resulted in a total loss of oxidase activity and formation of another structurally modified ceruloplasmin form containing 4 Ag atoms instead of copper (Pribyl et al., 1989).

Nearly identical results were reported recently (Ilyechova et al., 2011). Feeding rats a Ag-diet with 37.6 mg Ag/kg bw/d (as AgCl) for 4 weeks resulted in no detectable oxidase activity and a reduced copper concentration by 90% in serum, while the ceruloplasmin content in serum remained unchanged as evidenced by immunoblotting. The Ag-containing ceruloplasmin purified from serum of Ag-treated rats contained a molar ratio Ag:Cu of 4.5:1 and was shown to exist in a drastically misfolded tertiary/secondary structure. However, ceruloplasmin gene expression in liver was not affected by Ag feeding as measured by RT PCR analysis. Single injection of CuSO4 (2.6 mg Cu2+/kg bw) to Ag-treated rats and mice resulted in recovery of the oxidase activity of serum within 4h in both species presumably due to de novo synthesis of copper-containing ceruloplasmin.

Feeding female rats a very high dose of estimated 188 mg Ag/kg bw/d (as AgCl) during entire gestational period (gd 1-20) resulted in the absence of copper and oxidase activity in serum and a markedly reduced copper content in liver, heart, and kidney, while copper was completely absent in placenta and embryonic tissues (Shavlovski et al., 1995). Thus, besides the Ag-caused alterations in serum copper status the markedly reduced copper content in maternal tissues has to be considered as indication of strongly disrupted copper homeostasis in dams, obviously without any clinical signs of copper deficiency or maternal toxicity as stated by the authors.

A 3-week feeding study in mice showed that AgCl diet corresponding to 37.6 mg Ag/kg bw/d caused a reduction in the oxidase activity and copper content in serum indicating likewise a serum copper deficiency (Zatulovskiy et al., 2012). Again, as in rats, no changes in ceruloplasmin gene expression in liver occurred. In addition, the SOD1 and COX activity were not decreased. Also Ctr1 and ATP7B gene expression and level of these proteins were not affected. Apparently, copper metabolism in liver cells was not disturbed. The serum copper status (Cu content and oxidase activity) was clearly restored in the Ag-treated mice within three days after the removal of silver from diet.

Copper uptake and metabolism

For comparative purposes, some information on copper metabolism is also presented here:

Intracellular copper concentrations are tightly regulated by copper transporter (Ctr) proteins and copper-transporting ATPases (ATP7A and ATP7B; cf. Nevitt et al., 2012). Dietary copper (Cu2+) is reduced to Cu+ and transported into enterocytes primarily via the so-called high-affinity copper transporter 1 (Ctr1) localized on the apical membrane of enterocytes within the lumen of the intestine. Silver is a potent inhibitor of copper uptake via Ctr1 as shown by metal competition experiments in a human embryonic kidney (Hek293) cell line suggesting that Ctr1 may also function in importing silver into cells (Lee et al., 2002). Thus, it can be expected that in the presence of silver salts the import of copper by enterocytes is diminished due to the concomitant import of Ag resulting in copper deficiency within these cells leading to an impairment of intracellular copper-dependent processes and to a decreased enterocyte copper efflux thus lowering the copper levels in blood for further distribution to tissues.

Tracer studies in rats have shown that radioactive copper entering the blood initially binds to albumin and transcuprein forming the “exchangeable copper pool” (cf. Hanson et al., 2001; Donley et al., 2002). Radioactive copper transported mainly by transcuprein then rapidly leaves the blood for the liver and kidney.

Lower copper concentrations in blood are of special importance to the liver, which is the main storage organ for copper and fulfils the decisive function in the regulation of systemic copper homeostasis, especially incorporation of copper into ceruloplasmin and other Cu-dependent proteins (Nevitt et al., 2012). With respect to ceruloplasmin synthesis, the hepatic copper pool is not rate-limiting under normal circumstances, as the serum ceruloplasmin level increases rapidly during infection, trauma, and three- to fourfold in pregnancy (cf. Hellman & Gitlin, 2002). However, a decrease in the hepatic copper pool, e.g. as occurs in nutritional copper deficiency, results in a marked decrease in serum ceruloplasmin.

Tracer studies in female rats with radioactive silver (110Ag, as AgNO3) have shown that the transport and distribution of silver resemble those for copper in some aspects, particularly with regard to its rapid accumulation in liver (Hanson et al., 2001). However, silver transport in plasma was mainly carried out by α1-macroglobulin. A small proportion of silver was also incorporated into ceruloplasmin. No association of radioactive silver to albumin and transcuprein was detected indicating the carrier function of another macroglobulin than that involved in copper transport.

While administration of silver salts result in decreased copper content, this is not the case for silver powder. For a micron-sized silver powder (AgMP), there was no indication of any depletion in circulating Cu levels after 28 days treatment via the oral route, even with treatment corresponding to a limit dose (1000 mg/kg bw/d). In contrast, a depression in circulating Cu levels was apparent from a quite low exposure in the case of the ionic reference compound (being evident from 35 mg Ag/kg bw/d) (Melvin et al. 2021).

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Dose level	Mean 6h blood [Ag]		Mean [Ag]_6h/
(mg/kg Test Item bw/d)	ng/mL		Ag Equiv. Dose
			ng/mL
	♂	♀	♂	♀
Control	<LLOQ	<LLOQ	--	--
3.6	67	81	19	23
	± 21	± 8
36	175	268	4.9	7.4
	± 46	± 41
360	226	434	0.63	1.2
	± 46	± 71

Dose (mg/kg bw/d) /Sex		Bone marrow	Brain	GI tract	Spleen	Testis	Liver

Control M	Mean	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
	SD	NC	NC	NC	NC	NC	NC

3.6 M	Mean	BLQ	77.5	156	77.0	66.1	BLQ
	SD	NC	13.9	42.8	38.6	26.6	NC

36 M	Mean	181	226	3888	938	452	154
	SD	107	46.5	1799	920	173	114

360 M	Mean	NC	575	11466	14928	1310	1949
	SD	NC	136.9	8252	5257	152	900

Dose(mg/kg bw/d) /Sex		Bone Marrow	Brain	GI Tract	Spleen	Ovaries	Uterus	Liver

Control F	Mean	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
	SD	NC	NC	NC	NC	NC	NC	NC

3.6 F	Mean	BLQ	64.8	415	53.2	116	BLQ	77.2
	SD	NC	12.5	196	21.8	93.7	NC	50.4

36 F	Mean	NC	221	5784	2233	5957	225	382
	SD	NC	13.4	726	1069	2255	133	233

360 F	Mean	NC	604	9630	28081	19170	3865	4325
	SD	NC	36.8	3721	7067	6142	1915	1544