Silver

Endpoint summary

Description of key information

Silver metal massive and powder:

Oral: no adverse effects via repeated exposure via oral route is expected for silver (massive and powder).

Inhalation: a strong weight of evidence using the available data demonstrates that adverse effects via repeated
exposure via inhalation route are not expected for silver metal (massive and powder). The repeated dose inhalation
toxicity studies performed with nanosilver only identified adverse local effects and inflammation, which in certain
is reversible but any case considered relevant for nanoparticles only. There is no systemic / local effect expected via
repeated exposure to silver metal (massive and powder) inhalation route.

Dermal: route considered to be less relevant

For further information, please refer to section 13 documents "CSR Annex 11 - Weight of Evidence Justification for 
Silver metal - human health endpoints.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

The aim of this study was to investigate the differential accumulation, distribution, and potential toxicity of silver in rats following daily oral exposure to nano silver AgNP 10, 75 and 110 nm.
Data of this robust study summary refers only to AgNP-110nm (thin metal powder).

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

In order to determine the amount of silver in the AgNP stock suspensions that was in ionic form, filtrates of the AgNP suspensions were prepared by centrifugation through a microcentrifuge cellulose filter that had a molecular weight cut-off of 3 kDa (AmiconVR Ultra-4 centrifugal filter units; Millipore Corporation, Billerica, Massachusetts). Subsamples (1 ml) of the pre-mixed AgNP stock suspensions were applied to the Amicon filter units and centrifuged at 4000g for 30 min in an Eppendorf 5810 R centrifuge that was equipped with an Eppendorf A-4-81 swinging bucket.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- 3-week-old male and female Sprague Dawley/CD-23 rats with specific pathogen-free health status were obtained from the NCTR breeding colony.
- At 6 week of age, the rats were weight-ranked and randomly assigned to treatment groups. Male and female rat were housed conventionally in separate animal rooms with 2 animals per cage.
- The environment of the animal rooms was set to maintain a 12h light cycle, temperature of 22+/-4°C, relative humidity of 40%-70%, and air changes of 10-15 per hour.
- The animals were provided NIH-41 gamma-irradiated pellets and Millipore-filtered drinking water ad libitum.
- Rats were dosed initially at 7 weeks of age.

Route of administration:

oral: gavage

Details on route of administration:

Gavage dosing was conducted using computer-controlled MicroLab 500 series dispensers (Hamilton Co., Reno, Nevada) equipped with gastight syringues and capable of dispensing 1µL to 50mL.
Syringue were fitted with flexible plastic gavage needles, and the rats were provided equal volume doses based on the daily body weight of the individual rats.
The MicroLab dispersion were programmed to administer the total daily dose in 2 daily gavage administrations per day, with half of the dose administered just prior to start of the dark cycle.


Route Exposure: Ingestion is the primary route of exposure in humans for silver, silver compounds, and colloidal silver (Silver, 2003). Daily intakes for silver in humans are estimated in the range 0.4–27 mg/day (Clemente et al., 1977; Gibson and Scythes, 1984; Hamilton and Minski, 1972/1973). The Environmental Protection Agency lists the reference dose for silver as 0.5 mg/kg/day or 35 mg/day for an average 70 kg adult (EPA, 2014).

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

The high-dose formulation (0.9 mg/ml) for each size of AgNP was prepared in the CIT/CMC vehicle (2mM sodium citrate/ 0.1% CMC; final concentration; wt/wt), CIT= trisodium citrate dihydrate

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared weekly and were based on the total silver mass concentrations obtained by ICP-MS for the batch stock AgNP suspensions. The high-dose formulation (0.9 mg/ml) for each size of AgNP was prepared in the CIT/CMC vehicle (2mM sodium citrate/ 0.1% CMC; final concentration; wt/wt),

- The total silver concentrations of the high-dose formulations were confirmed by ICP-MS and, subsequently, the AgNP high-dose formulations were serially diluted in the CIT/CMC vehicle to achieve the 0.45 and 0.225 mg/ml concentrations for the mid- and low-dose formulations, respectively.

DOSE CHARACTERIZATION: The total silver concentration by mass of each of the prepared dose formulations (high-, mid-, and lowdose) and the controls (CIT/CMC and water/MC) was determined by ICP-MS in acid- and microwave-digested samples. The hydrodynamic sizes of particles in the dosed solutions were determined by DLS, and the core diameters and aspect ratios of the AgNP were evaluated by TEM. The median number of particles
evaluated by TEM per sample was 214 (range 44–498). Characterization analyses were conducted on triplicate samples. Dose concentration acceptability was set at 610% targeted
values.

VEHICLE
Carboxymethyl cellulose (CMC) and trisodium citrate dihydrate (CIT) were purchased from Sigma Aldrich (St. Louis, Missouri), and methyl cellulose (MC) was purchased from Fisher Scientific (Fair Lawn, New Jersey). The water used in dose formulations was 18 megaohm and autoclave-sterilized.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In order to determine the amount of silver in the AgNP stock suspensions that was in ionic form, filtrates of the AgNP suspensions were prepared by centrifugation through a microcentrifuge cellulose filter that had a molecular weight cut-off of 3 kDa (AmiconVR Ultra-4 centrifugal filter units; Millipore Corporation, Billerica, Massachusetts). Subsamples (1 ml) of the pre-mixed AgNP stock suspensions were applied to the Amicon filter units and centrifuged at 4000g for 30 min in an Eppendorf 5810 R centrifuge that was equipped with an Eppendorf A-4-81 swinging bucket.

Duration of treatment / exposure:

Groups of rats (10 males and 10 females) were exposed daily by oral gavage to dose formulations of AgNP-110 nm at 9, 18, and 36 mg/kg bw; or to the respective control formulations (CIT/CMC) for a period of 13 weeks.

Frequency of treatment:

Gavage dosing was conducted using computer-controlled MicroLabVR 500 series dispensers (Hamilton Co., Reno, Nevada) equipped with gastight syringes and capable of dispensing 1 ml to 50 ml. The syringes were fitted with flexible plastic gavage needles, and the rats were provided equal volume doses based on the daily body weight of the individual rats. The MicroLab dispensers were programmed to administer the total daily dose in 2 daily gavage administrations per day, with half of the dose administered at the start of the light cycle and half of the dose administered just prior to start of the dark cycle. The dose volumes did not exceed 20 ml/kg bw (OECD, 1998). Animals were dosed 7 days each week and the study period was 13 weeks.

Dose / conc.:

9 mg/kg bw/day (actual dose received)

Dose / conc.:

18 mg/kg bw/day (actual dose received)

Dose / conc.:

36 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The aim of this study was to investigate the potential toxicity of AgNP at doses selected to provide the maximal concentrations that could be achieved with the citrate stabilize particles
and that would also ensure stability of the nanoparticles. The nominal concentrations (1 mg/ml) of the AgNP stock suspensions and the maximum gavage dose volume of 20 ml/kg bw per dosing episode (OECD, 1998) set the high dose level for AgNP at 36 mg/kg bw (0.9mg/ml40 ml). Oral gavage, rather than drinking water or dosed feed, was the selected method of dose administration to ensure that the suspensions of AgNP were dispersed at the time of introduction to the animals and to ensure the precision of the dosed amount.

- Fasting period before blood sampling for clinical biochemistry: terminal sacrifices were conducted on over-night fasted rats.

Positive control:

An ionic form of silver (silver acetate), served as a positive control in this study and as a marker for silver ion toxicity

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly clinical observations on individual animals were recorded in the animal records database (NCTR Multi-generation Computer Support System; MGSS).

BODY WEIGHT: Yes
- Time schedule for examinations: body weights recorded daily in MGSS for dose administration.

FOOD CONSUMPTION AND COMPOUND INTAKE: Feed and water consumptions were measured and recorded weekly in MGSS.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at terminal and moribund sacrifices
- Anaesthetic used for blood collection: Yes: Animals were euthanized humanely by carbon dioxide asphyxiation after the 72h blood sample collection.
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at terminal and moribund sacrifices
- Animals fasted: Yes

IMMUNOLOGY: A section of the ileum and scrapping of the ileal mucosa were collected for intestinal microbiota and immune response evaluations (Williams et al., 2015).

Sacrifice and pathology:

At necropsy, organs and tissues were examined for grossly visible lesions, and protocol designated tissues and organs were weighed and preserved in 10% neutral buffered formalin (NBF), with the exception that modified Davidson’s fixative was used for the right testes and eyes. Femur bone marrow was collected for histopathology (left) and ICP-MS (right). The testes and epididymides were collected for sperm analysis (left) and histopathology (right).

Statistics:

A Cox proportional hazard model was used to analyze survival data and to test the effect of treatment relative to control. All tests were conducted as 2-sided with significance at the .05 probability level.
Pharmacokinetic parameters were determined through the use of PK Solutions 2.0 software (Summit Research Services, Montrose, Colorado). The parameters included maximum observed concentration (Cmax), the area-under-the concentration- versus-time curve (AUC), absorption/distribution half-life (t1=2 A/D), and elimination half-life (t1=2 E). The AUC was estimated to the last sampling time using the trapezoidal rule and further extrapolated to infinity (AUC0-1).

Body weights, feed consumption, water intake, and blood were analyzed using a 1-way repeated measures, mixed model ANOVA for each sex, with terms for dose, week, and all interactions.
Week was treated as the repeated measure. For body weight analyses, the last body weight obtained for each dose week was used as the weekly body weight. Within-group correlations
were modeled using a heterogenous first-order autoregressive correlation structure, and pairwise comparisons of the 2 control groups and each of the treatment groups to the appropriate control group were performed with Bonferroni adjustments.

The statistical analysis of hematology and clinical chemistry data was performed using a non-parametric method with midranks (Brunner et al., 2002). Pairwise comparisons were performed with Bonferroni adjustments. All tests were conducted as 2-sided with significance at the .05 probability level.

The organ weights were analyzed using a 1-way analysis of covariance with necropsy body weights as a covariate. Pairwise comparisons were performed with Bonferroni adjustments.

Further information on statistical analysis under section "Any other information on materials and methods incl. tables"

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant differences in survival between the vehicle and water controls or between any AgNP group and the vehicle control group in either the female or male rats.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Five animals in this study were either moribund or died with severe acute nephropathy. These types of renal changes have been noted in Sprague Dawley rats in previous studies conducted at NCTR and are considered spontaneous findings and not treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights did not differ significantly between the vehicle and water controls or between the different doses and sizes of AgNP and the vehicle controls in female rats. Sporadic statistically significantly higher body weights were observed in male rats administered the 110nm AgNP, but body weight never exceeded 113% and were not considered biologically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences in feed and water intakes between the vehicle and water controls. When compared with respective control animals, sporadic differences in feed and water intakes were observed in female and male rats administered AgNP or AgAc.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematological and plasma clinical chemistry parameters recorded for rats exposed to AgNP show no significant differences in the values for these parameters between the vehicle and
water controls, and values recorded for the AgNP-treated groups did not differ significantly from the vehicle controls.

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The effects of AgNP on the absolute organ weights and relative organ weights (%; organ weight/necropsy body weight) of rats exposed for 13 weeks to these formulations were no meaningful treatment-related differences in the absolute or relative organ weights of female and male rats exposed to AgNP when compared with control groups.

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A notable observation by light microscopy was the occurrence of diffuse brown pigmentation in numerous organs and tissues of rats exposed to AgNP.

In many respects, the detection of pigmentation was more a measure of silver mobility rather than toxicity, and pigmentation was graded for severity as (1) minimal, (2) mild, (3) moderate, or (4) marked.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

36 ca. mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

haematology

histopathology: neoplastic

histopathology: non-neoplastic

Remarks on result:

other: highest dose tested

Critical effects observed:

no

Characterization of AgNP stock suspension and dose formulation:

The proportion of silver in the AgNP stock solutions that was present in
dissolved ionic form was determined by means of ultrafiltration over a 3-kDa membrane. TEM examination of filtrates confirmed that AgNP did not cross the membrane, and ICP-MS determinations of the pre-filtered samples, filtrates, and resuspensions of the concentrated solutes and filtrates indicated an average recovery>98%, irrespective of AgNP size (data not shown). The mean concentration of ionic silver in the AgNP suspensions was similar across nanoparticle sizes and averaged 0.01%–0.02% of the AgNP stock suspensions. The size, morphology, and distribution parameters of the AgNP stock suspensions were determined by TEM and DLS. AgNP size derived from TEM determinations indicated that mean core sizes for the 110 nm particles were 101.56 +/-15.8 nm. These results closely corroborate the data provided by the manufacturer with each shipment of the AgNP suspensions. The morphological characteristics of the AgNP were also evaluated by TEM, and the mean aspect ratios confirmed that the AgNP particles were mostly spherical in shape.

The hydrodynamic size was determined for the AgNP by DLS (z-average). In the examples, the largest peak in the graph represents 100% intensity, with z-average values of 103.7nm for the 110 nm AgNP. The size determinations by DLS were larger than those measured by TEM for the same particle suspension. The AgNP used in this study were stabilized with the citrate capping agent, likely accounting for the larger hydrodynamic size when measured by DLS, suggesting that the actual size of the particles reside somewhere within the upper TEM and lower DLS range of values.

The concentrations of total silver for the AgNP dose and control formulations
were determined by ICP-MS. The levels of total silver for each of the control formulations were below the limit of quantitation (40 ng/mL) by ICP-MS thourghout the study. Dose formulations were prepared on a weekly basis at 3 concentrations level for each size of AgNP (0.225, 0.45 and 0.9 mg/mL) and the toal silver concentration of each formulation was determined by ICP-MS prior issuance of the dose formulation to animals on the study. The mean percentage of targeted dose for all doses over the study period was 104+/-2% for the 110nm AgNP formulations.

Homogeneity and Stability of the AgNP Suspensions
The results of homogeneity and stability tests indicate that the mixing procedure provided evenly dispersed particles throughout the vessel. Stability testing by
DLS and TEM was conducted on the same combined lot for each particle size suspension and by ICP-MS on filtrates for ionic silver determinations for 90 days. The hydrodynamic size of the particles determined by DLS and the core size of the particles determined by TEM remained stable over the testing period. ICP-MS analysis of filtrate samples showed only a very slight increase in soluble silver formed over the 90-day period.

Dose formulations for the AgNP were prepared in their respective control formulations that contained either 0.1% CMC. CMC was used in this study as
bulking agents to inhibit somewhat the gastrointestinal passage of the mostly aqueous dose formulations. This compounds is not toxic and do not promote allergic reactions in humans or rodents (Frawley et al., 1964; Shelanski and Clark, 1948). The hydrodynamic size of particles were higher in the dose formulations,
when compared with the stock solutions, and the CMC was likely responsible for the increase in particle size, as determined by DLS. The mean core diameters
and aspect ratios of the particles were similar to those of the stock solutions, suggesting that little change occurred in the particle size and shape during the preparation of dose formulations.

Histopathology further details:

The incidences of pigmentation observed by light microscopy among rats that were administered the AgNP treatments conveyed a similar story as told by the results of silver distribution obtained by ICP-MS. The severity of pigmentation in AgNP treatment groups was graded as minimal, with only a few instances of mild pigmentation. Pigmentation was more prevalent in female rats than male rats, especially in the mesenteric lymph nodes, the large intestine (except the rectum), the stomach, kidneys, and spleen. However, the high incidences
(~80%–100%) of pigmentation in most every tissue of female rats tended to mask any differences due to AgNP size. Male rats administered AgNP showed a similar pattern of pigmentation as female rats; however, incidences of pigmentation were much lower and demonstrated a size-dependent prominence, with the AgNP-10 nm having greater prominence than either the AgNP-75nm or 110 nm treatments. Evaluations by light microscopy were able to discern the presence or absence of silver pigmentation in tissue and organ specimens; however, this method could not quantify differences in the intensity of the pigmentation at the levels of accumulation observed for AgNP-exposed rats.

Disposition and accumulation of silver:

The analysis of silver concentrations by ICP-MS in tissue and organ samples collected from the control groups showed no significant differences in silver concentrations between the water and vehicle controls for any reported parameter; however, all tissues and organs analyzed from rats exposed to AgNP for 13 weeks, with the exception of bone marrow, revealed statistically significant
(P<.05) dose-dependent increases in silver concentrations. 

The silver content in blood and bone marrow averaged 3–4 times lower than the silver content of the heart, which had the lowest content of silver among the major organs analyzed and indicated that the contribution of silver accumulation in the blood and bone marrow was minimal. Furthermore, a repeated measures statistical analysis of these data found no significant differences for silver concentrations in rat blood collected at weeks 1 and week 12. As blood levels of silver were not significantly higher after 12 weeks of dosing than levels found after only 1 week of dosing, these results suggest rapid clearance of silver from the blood for all groups irrespective of silver form. In preliminary studies, the half-time for elimination of silver from the blood
was ~24 h regardless of sex of form of silver.

Results of pairwise comparison tests indicated that silver concentrations were significantly higher in the blood and bone marrow of rats exposed to AgNP-10 nm, especially at the 2 higher doses, when compared with those of rats exposed to
AgNP- 75 or 110nm and suggested that the concentration of silver in these samples was inversely related to particle size. 

In female and male rats exposed to AgNP the kidney and spleen represented sites of significant silver accumulation. As observed in the intestinal tract, sex differences in silver concentrations were observed. The concentrations of silver in all organs were significantly lower in male rats exposed to AgNP. In addition, the pattern of distribution in the kidneys and spleens of male rats differed from that
of female rats.

The concentrations of silver in all organs were several folds higher in rats exposed to AgAc, and, as observed with rats exposed to AgNP, the concentrations of silver in all organs appeared to be higher in females than males.

Conclusions:

The results presented in this study demonstrate that AgNP are absorbed and translocate to organs and tissues as mostly intact particles. This is the first direct comparison of the morphological differences between the accumulation of AgNP and soluble silver in organs and tissues of rats following daily oral administration (7 days/week) of AgNP and AgAc for a period of 13 weeks.
No NOAEL has been defined by the authors, however, based on the results obtained from the AgNP-110nm (thin silver powder), a No Adverse Effect Level could be set up at 36 mg/kg bw/day (highest dose tested).

Executive summary:

Statistically significant sex differences in the distribution and accumulation of silver were noted for number of tissues and organs in this study.

Microscopic examination found no treatment-related histopathological findings in rats administered AgNP. Pigmentation, assumed silver accumulation, was observed in a number of tissues, with the highest levels of pigmentation detected in the small and large intestine, kidney, liver, and mesenteric lymph nodes; however, the presence of pigmentation in most tissues was minimal in both sexes or rats exposed to AgNP, and the presence of pigmentation did not induce lesions that could be
detected by light microscopy.

Statistically significant sex differences in the distribution and accumulation of silver were noted for number of tissues and organs in this study.
Sex differences in silver accumulation were observed in nearly every tissues in this study. 

TEM micrographs also revealed that AgNP appeared to be located predominantly within cells (intracellular), in particular epithelial cells; whereas AgAc appeared to have an affinity for the basement membranes (extracellular) of tissues. 

EDS was used in this study to confirm the presence of silver in the observed granules within rat tissues and organs, however, there were no qualitative differences between the elemental composition of particles or ganules in any of the examined tissues of rats exposed to either AgNP or AgAc.

The presence of sulfur and selenium in association with the AgNP and AgAc suggests protein-binding of the granules, either at the site of absorption or at the site of accumulation, due to the high affinity of silver for sulfur and selenium. The observation of sulfur, selenium, and chlorine in association with silver
granules derived from an ionic silver form was anticipated, due to the high affinity of silver ions for these minerals; however, the similarity in elemental composition between AgAc and AgNP is difficult to explain.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 January 2021-13 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

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

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guideline on Bioanalytical Method Validation, European Medicines Agency (EMA), EMEA/CHMP / EWP/192217/2009

Version / remarks:

21 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance for industry: Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM)

Version / remarks:

May 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 417 Toxicokinetics

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Silver purity: 100%
Silver content: 64.56%
nitrate (NO3): 95 ppm
Copper: 1 ppm
Iron: 1 ppm
Sodium: 3 ppm
Magnesium: 1 ppm

Species:

rat

Strain:

other: Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent
species for nonclinical toxicity test by regulatory agencies.
Species: Rat
Strain: Crl: WI(Han)
Condition: Outbred, SPF-Quality
Source: Charles River Deutschland, Sulzfeld, Germany or Charles River
Laboratories France, L'Arbresle Cedex, France.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks old
- Weight at study initiation: 165-240 g (males), 127-170 g (females
- Fasting period before study: not applicable
- Housing:
Caging: Polycarbonate cages (Makrolon type IV, height 18 cm or Makrolon type 2000P, height 21.5 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
Up to 5 animals of the same sex and same dosing group together. During locomotor activity monitoring, animals are housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
These housing conditions will be maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the animals will be kept will be documented in the study records.
Cage Identification:
Color-coded cage card indicating at least Test Facility Study No., group, animal identification number(s).
- Diet (e.g. ad libitum): Animals will have access to the test diets from Day 1 onwards.
During the acclimatization period, animals will have free access to pellets without the test item (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals will not have access to food for a maximum of 2 hours.
The test item diets remain in the food hopper for a maximum of one week. On the day of weighing the remaining food in the food hopper, is replaced with new diet.
Type: Pellets.
Frequency: Ad libitum, except during designated procedures.
- Water (e.g. ad libitum):Municipal tap water, freely available to each animal via water bottles.
- Acclimation period: The animals will be allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
-animal enrichment:
Animals will be socially housed for psychological/environmental enrichment and may be provided with items such as devices for hiding in, paper and/or objects for chewing, except
when interrupted by study procedures/acivities. Results of analysis for contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants that would interfere with the objectives of the study.

DETAILS OF FOOD AND WATER QUALITY:
Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 °C
- Humidity (%): 40-70%
- Air changes (per hr): ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h light and 12 h dark (except during designated procedures

IN-LIFE DATES:
From: 20 Jan 2021 (Animal arrival, first date data are collected from the study)
To: 7 May 2021 (last date of necropsy

Route of administration:

oral: feed

Details on route of administration:

The oral route of exposure via dietary inclusion was selected because this is a possible route
of human exposure during manufacture, handling or use of the test item.

Vehicle:

unchanged (no vehicle)

Remarks:

oral administration via the diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
Stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) over the concentration range 50
to 10000 ppm was confirmed for at least 4 weeks at room temperature (Test Facility Study
No. 20274169. The test item diets remain in the food hopper for a maximum of one
week. On the day of weighing the remaining food in the food hopper, is
replaced with new diet. As such, diets may be prepared up to 3 weeks in advance of first use.

- Mixing appropriate amounts with (Type of food):
Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest,
Germany) will be used to prepare pelleted diets. The test item will be grinded using a mortar
and pestle and mixed with some powder feed (premix) in multiple mixing steps without the
use of a vehicle, and subsequently mixed with the bulk of the diet. Water (approximately
15% in total) will be added to aid pelleting. The pellets will be dried for approximately 24
hours at 35°C before storage. The control animals will receive similarly prepared pellets but
without the test item. The diets will be prepared under yellow light.
- Storage temperature of food: room temperature (protected from light)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet preparation samples (week1, week 6 and week 13) will be collected directly after preparation for analysis:
-all groups for concentration
-group 2 and 4 for homogeneity.
All samples are transferred (at room temperature) to the analytical laboratory at the Test Facility for same day analysis, where possible or stored for analysis (at 18-22 °C)within known formulation stability period.
Analyses are performed by using a validated analytical procedure (Test Facility Study No. 20274169).
Acceptance Criteria:
-For concentration: mean sample concentration results within or equal to +/- 20% of theoretical concentration.
-For homogeneity, relative standard deviation (RSD) of concentrations of +/- 10% for each group.
Backup Samples: Duplicate middle samples for Groups 1 and 3 (concentration analysis
only) and duplicate top, middle, and bottom samples for Groups 2 and 4
(concentration and homogeneity analysis) are taken on the same day. Samples are collected for analyses and stored at the Test Facility at ≤-15ºC for possible future analysis.

Results:
Accuracy
The concentrations analyzed in the diets of Groups 2-4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).
The analysis performed on Day 2 with the diets prepared for use in Week 1 was not accepted
because the QC samples were not prepared on the correct target levels. Therefore, new QC
samples were prepared and analyzed on Day 8 together with re-analysis of the Week 1 diet
samples, which were acceptable.
For the diets of Group 2 Females (570 ppm) prepared for use in Week 13, the mean accuracy
was above the target concentration (i.e. 148% of target). Therefore, reserve samples of
Group 2 Females prepared for use in Week 13 were analyzed after the end of in-life and these
results were within specification. It was therefore concluded that the diet was accurately
prepared and the initial results to be erroneous.
Samples of Group 1 prepared in Weeks 1, 6 and 13 had a comparable response with the QC
sample blanks response. Hence, it can be concluded that no test item was present in Group 1
diet samples.

Homogeneity
The diets of Groups 2 and 4 prepared for use in Weeks 1 and 6 were homogeneous (i.e.
coefficient of variation ≤ 10%). Furthermore, the diet of Group 4 Males (4960 ppm) prepared
for use in Week 13 was also homogenous. The diets of Group 2 Females (570 ppm) prepared
for use in Week 13 were not homogeneous (i.e. 27% of target). Therefore, reserve samples of
Group 2 Females prepared for use in Week 13 have been analyzed after the end of in-life and
these results were within specification (i.e. coefficient of variation ≤ 10%). It was therefore
concluded that the diet was accurately prepared and the original result was considered to be
erroneous.

Duration of treatment / exposure:

90 days

Frequency of treatment:

ad libitum access to food (dietary administration of the test item), except during designated procedures

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

'group 1'

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

'group2'
Actual Mean over Means Intake [mg test item/kg body weight/day] with (mean range indicated within brackets):
-Males: 41 (33-48)
-females: 42 (37-48)

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

'group 3'
Actual Mean over Means Intake [mg test item/kg body weight/day] with (mean range indicated within brackets):
-Males: 122 (108-141)
-females: 126 (109-142)

Dose / conc.:

320 mg/kg bw/day (nominal)

Remarks:

'group 4'
Actual Mean over Means Intake [mg test item/kg body weight/day] with (mean range indicated within brackets):
-Males: 287 (270-328)
-females: 319 (299-354)

No. of animals per sex per dose:

10 males + 10 females per dosing group, plus 3 male + 3 females for toxicokinetics assessment, plus two spare animals.
Total: 52 males + 52 females

Control animals:

yes

Details on study design:

- Dose selection rationale:
The 90-day study will be performed with a readily soluble silver salt to maximise read-across
possibilities. Silver acetate was selected as the representative silver salt as it was considered
the best option as a soluble silver compound. This selection is based on other sub-chronic
toxicity testing like Price et al (2002), Sprando et al (2017), van den Brule et al (2019). The
alternative candidate, Ag nitrate, has a higher corrosive potential. The EPMF is generating
comparative in vivo TK data for different forms of silver/silver salts and this work is
anticipated to confirm that AgAc can be considered the most bioavailable form of Ag.
The dose levels are based on the results of a 14 day dietary toxicity study in the rat (Test
Facility Study No. 20274197). In the 14 days study groups of 3 males and females were fed
diets containing 1680 ppm (low dose) or 4480 ppm (high dose). The target doses were 120
and 320 mg/kg bw/day respectively. Achieved doses were 150 and 170 mg/kg bw/day for
low dose males and females respectively and 378 and 426 mg/kg bw/day for high dose males
and females respectively. No effects were observed in the low dose group animals and this
has been used to represent the control for assessing effects in the high dose group animals. A
lower body weight gain was observed in high dose males; difference in body weight at
termination was -8.6%. Food consumption was also reduced in males of the high dose (-12%
overall). There were no effects in females. There were also no test item-related clinical
observations, macroscopic post mortem findings or effects on organ weights.
The target high dose of 320 mg/kg bw/day in the 14 day study is considered equally
appropriate for the high dose of the 90 day study. It is anticipated that there will be an effect
on body weight in males and possibly also in females with the extended duration of treatment
for this study. This dose level should ensure that an MTD is not exceeded if the body weights
continue to diverge from control during the course of the 90 day study and is expected to
achieve the requirements of OECD 408 for the highest dose level. The mid dose target level
recommended is 120 mg/kg bw/day, which aims to achieve at least 100 mg/kg bw/day and
this is important for classification and labelling for STOT-RE. It also fulfills the suggested
fold intervals in OECD 408. Based on the recommended fold interval above doses the low
dose suggested is 40 mg/kg/bw/day. Target doses will be achieved by adjusting the dietary
incorporation levels based on body weight and food consumption in each treated group.

- Rationale for animal assignment (if not random):
random assignment

- Fasting period before blood sampling for clinical biochemistry:
Yes (overnight with a maximum of 24 hours)

- Rationale for selecting satellite groups:
not applicable

-Veterinary Care:
Veterinary care will be available throughout the course of the study and animals will be
examined by the veterinary staff as warranted by clinical signs or other changes. In the event
that animals show signs of illness or distress, the responsible veterinarian may make initial
recommendations about treatment of the animal(s) and/or alteration of study procedures,
which must be approved by the Study Director. Treatment of the animal(s) for minor injuries
or ailments may be approved without prior consultation with the Sponsor representative when
such treatment does not impact fulfillment of the study objectives. If the condition of the
animal(s) warrants significant therapeutic intervention or alterations in study procedures, the
Study Monitor will be contacted, when possible, to discuss appropriate action. If the
condition of the animal(s) is such that emergency measures must be taken, the Study Director
and/or attending veterinarian will attempt to consult with the Study Monitor prior to
responding to the medical crisis, but the Study Director and/or veterinarian has authority to
act immediately at his/her discretion to alleviate suffering. The Sponsor representative will be
fully informed of any such events.

Positive control:

no positive controls

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (all main animals)
- Time schedule:
At least once daily from start of administration onwards, up to the day prior to necropsy.
Animals will be observed within their cage unless necessary for identification or confirmation of
possible findings.
For observations that cannot be attributed to an individual animal due to social housing (e.g., watery feces), the observation will be recorded to each animal in the socialized group.
No cage side observations reported since no major observations noted.

DETAILED CLINICAL OBSERVATIONS: Yes (all main animals)
- Time schedule: Weekly; from Week 1 and throughout the study, and on the day of necropsy.

BODY WEIGHT: Yes (all main and TK animals)
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study. In order to monitor the health status, animals may be weighed more often. (Fasted weight on the day of
necropsy.)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-All Main and TK animals
-Weekly; from at least Day 1 and throughout the study
- Quantitatively measured per cage
- Compound intake calculated based on nominal concentration of test item in diet against body weight and food consumption.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes (all main animals)
- Time schedule for examinations: on regular basis throughout the study. Water consumption is monitored by visual inspection of the water bottles. If inter group differences are noted, consumption is assessed by weight.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretreatment Period (all Main Study animals once (including spare animals) and Administration Period (All Group 1 and 4 Main Study animals during
Week 13. If treatment-related findings are noted, animals in Groups 2 and 3 will also be examined.)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment (all main animals), prior to necropsy
- Anaesthetic used for blood collection: Yes (Sampled prior to necropsy from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands).
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all main animals (4 groups x (10 males + 10 females))
- Parameters checked:
White Blood Cells (WBC)
Neutrophil (absolute)
Lymphocyte (absolute)
Monocyte (absolute)
Eosinophil (absolute)
Basophil (absolute)
Large unstained cells (LUC) (absolute)
Red Blood Cell
Reticulocyte (absolute)
Red Blood Cell Distribution Width (RDW)
Hemoglobin
Hematocrit
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet
A blood smear will be prepared from each hematology sample. Blood smears are labeled, stained, and stored. These smears will not be examined, but may be evaluated when required to confirm analyzer results.

CLINICAL CHEMISTRY & PLASMA/SERUM HORMONES/LIPIDS: Yes (all main animals)
- Time schedule for collection of blood: end of treatment (Sampled between 7.00 and 10.30 from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The
Netherlands)
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: all main animals (4 groups x (10 males + 10 females))
- Parameters checked
Alanine aminotransferase (ALT)
Triglycerides
Aspartate aminotransferase (AST)
HDL and LDL Cholesterol
Alkaline Phosphatase (ALP)
Sodium
Total protein
Potassium
Albumin
Chloride
Total Bilirubin
Calcium
Urea
Inorganic Phosphate (Inorg. Phos)
Creatinine
Triiodothyronine (T3)
Glucose
Thyroxine (T4)
Cholesterol
Thyroid-Stimulating Hormone (TSH)

After receipt of the serum for T3, T4 and TSH analysis it will be divided in two aliquots.
One aliquot will be used for measurement of thyroid hormones TSH using the IMMULITE®
1000 analyser. These aliquots for TSH will be stored in an ultra-low freezer (≤ -75°C) until
analysis. Any sample remaining after TSH analysis will be discarded. The other aliquot will
be used for measurement of T3 and T4 using LC-MS. The aliquots for T3 and T4 will be
collected in uniquely labelled clear 1.4 mL V-bottom Micronic polypropylene tubes and
stored in a freezer (≤ -15°C) until analysis. Measurement of T3 and T4 will be performed
according to the bioanalytical method validated in Test Facility Study No. 20213516. Any
samples remaining after the LC-MS analysis will be returned to storage for the retention
period.
The LC-MS analysis will be based on the following guidelines:
European Medicines Agency (EMA). Guideline on Bioanalytical Method Validation.
EMEA/CHMP/EWP/192217/2009, 01 February 2012.
Guidance for industry: Bioanalytical Method Validation, U.S. Department of Health and
Human Services, Food and Drug Administration, Center for Drug Evaluation and Research
(CDER) and Center for Veterinary Medicine (CVM), May 2018.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the Administration Period. All Main animals during Week 12-13. These tests are performed after clinical observations (including arena observation, if applicable).
- Dose groups that were examined: all main animals
- Battery of functions tested:
• hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength will be recorded as the mean of three measurements.
• locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations will be reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

IMMUNOLOGY: Yes
- Time schedule for examinations: end of treatment (Sampled between 7.00 and 10.30 from the
retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands)
- How many animals: all main animals (4 groups x (10 males + 10 females))
- Dose groups that were examined: all groups
- Parameters checked
On the day of sampling, peripheral blood mononuclear cells (PBMC) will be isolated from
blood samples by gradient density centrifugation using Ficoll Paque™ PREMIUM 1.084.
PBMCs will be counted with a Coulter Counter Z1 (Beckman Coulter Inc, Fullerton, United
States) and diluted to 1 x 106 cells/mL.
The following parameters will be determined in isolated PBMCs using a FACSCanto™ flow
cytometer system (BD Biosciences, Erembodegem, Belgium) according to a validated
method (Project 499468):
Parameter (Abbreviation) // Unit // Markers for identification
T-cells // % Lymphoid cells // CD3+/CD45RA-
T-helper cells // % Lymphoid cells // CD3+/CD4+/CD8-
T-cytotoxic cells // % Lymphoid cells // CD3+/CD4-/CD8+
B-cells // % Lymphoid cells // CD3+/CD45RA+
NK-cells // % Lymphoid cells // CD3-/CD161a+
Ratio T-helper cells/ T-cytotoxic cells (Th/Tc)

The % lymphoid cells of PBMC will be determined using the Forward Scatter and Side
Scatter.

OPHTHALMIC EXAMINATIONS
-Frequency: Pretreatment Period = All Main Study animals once (including spare animals), administration Period = All Group 1 and 4 Main Study animals during Week 13. If treatment-related findings are noted, animals in Groups 2 and 3 will also be examined.
-Procedure: The eyes will be examined using an ophthalmoscope after application of
a mydriatic agent (tropicamide 0.5%).

ESTROUS STAGE DETERMINATION
-Frequency: End of Treatment (on the day of necropsy) a vaginal smear will be taken to determine the stage of estrus from all Main Study animals. This will be done for all females, except for females that have to be euthanized in extremis or die spontaneously.
-Procedure: Estrous stage will be evaluated by examining the vaginal cytology of
the samples obtained by vaginal smears procedures.

COAGULATION
-all main animals
-end of treatment
-overnight fasting (with a maximum of 24 h)
-Sampled prior to necropsy from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands).
-Coagulation Parameters:
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)

BIOANALYSIS AND TOXICOKINETIC EVALUATION
-Bioanalytical Sample Collection from TK animals on day 1 and week 13
-group1 TK animals: between 2-3 am and between 8-9 am, group 2-3-4 TK animals: between 8-9pm, 2-3 am, 8-9 am and 2-3 pm
-0.3 mL sample from jugular vein, K2ADTA as anticoagulant, collection on ice.
-Whole blood samples will be collected and stored within 1 hour in a freezer set to maintain
-75°C. After transfer on dry ice to the bioanalytical laboratory the samples will be stored in a
freezer set to maintain -75°C until analysis.
-Whole blood will be analyzed for concentration of the total Silver (Ag) concentration using a
validated analytical procedure. Analysis will be performed under Analytical Procedure AP.20274188.ICP-R-WB. Statistical analyses including regression analysis and descriptive statistics including arithmetic means and standard deviations, accuracy and precision will be performed.
-Toxicokinetic Evaluation
Toxicokinetic parameters will be estimated using Phoenix pharmacokinetic software. A
non-compartmental approach consistent with the oral route of administration will be used for
parameter estimation. All parameters will be generated from Silver individual concentrations
in whole blood from Days 1 and Week 13 whenever practical.
-Parameters to be Estimated
Parameter // Description of Parameter
AUClast // The area under the concentration versus time curve from the start of dose administration to the last observed quantifiable concentration calculated using the linear trapezoidal method.
AUClast/Dose // The AUClast divided by the dose administered.
AUC(0-24h) // The area under the concentration versus time curve within a 24 hours’ time interval calculated using the linear trapezoidal method.
AUC(0-24h)/Dose // AUC(0-24h) divided by the dose administered.

Based on the data, additional secondary parameters may be determined, if appropriate.
Partial AUCs (between two defined sample times), and corresponding dose-normalized
values, may be derived and reported to aid interpretation. Descriptive statistics (e.g. number,
arithmetic mean, median, standard deviation, standard error, coefficient of variation) will be
reported as deemed appropriate. TK table and graphs will also be generated.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
artery, aorta; bone marrow, sternum; bone, sternum: brain; epididymis; esophagus; eye; adrenal gland; mammary gland; parathyroid gland; pituitary gland; prostate gland; submandibular salivary gland; sublingual salivary gland; seminal vesicle gland (including coagulation gland); thyroid gland; gut associated lymphoid tissue (Examine only if detectable in routine section of intestine); heart; kidney; large intestine, cecum; large intestine, colon; large intestine, rectum; liver; lung; mandibular lymph node; mesenteric lymph node; skeletal muscle; optic nerve (Examine only if detectable in the routine section of the eye); sciatic nerve; ovary; pancreas; skin; small intestine, duodenum; small intestine, ileum; small intestine, jejenum; spinal cord; spleen; stomach; testis; thymus; trachea; urinary bladder; uterus/cervix; vagina

HISTOPATHOLOGY: Yes
artery, aorta; body cavity, nasal; bone marrow, sternum; bone, femur; bone, sternum: brain; epididymis; esophagus; eye; adrenal gland; clitoral gland; harderian gland; lacrimal gland; mammary gland; parathyroid gland; pituitary gland; preputial gland; prostate gland; submandibular salivary gland; sublingual salivary gland; parotid salivary gland; seminal vesicle gland (including coagulation gland); thyroid gland; gut associated lymphoid tissue (Examine only if detectable in routine section of intestine); heart;, kidney; large intestine, cecum; large intestine, colon; large intestine, rectum: larynx; liver; lung; mandibular lymph node; mesenteric lymph node; skeletal muscle; optic nerve (Examine only if detectable in the routine section of the eye); sciatic nerve; tibial nerve; ovary; pancreas; skin; small intestine, duodenum; small intestine, ileum; small intestine, jejenum; spinal cord; spleen; stomach; testis; thymus; tongue; trachea; urinary bladder; uterus/cervix; vagina
Macroscopic abnormalities in the organs listed and in other organs will be sampled at necropsy, processed for histology and examined microscopically.
Tissues are embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Tissues are evaluated histopathologically by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

Optional endpoint(s):

Optional endpoints: No

Statistics:

-Constructed Variables:
Body Weight Gains: Calculated between each scheduled interval.
Food Consumption: Calculated between each scheduled interval.
Test Item Intake: Calculated based on nominal concentration of test item in diet against body weight and food consumption.
Organ Weight Relative to Body Weight: Calculated based on the terminal body weight.

-Descriptive Statistical Analyses
Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences are reported as appropriate by dataset.
-Inferential Statistical Methods
All statistical tests will be conducted at the 5% significance level. All pairwise comparisons are conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted.
-Parametric/Non-parametric
Levene’s test is used to assess the homogeneity of group variances.
The groups are compared using an overall one-way ANOVA F-test if Levene’s test is not
significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis
test is found to be significant, then pairwise comparison sare conducted using Dunnett’s
or Dunn’s test, respectively.
-Non-Parametric
The groups are compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons are conducted using Dunn’s test.
-ANCOVA
The data corresponding to a response variable of interest and to a related covariate will be
submitted to an analysis of covariance, including only groups with at least three
non-missing paired values and if found to be significant, then pairwise comparisons will be
conducted using Dunnett’s test.
[cfr. section 'any other information on materials and methods incl.tables' for continuation]

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted during daily detailed observations and no findings were noted during the arena observations in this study.
On Days 92 or 93 erected fur was noted in two males at 320 mg/kg body weight/day and in one female at 40 mg/kg body weight/day, which also showed hunched posture. Based on the incidental nature of these clinical signs, they were considered not toxicologically relevant.
The other clinical signs noted during the administration period were scabs, which occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose related trend. At the incidence observed, these were considered to be unrelated to the test item.

Mortality:

no mortality observed

Description (incidence):

No deaths occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
40 and 120 mg/kg body weight/day: slightly lower body weight and body weight gain between Days 1 and 36 (mean body weights 3.9% and 4.3% lower than control on Day 36 at 40 and 120 mg/kg body weight/day, respectively), which did recover thereafter.
320 mg/kg body weight/day: lower body weight and body weight gain throughout the administration period (mean body weight 14.9% lower and mean body weight gain 33% lower than control at the end of the administration period).

Females:
40 mg/kg body weight/day: similar body weight growth curve to those at 320 mg/kg body
weight/day during the initial part of the study. However, a -3.1% difference in mean body
weight was noted between females at 40 mg/kg body weight/day and the control group on
Day 1. When taking this difference on Day 1 into account, the maximum mean difference
between this group and the control group was only 3.3%, which is only minimal and
considered to be not toxicologically relevant.
120 mg/kg body weight/day: no clear effects on body weight and body weight gain
320 mg/kg body weight/day: slightly lower body weight gain seen starting
at Day 50 onwards (mean body weight up to 6.1% lower than control on Day 85
(not statistically significant) and a 14.5% lower mean body weight gain than control at the
end of the administration period).

cfr. Table in section 'Any other information on results incl. tables'

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males:
40 mg/kg/body weight/day: although food consumption was also slightly decreased between Days 8 and 50 (up to 7.0% lower than of control), this is considered not to be related to treatment with the test item in the absence of a similar finding at 120 mg/kg body weight/day.
120 mg/kg body weight/day: no effects on food consumption
320 mg/kg body weight/day: food consumption was decreased throughout the administration period with a maximum of 19.6% lower than of control between Days 57 and 64.

Females:
40 mg/kg body weight/day: food consumption decreased from Day 8 onwards, up to 10% lower than control between Days 22 and 29 and 85 and 91. These differences are considered not to be related to treatment in the absence of a similar finding at 120 mg/kg bw/day.
120 mg/kg body weight/day: no clear effects on food consumption
320 mg/kg body weight/day: food consumption was decreased on several occasions during the administration period (between Days 8 and 15, 36 and 57, 64 and 71 and 78 and 85). The maximum difference from the control group was reached between Days 64 and 71 (11.2% lower than control).

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

visual inspection of water consumption throughout the assay did not suggest any effect at any dosing level

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test item-related ophthalmology findings in Week 13.
The nature and incidence of ophthalmology findings noted during the Pre-treatment Period and in Week 13 was similar among the groups and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
120 and 320 mg/kg body weight/day: an increase in red blood cell distribution width (RDWG) and decrease in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) was observed. The MCV and MCHC changes did not, however, show a clear dose response. At 320 mg/kg body weight/day, hemoglobin concentration (HGB) was also decreased.

Females:
120 and 320 mg/kg body weight/day: an increase in eosinophil (EOS) count and a decrease in MCV (no dose response) and MCH was seen. Furthermore, an increase in RDWG was observed in females at 320 mg/kg body weight/day.

Other values in treated males and females achieving a level of statistical significance, when compared to controls, were considered to have arisen as a result of slightly high or low control values, occurred in the absence of a dose-related distribution and/or were, given the magnitude of change (e.g. MCV in females at 40mg/kg body weight/day), considered to be of no toxicological significance.

cfr Table 'Ratio Hematology Differences from Control Groups' in section 'Any other information on results incl. tables'

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
40 mg/kg/d: Increase in HDL cholesterol
120 mg/kg/d: increases in cholesterol (CHOL) and HDL cholesterol concentration, increase in calcium concentration (not statistically significant)
320 mg/kg/d: increase in alanine aminotransferase (ALT) activity and urea concentration, increases in cholesterol (CHOL) and HDL cholesterol concentration, increase in triglyceride (TRIG) concentration (not statistically significant), increase in calcium concentration (not statistically significant)

Females:
40 mg/kg/d: increase in alkaline phosphatase (ALP) activity
120 mg/kg/d: increase in alkaline phosphatase (ALP) activity, increases in cholesterol (CHOL) and HDL cholesterol concentration, increase in calcium concentration
320 mg/kg/d: increase in alkaline phosphatase (ALP) activity, increases in cholesterol (CHOL) and HDL cholesterol concentration, increase in triglyceride (TRIG) concentration (not statistically significant), increase in calcium concentration

Other remaining differences in clinical chemistry parameters, despite the statistical significance, were considered not to be test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and were of a magnitude of change commonly observed in rats under similar study conditions.

cfr Table 'Ratio Clinical Chemistry Differences from Control Groups' in section 'Any other information on results incl. tables'

Endocrine findings:

no effects observed

Description (incidence and severity):

No test item-related effects were seen on the thyroid hormones (T3, T4 and TSH).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between control and the test item groups.
All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

no effects observed

Description (incidence and severity):

There were no clear test item-related immunophenotype alterations.
Differences in immunophenotype results, despite the statistical significance, were considered not test item-related based on the absence of a dose response and the magnitude of change observed.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Differences in mean weights were noted for a few organs starting at 120 mg/kg body weight/day.

Males:
40 mg/kg/d: /
120 mg/kg/d: Slightly higher liver weight (relative to body weight only, no histological correlate)
320 mg/kg/d: Slightly higher liver weight (relative to body weight only, no histological correlate), lower pituitary gland weight (absolute and relative to body weight, no histological correlate), lower absolute epididymis weight (considered secondary to the lower body weight, no histological correlate in the testis or epididymis, the testis weight was not significantly different from the control
animals)

In males at 120 and/or 320 mg/kg body weight/day apparent higher brain, heart and testis weights were noted (statistically significant when expressed relative to body weight only). This was regarded secondary to lower terminal body weight in these groups (-5%, -15%, respectively). Absolute values of these three organs, were not statistically significant from the control animals.

Females:
40 mg/kg/d: /
120 mg/kg/d: /
320 mg/kg/d: Slightly higher liver weight (relative to body weight only, no histological correlate)

Cfr Table 'Mean percent organ weight differences from control groups' in section 'Any other information on results incl. tables'

Gross pathological findings:

no effects observed

Description (incidence and severity):

Dark gray discoloration was noted in all animals in all dose groups in the kidneys, pancreas, and Harderian glands and in most animals in all dose groups in the clitoral/preputial glands (including gray discoloration and gray foci), thymus, urinary bladder, liver, thyroid gland, stomach, small intestine (duodenum, jejunum, and/or ileum), large intestine (cecum, colon, and/or rectum).

More variable response was observed in other tissues including the mandibular salivary gland at all dose levels, spleen (males all dose levels, females starting at 120 mg/kg body weight/day), and the mandibular lymph node starting at 120 mg/kg body weight/day.

In males, a low incidence of dark gray discoloration was noted in the prostate at all dose levels, and the pituitary gland at 320 mg/kg body weight/day only. In females, dark gray discoloration was noted in the lacrimal gland and uterus at all dose levels, and in the skin starting at 120 mg/kg body weight/day. Dark gray discoloration was also noted in a few non-study plan tissues including several lymph nodes in males dosed at 320 mg/kg body weight/day, including axillary, bronchial, hepatic, iliac, inguinal, pancreatic, and popliteal lymph nodes, and the rib from a female dosed at 120 mg/kg body weight/day.

For most tissues, macroscopic gray discoloration correlated well with the presence of blackbrown pigment microscopically. However, for a few tissues, there was no microscopic correlate detected (thyroid gland, mandibular salivary gland, pituitary gland, prostate gland,
and uterus).

The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not specified

Description (incidence and severity):

cfr. gross pathology section

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related black-brown pigment was observed microscopically in all animals at all dose levels in one or more of the following organs: brain (area postrema and subfornical organ), gastrointestinal tract (stomach, small and large intestines), kidney, liver, lymph nodes (mesenteric, mandibular), pancreas, skin, thymus, urinary bladder, harderian gland, preputial/clitoral gland, and lacrimal gland. In the spleen, increased pigment was noted in males at 320 mg/kg body weight/day. In the spleen of females, increased extramedullary hematopoiesis was noted at 320 mg/kg body weight/day. Details of the incidence, severity, and histologic characterization are described by organ below.

Brain: In the brain, pigment (minimal or mild) was noted at all dose levels in the area postrema in all treated animals for which this structure was present in section, and in the subfornical organ of four animals (2 males at 40 mg/kg body weight/day, and one female each at 40 and 320 mg/kg body weight/day) for which this structure was present in section. Both of these structures are circumventricular organs and thus have a specialized capillary structure necessary for their function and are considered to be outside the blood brain barrier, which likely explains the selective presence of the pigment at these locations. Pigment in the area postrema was characterized by the presence of black-brown pigment in/around the blood vessel walls, highlighting these structures. In the subfornical organ, the pigment was less obvious, and was sparce and finely granular black-brown and not clearly associated with the interstitium/blood vessels.
These substructures are not required for evaluation of the brain and there is a normal and acceptable degree of variability in brain sections. These substructures were only available for several animals and noted under a separate organ in the data when present (Brain, area postrema; Brain, subfornical organ).

Gastrointestinal tract: Minimal to moderate pigment was present in at least one gastrointestinal segment (most often several) in all males and females at all dose levels.

Stomach: Minimal (and occasionally mild) pigment was present in the superficial lamina propria of the glandular mucosa and correlated with macroscopic dark gray discoloration.

Small intestine: Pigment was present in all segments of the small intestine with a fairly consistent gradation such that the duodenum was the most affected (mild to moderate), then the jejunum (minimal to mild), and lastly the ileum (minimal). A dose related increase in severity was noted for the duodenum.

Large intestine: Pigment was present in all segments of the large intestine with a fairly consistent gradation such that the cecum and colon were approximately equally affected (minimal and mild) and the rectum was the least affected (minimal). This was characterized by the presence of black-brown pigment in the lamina propria.

Kidney: Pigment was noted in the kidney of all animals at all dose levels (mild or moderate) and correlated with macroscopic dark gray discoloration. This was characterized by the presence of black-brown pigment in the interstitial spaces of the outer stripe of the outer medulla, strikingly highlighting this region. To a lesser degree, pigment was also noted in the cortical tubules as rare black brown granules present near the surface of the tubular epithelium, and less commonly, light brown pigment was also present diffusely in the glomerulus.

Liver: Pigment was noted in the liver (minimal to mild) of all animals at all dose levels and correlated with macroscopic dark gray discoloration with a dose-related increase in severity. This was characterized by the presence of finely granular black-brown pigment in the interstitium of the portal areas in and around the blood vessel walls, in the sinusoidal lining cells (mostly of the periportal/midzonal regions), and occasionally within hepatocytes.

Lymph nodes: Pigment was noted in the routinely examined lymph nodes (mesenteric and mandibular) of most animals at all dose levels (minimal to moderate). This was characterized by the presence of finely granular pigment in macrophages within the subcapsular and medullary sinuses. The macrophages containing the pigment were not notably enlarged nor,were the affected lymph nodes enlarged. The mesenteric lymph node (minimal to moderate) was consistently affected more than the mandibular lymph node (minimal to mild) and showed a dose related increase in severity.

Pancreas: Pigment was noted in the pancreas of all animals (minimal) and correlated with macroscopic dark gray discoloration. This was characterized by the presence of black, finely granular pigment present diffusely throughout the interstitium of the exocrine and endocrine pancreas.

Skin: Pigment (minimal) was noted in the skin in of males and females at all dose levels. The pigment was located in the dermis, specifically around the hair bulb, and this correlated with macroscopic dark gray discoloration noted in females. The incidence was somewhat variable in part because the detection depended on the presence of the hair bub in section.

Spleen: In most tissues pigment is not normally present in albino rats making the detection of even small amounts of the test item-related black-brown pigment possible in this study. In the spleen, however, hemosiderin pigment is normally present and the detection of the test item related black-brown pigment on this normal background of brown pigment was not possible. Thus, in the spleen, the diagnosis of pigment was graded on the overall amount, acknowledging it is not possible to clearly differentiate the two types (background pigment vs. test item-related pigment) in routinely stained (hematoxylin and eosin) sections.
In males, there was a slightly higher severity of spleen pigment (up to moderate degree) at 320 mg/kg body weight/day, compared to the control animals (minimal to mild). This could represent either increased amount of the typical pigment present (hemosiderin) or the addition of the test item-related pigment, thus there was no clear microscopic correlate to the gray discoloration noted macroscopically. Minor variation in the amount of extramedullary hematopoiesis was not clearly test item-related and may represent normal biologic variability.
The amount of pigment noted histologically in the spleen of females was comparable between control and treated animals and there was no correlate to the gray discoloration noted macroscopically.

Thymus: Pigment (minimal) was noted in the thymus of most animals at all dose levels. This was characterized by a small amount of finely granular black-brown pigment most often detected in the capsule and correlated with macroscopic dark gray discoloration.

Urinary bladder: Pigment (minimal) was noted in the urinary bladder of all animals at all dose levels. This was characterized by a small amount of finely granular black-brown pigment just below the epithelium in the region of the epithelial basement membrane/superficial lamina propria and correlated with macroscopic dark gray discoloration.

Non-Study plan tissues (examined due to macroscopic observations):
Preputial/Clitoral glands: Pigment (minimal or mild) was present in all dose groups in nearly all animals for which it was examined and correlated with macroscopic dark gray discoloration. This was characterized finely granular black-brown pigment diffusely in the interstitial tissues.

Harderian gland: Pigment was noted in the Harderian gland of all animals histologically (minimal or mild) and correlated with macroscopic dark gray discoloration. This was characterized finely granular black-brown pigment diffusely in the interstitial tissues.

Lacrimal glands: Pigment (minimal) was noted in the lacrimal glands of females at all dose levels for which this tissue was examined and correlated with macroscopic dark gray discoloration. This was characterized finely granular black-brown pigment diffusely in the interstitial tissues.

Lymph nodes: Several non-study plan lymph nodes were examined from the males at 320 mg/kg body weight/day (collected because of macroscopic dark gray discoloration - axillary, bronchial, hepatic, iliac, inguinal, pancreatic, and popliteal) and females at 320 mg/kg body weight/day (popliteal) which also correlated with microscopic pigment, similar to the mandibular and mesenteric lymph nodes described above.

Remaining test item-related findings:
Spleen: In the spleen of females at 320 mg/kg body weight/day a slightly higher incidence and severity of extramedullary hematopoiesis was observed compared to the control females, up to mild degree. In males, the extramedullary hematopoiesis was comparable in control and test item-treated animals.

There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings or were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no neoplastic, test item-related histologic changes. Changes were considered to be incidental findings or were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of these.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Coagulation:
There were no test item-related findings in males at 40 mg/kg body weight/day and females at
40, 120 and 320 mg/kg body weight/day.
Prothrombin time (PT) was slightly shortened in males at 120 and 320 mg/kg body weight/day (0.95 and 0.94x of control respectively).

Details on results:

Toxicokinetic Evaluations:
The exposure to Silver increased in a less than dose-proportional manner for males and females on Day 1 and in Week 13. On Day 1, an increase in target dose from 40 to 320 mg/kg body weight resulted in a 0.2/0.2-fold increase in AUClast/Dose (expected ratio of 1.0), for males/females respectively. In Week 13, an increase in target dose from 40 to 320 mg/kg body weight/day resulted in a 0.4/0.3-fold increase in AUC(0-24h)/Dose (expected ratio of 1.0), for males/females respectively.
At all dose levels, (slightly) higher AUClast/Dose values were observed in Week 13 compared to Day 1. Therefore, it can be concluded that slight accumulation of Silver seemed to occur after administration with Silver acetate. This effect was most pronounced at a target dose level of 320 mg/kg body weight/day.
On Day 1, a slight trend towards higher exposure levels in females compared to males was observed, whereas in Week 13, no clear sex differences were noted for AUClast/Dose at all dose levels.
The whole blood concentrations of Silver in the control animals were all below the LLOQ, confirming the absence of exposure to silver acetate in control animals.
cfr. detailed values in table 'Summary of Silver Toxicokinetic Parameters in Male and Female Wistar Han Rat Whole Blood Following Dietary Administration of Silver Acetate on Day 1 and in Week 13' in section 'Any other information on results incl. tables'

Dose descriptor:

NOAEL

Effect level:

>= 320 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Critical effects observed:

no

Table: summary of body weight evolution of male and female animals (in g)

Male	Mean body weight (day1)	Mean body weight (day91)	% difference compared to control
0 mg/kg bw/day	203.1 (SD 13.6, N=10)	413.9 (SD 37.4, N=10)	-
40 mg/kg bw/day	207.9 (SD 19.6, N=10)	400.3 (SD 19.6, N=10)	-3.3%
120 mg/kg bw/day	212.6 (SD 7.6, N=10)	396.7 (SD 27.2, N=10)	-4.2%
320 mg/kg bw/day	211.0 (SD 7.8, N=10)	352.3 (SD 27.3, N=10)**	-14.9%
Female
0 mg/kg bw/day	151.2 (SD 9.1, N=10)	241.1 (SD 19.1, N=10)	-
40 mg/kg bw/day	146.5 (SD 10.4, N=10)	229.1 (SD 17.1, N=10)	-5.0%
120 mg/kg bw/day	153.9 (SD 9.0, N=10)	240.9 (SD 20.5, N=10)	-0.1%
320 mg/kg bw/day	149.9 (SD 10.6, N=10)	226.8 (SD 16.9, N=10)	-5.9%
Anova & Dunnett: ** = p ≤ 0.01

 

Table: Ratio Hematology Differences from Control Groups (Numerical values indicate fold change of the treated group mean value relative to the control group mean)
Dose	40 mg/kg bw/day		120 mg/kg bw/day		320 mg/kg bw/day
Sex (M(ale), F(emale))	M	F	M	F	M	F
eosinophil (EOS)	-	-	-	1.6*	-	1.8**
red blood cell distribution width (RDWG)	-	-	1.08*	-	1.11**	1.14**
mean corpuscular volume (MCV)	-	0.97**	0.93**	0.95**	0.93**	0.95**
mean corpuscular hemoglobin (MCH)	-	-	0.91**	0.95**	0.91**	0.94**
mean corpuscular hemoglobin concentration (MCHC)	-	-	0.98*	-	0.97*	-
hemoglobin concentration (HGB)	-	-	-	-	0.95**	-
Legend: - = no test item effect, *p<=0.05, **p<=0.01

 

Table: Ratio Clinical Chemistry Differences from Control Groups (Numerical values indicate fold change of the treated group mean value relative to the control group mean)
Dose	40 mg/kg bw/day		120 mg/kg bw/day		320 mg/kg bw/day
Sex (M(ale), F(emale))	M	F	M	F	M	F
alanine aminotransferase (ALT) activity	-	-	-	-	1.49**	-
alkaline phosphatase (ALP) activity	-	1.45*	-	1.75**	-	1.84**
urea concentration	-	-	-	-	1.34**	-
cholesterol (CHOL) concentration	-	-	1.46**	1.39*	1.68**	2.03**
HDL cholesterol concentration	1.22*	-	1.49**	1.42**	1.82**	2.15**
triglyceride (TRIG) concentration	-	-	-	-	1.32	1.36
calcium concentration	-	-	1.05	1.07*	1.07	1.05*
Legend: - = no test item effect, *p<=0.05, **p<=0.01

 

Table: Mean Percent Organ Weight Differences from Control Groups
Dose	40 mg/kg bw/day		120 mg/kg bw/day		320 mg/kg bw/day
Sex (M(ale), F(emale))	M	F	M	F	M	F
Terminal Body Weight	-3	-5	-4	0	-15	-6
PITUITARY GLAND
absolute	-9	-9	-7	-6	-24**	-11
relative to body weight	-5	-4	-2	-6	-10*	-5
LIVER
absolute	-4	-5	0	4	-1	8
relative to body weight	-1	0	6*	4	16**	14**
EPIDIDYMIDES
absolute	-5	/	-5	/	-14**	/
relative to body weight	-2	/	-1	/	1	/
Legend: *p<=0.05, **p<=0.01

 

Summary of Coagulation values
Male	PT (sec)	APTT (sec)
0 mg/kg bw/day	17.43 (SD 0.31, N=10)	21.63 (SD 0.77, N=10)
40 mg/kg bw/day	16.93 (SD 0.47, N=10)	21.81 (SD 1.31, N=10)
120 mg/kg bw/day	16.50 (SD 0.56, N=10)**	21.86 (SD 0.69, N=10)
320 mg/kg bw/day	16.41 (SD 0.83, N=10)**	20.46 (SD 1.80, N=10)
Female
0 mg/kg bw/day	16.76 (SD 0.94, N=10)	18.56 (SD 1.29, N=10)
40 mg/kg bw/day	15.99 (SD 0.63, N=10)	17.82 (SD 1.97, N=10)
120 mg/kg bw/day	15.97 (SD 0.29, N=10)	18.73 (SD 1.57, N=10)
320 mg/kg bw/day	16.04 (SD 0.40, N=10)	19.06 (SD 1.53, N=10)
Kruskal-Wallis & Dunn: ** = p ≤ 0.01

 

Summary of Silver Toxicokinetic Parameters in Male and Female Wistar Han Rat Whole Blood Following Dietary Administration of Silver Acetate on Day 1 and in Week 13

Parameters	Target dose level (mg/kg bw/day)
	40		120		320
Sex	M	F	M	F	M	F
Day 1	N=3	N=3	N=3	N=3	N=3	N=3
Average actual test item intake (mg/kg bw/day)	48	48	149	144	337	373
t(last) in h	18	18	18	18	18	18
AUC(last) in h*ng/mL	7610	11100	11000	13300	9830	17300
AUC(last)/Dose in h*kg*ng/mL/mg	158	231	74.1	92.2	29.2	46.4
Week 13	N=3	N=3	N=3	N=3	N=3	N=3
Average actual test item intake (mg/kg bw/day)	38	38	113	112	279	308
t(last) in h	18	18	18	18	18	18
AUC(last) in h*ng/mL	10400	11000	11800	14200	34100	28200
AUC(last)/Dose in h*kg*ng/mL/mg	274	291	105	127	122	91.5
AUC(0-24h) in h*ng/mL	13800	14700	15700	18900	45100	38200
AUC(0-24h)/Dose in h*kg*ng/mL/mg	362	387	139	169	162	124

Conclusions:

The administration of Silver acetate by dietary inclusion was well tolerated in
Wistar Han rats at target dose levels up to 320 mg/kg body weight/day in a 90-day study according to OECD 408. Non-adverse test item-related macroscopic gray discoloration and microscopic pigment were observed in several tissues and organs of all animals starting at 40 mg/kg body weight/day, which was regarded to be due to accumulation of silver. The only other histopathology finding was a non-adverse minor increase in incidence and severity of extramedullary hematopoiesis noted in the spleen of females at 320 mg/kg body weight/day. Finally, minimal and non-adverse changes in clinical pathology parameters were noted mainly at 120 and 320 mg/kg body weight/day.
At 320 mg/kg body weight/day, lower body weight gain and lower food consumption were observed in males and females, which were considered to be adverse in males only.
Based on these results, the No-Observed-Adverse-Effect level (NOAEL) was considered to be at a target dose level of 120 mg/kg body weight/day for males and a target dose level of at least 320 mg/kg body weight/day for females.

Executive summary:

A GLP-compliant study was performed to determine the potential toxicity of Silver acetate, when given via diet for 90 days to the Wistar Han rat (according to OECD 408).

Target dose levels for groups 1 to 4 were 0, 40, 120 and 320 mg silver acetate/kg bodyweight/day, respectively. The target dose level was reached for Group 2 and 3 animals and Group 4 females. Group 4 males had an actual mean dose level of 287 mg/kg body weight/day as a consequence of a dose related reduction in food consumption/palatability for Group 4 males. For the identification of Group 4 males in this report the target dose level (320 mg/kg body weight/day) is used.

Chemical analyses of dietary preparations were conducted in Weeks 1, 6 and 13 to assess accuracy and homogeneity. Dietary analyses confirmed that formulations of test item in diets were prepared accurately and homogeneously.

 

The following parameters and endpoints were evaluated in this study: mortality, clinical signs, functional observation tests, body weights, food consumption, ophthalmology, estrus stage determination, clinical pathology parameters (hematology, coagulation, clinical chemistry, thyroid hormones and immunophenotyping), toxicokinetic parameters, gross necropsy findings, organ weights, and histopathologic examinations.

 

At 40 mg/kg body weight/day, a non-adverse test item-related increase in alkaline phosphatase activity (ALP) in females and HDL cholesterol concentration in males was noted.

 

At 120 mg/kg body weight/day, non-adverse test item-related changes in clinical pathology included increased eosinophil count in females, increased red blood cell distribution width in males, along with a decreased mean corpuscular volume and mean corpuscular hemoglobin in both sexes. In addition, mean corpuscular hemoglobin concentration was decreased in males only. Moreover, slightly shortened prothrombin time (PT) was observed in males. Furthermore, increased ALP activity in females and increased (HDL) cholesterol and calcium concentrations in males and females were noted. At necropsy, a test item-related higher relative liver weight was observed in males, which was without microscopic correlate and therefore considered to be non-adverse.

 

At 320 mg/kg body weight/day, a test item-related lower body weight and body weight gain was seen in males throughout the administration period. In females, a slightly lower body weight and/or body weight gain was seen starting at Day 50 onwards. The food consumption in males was decreased throughout the administration period, while in females it was decreased during six out of thirteen weeks. The effects on food consumption at 320 mg/kg body weight/day and consequently on body weight are at the severity observed considered to be adverse for males and not adverse for females. At clinical pathology, non-adverse changes included increased eosinophils (females), increased red blood cell distribution width (both sexes) and decreased mean corpuscular volume (both sexes), mean corpuscular hemoglobin (both sexes), mean corpuscular hemoglobin concentration (males) and hemoglobulin concentration (males). In addition, a slightly shortened PT was observed in males. Clinical chemistry analysis revealed increased alanine aminotransferase activity and urea concentration in males, ALP activity in females and (HDL) cholesterol, triglyceride and calcium concentrations in males and females. Test item-related non-adverse organ weight differences consisted of slightly higher liver weight in males and females and slightly lower pituitary gland and epididymis weight in males, which were without a microscopic correlate. Microscopic evaluation showed a slightly higher incidence and severity of extramedullary hematopoiesis in the spleen in females compared to the control females. At the severity observed, this finding was considered to be not adverse.

 

At necropsy, test item-related non-adverse macroscopic gray discoloration was present in all animals at all dose levels. Affected organs included: kidneys, harderian gland, clitoral or preputial gland, thymus, urinary bladder, liver, thyroid gland, stomach, small and large intestines, mesenteric and mandibular lymph nodes, mandibular salivary gland, skin, lacrimal gland, pituitary gland, uterus, and prostate gland. Test item-related microscopic black-brown pigment (ranging from minimal to moderate degree) was present in tissues from all animals at all dose levels. Affected organs included the brain (area postrema and subfornical organ), kidney, liver, lymph nodes (mesenteric, mandibular), pancreas, skin, stomach, small and large intestines, thymus, urinary bladder, harderian gland, preputial/clitoral gland, and lacrimal gland. In the spleen at 320 mg/kg body weight/day increased pigment was noted in males. The presence of the pigment microscopically was caused by the silver accumulation and was not associated with any other tissue alterations and was therefore considered to be a non-adverse change.

 

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical signs, functional observation, ophthalmoscopy, thyroid hormones and immunophenotype parameters).

 

There was no evidence of exposure to Silver in control animals. All Silver acetate-treated animals were exposed to Silver confirming administration and absorption. Exposure to Silver, expressed as AUC/Dose, increased less than dose proportionally over the used target dose accumulation of Silver seemed to occur after administration with Silver Acetate. This effect was most pronounced at a target dose level of 320 mg/kg body weight/day. On Day 1, a slight trend towards higher exposure levels in females compared to males was observed, whereas in Week 13, no clear sex differences in exposure were observed during the study.

 

In conclusion, administration of Silver acetate by dietary inclusion was well tolerated in Wistar Han rats at target dose levels up to 320 mg/kg body weight/day. Non-adverse test item-related macroscopic gray discoloration and microscopic pigment were observed in several tissues and organs of all animals starting at 40 mg/kg body weight/day, which was regarded to be due to accumulation of silver. The only other histopathology finding was a non-adverse minor increase in incidence and severity of extramedullary hematopoiesis noted in the spleen of females at 320 mg/kg body weight/day. Finally, minimal and non-adverse changes in clinical pathology parameters were noted mainly at 120 and 320 mg/kg body weight/day.

At 320 mg/kg body weight/day, lower body weight gain and lower food consumption were observed in males and females, which were considered to be adverse in males only.

Based on these results, the No-Observed-Adverse-Effect level (NOAEL) was considered to be at a target dose level of 120 mg/kg body weight/day for males and a target dose level of at least 320 mg/kg body weight/day for females.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

The aim of this study was to investigate the differential accumulation, distribution, and potential toxicity of silver in rats following daily oral exposure to silver acetate (AgAc) for 13 weeks

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Test item was purchased 99% pure as a single lot from Gelest, Inc. (Morrison, Pennsylvania), and batches of stock aqueous solutions (nominal concentration 10.4 mg/mL) were prepared in 18 megaohm and autoclave-sterilized water on an "as needed" or bi-weekly basis and stored at room temperature. Samples were submitted to inductively coupled mass spectrometry (ICP-MS) analysis to determine the actual silver mass concentrations. The test item solutions remained clear and colorless throughout the useful life of the solution of 2 weeks, indicating that the silver did not precipitate or become reduced.

Characterization of test item solutions:
Test material characterisation was conducted weekly at the National Center for Toxicology Research (NCTR) on freshly prepared test item solutions.

The samples were diluted to 50 mL with and acid mixture of 1 N each of nitric and hydrochloric acids.
For the stability study, total silver concentrations were determined by ICP-MS, initially for the test item stock suspensions and filtrates, and subsequently for filtrates collected from the same stock suspensions after storage in the dark at 4C–8C for 1 and 90 days. Homogeneity testing was conducted on test item stock suspensions from the same shipment as the stability study; however, triplicate subsamples were collected from the top, middle, and bottom of each vessel and analyzed in triplicate.

The average total silver concentrations by mass of the test item samples were determined with a Thermo Scientific WSERIES 2 Quadrupole ICP-MS (Franklin, Massachusetts), using 107Ag, 109Ag, and 103Rhodium (Rh) at 50 ng/ml and 115Indium (In) at 100 ng/ml as internal standards.

The concentration of Ag was quantified against an external calibration curve (NIST traceable silver samples). The limit of quantification (LOQ) for the test item suspensions and filtrates was estimated to be 40 ng/ml.

The identity of the test item was confirmed by 1H nuclear magnetic resonance spectroscopy on an AVANCE III spectrometer equipped with a BBFO Plus Smart Probe (Bruker Instruments, Billerica, Massachusetts) operating at 500 MHz. The samples were dissolved in D2O and the acquisition was conducted at room temperature using a standard 1H acquisition sequence.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- 3-week-old male and female Sprague Dawley/CD-23 rats with specific pathogen-free health status were obtained from the NCTR breeding colony.
- At 6 week of age, the rats were weight-ranked and randomly assigned to treatment groups. Male and female rat were housed conventionally in separate animal rooms with 2 animals per cage.
- The environment of the animal rooms was set to maintain a 12h light cycle, temperature of 22+/-4°C, relative humidity of 40%-70%, and air changes of 10-15 per hour.
- The animals were provided NIH-41 gamma-irradiated pellets and Millipore-filtered drinking water ad libitum.
- Rats were dosed initially at 7 weeks of age.

Route of administration:

oral: gavage

Details on route of administration:

Gavage dosing was conducted using computer-controlled MicroLab 500 series dispensers (Hamilton Co., Reno, Nevada) equipped with gastight syringues and capable of dispensing 1µL to 50mL.
Syringue were fitted with flexible plastic gavage needles, and the rats were provided equal volume doses based on the daily body weight of the individual rats.
The MicroLab dispersion were programmed to administer the total daily dose in 2 daily gavage administrations per day, with half of the dose administered just prior to start of the dark cycle.


Route Exposure: Ingestion is the primary route of exposure in humans for silver, silver compounds, and colloidal silver (Silver, 2003). Daily intakes for silver in humans are estimated in the range 0.4–27 mg/day (Clemente et al., 1977; Gibson and Scythes, 1984; Hamilton and Minski, 1972/1973). The Environmental Protection Agency lists the reference dose for silver as 0.5 mg/kg/day or 35 mg/day for an average 70 kg adult (EPA, 2014).

Vehicle:

methylcellulose

Remarks:

water/0.1%methyl cellulose. Methylcellulose was used in this study as bulking agents to inhibit somewhat the gastrointestinal passage of the mostly aqueous dose formulation. The compound is not toxic and do not promote allergic reactions in rodents.

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared weekly and were based on the total silver mass concentrations obtained by ICP-MS for silver acetate solution. The high-dose formulation of test item (10 mg AgAc/mL or approximately 6.46 mg Ag/mL) was prepared in water/0.1% methylcellulose (final concentration wt/wt).

- The silver concentration of the high-dose formulation were confirmed by ICP-MS and, then serially diluted in water/0.1% methylcellulose to achieve the 3.23 and 1.62 mg Ag/mL concentrations for the mid- and low-dose AgAc formulation respectivelly.

DOSE CHARACTERIZATION: The total silver concentration by mass of each of the prepared dose formulations (high-, mid-, and lowdose) and the controls ( water/MC) was determined by ICP-MS in acid- and microwave-digested samples. Characterization analyses were conducted on triplicate samples. Dose concentration acceptability was set at 610% targeted values.

VEHICLE
Methylcellulose was purchased from Fischer Scientific (Fair Lawn, New Jersey). The water used in dose formulations was 18 megaohm and autoclave-sterilized.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The total silver concentration by mass of each of the prepared dose formulations (high-, mid-, and lowdose) and the controls (water/MC) was determined by ICP-MS in acid- and microwave-digested samples.

The AgAc solutions remained clear and colorless throughout the useful life of the solution of about 2 weeks, indicating that the silver did not precipitate or become reduced.

Duration of treatment / exposure:

Groups of rats (10 males and 10 females) were exposed daily by oral gavage to dose formulation of AgAc at 100, 200 and 400 mg/kg bw or to the respective control formulations (Water/MC) for a period of 13 weeks.

Frequency of treatment:

Gavage dosing was conducted using computer-controlled MicroLabVR 500 series dispensers (Hamilton Co., Reno, Nevada) equipped with gastight syringes and capable of dispensing 1 ml to 50 ml. The syringes were fitted with flexible plastic gavage needles, and the rats were provided equal volume doses based on the daily body weight of the individual rats. The MicroLab dispensers were programmed to administer the total daily dose in 2 daily gavage administrations per day, with half of the dose administered at the start of the light cycle and half of the dose administered just prior to start of the dark cycle. The dose volumes did not exceed 20 ml/kg bw. Animals were dosed 7 days each week and the study period was 13 weeks.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In studies with mice, Horner et al. (1983) determined that the oral lethal dose, 90% (LD90) of AgAc was 2505 mg/kg bw. In studies with rats to evaluate AgAc for developmental toxicity, the lowest observed adverse effect level by the oral
route was 30 mg/kg bw/day AgAc, and the no observed adverse effect level for development toxicity was 100 mg/kg bw/day (NTP, 2002). Based on the wide range of doses in toxicity tests a low dose of 100mg AgAc/kg bw/day (64.6mg Ag/kg bw/day) was selected.

- Fasting period before blood sampling for clinical biochemistry: terminal sacrifices were conducted on over-night fasted rats.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly clinical observations on individual animals were recorded in the animal records database (NCTR Multi-generation Computer Support System; MGSS).

BODY WEIGHT: Yes
- Time schedule for examinations: body weights recorded daily in MGSS for dose administration.

FOOD CONSUMPTION AND COMPOUND INTAKE: Feed and water consumptions were measured and recorded weekly in MGSS.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at terminal and moribund sacrifices
- Anaesthetic used for blood collection: Yes: Animals were euthanized humanely by carbon dioxide asphyxiation after the 72h blood sample collection.
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at terminal and moribund sacrifices
- Animals fasted: Yes

IMMUNOLOGY: A section of the ileum and scrapping of the ileal mucosa were collected for intestinal microbiota and immune response evaluations (Williams et al., 2015).

Sacrifice and pathology:

At necropsy, organs and tissues were examined for grossly visible lesions, and protocol designated tissues and organs were weighed and preserved in 10% neutral buffered formalin (NBF), with the exception that modified Davidson’s fixative was used for the right testes and eyes. Femur bone marrow was collected for histopathology (left) and ICP-MS (right). The testes and epididymides were collected for sperm analysis (left) and histopathology (right).

Statistics:

A Cox proportional hazard model was used to analyze survival data and to test the effect of treatment relative to control. All tests were conducted as 2-sided with significance at the .05 probability level.
Pharmacokinetic parameters were determined through the use of PK Solutions 2.0 software (Summit Research Services, Montrose, Colorado). The parameters included maximum observed concentration (Cmax), the area-under-the concentration- versus-time curve (AUC), absorption/distribution half-life (t1=2 A/D), and elimination half-life (t1=2 E). The AUC was estimated to the last sampling time using the trapezoidal rule and further extrapolated to infinity (AUC0-1).

Body weights, feed consumption, water intake, and blood were analyzed using a 1-way repeated measures, mixed model ANOVA for each sex, with terms for dose, week, and all interactions.
Week was treated as the repeated measure. For body weight analyses, the last body weight obtained for each dose week was used as the weekly body weight. Within-group correlations
were modeled using a heterogenous first-order autoregressive correlation structure, and pairwise comparisons of the 2 control groups and each of the treatment groups to the appropriate control group were performed with Bonferroni adjustments.

The statistical analysis of hematology and clinical chemistry data was performed using a non-parametric method with midranks (Brunner et al., 2002). Pairwise comparisons were performed with Bonferroni adjustments. All tests were conducted as 2-sided with significance at the .05 probability level.

The organ weights were analyzed using a 1-way analysis of covariance with necropsy body weights as a covariate. Pairwise comparisons were performed with Bonferroni adjustments.

Further information on statistical analysis under section "Any other information on materials and methods incl. tables"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings suggested severe gastrointestinal symptoms, loss of body weight and unthrifty appearance among these animals, likely due to the bactericidal activity of the silver ion on the intestinal microbiota.

Mortality:

mortality observed, treatment-related

Description (incidence):

Morbidity was pronounced in groups of rats exposed to the high (400 mg/kg bw/day) dose with a majority (70%) of female and (100%) male rats being removed prior to the scheduled terminal sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly lower mean body weights were observed in female rats administered test item at doses of 100 and 400 mg/kg bw/day; the overall mean body weights were 88.5% and 74.4% of the control group, respectively.
Male rats administered 400 and 200 mg/kg bw of test item demonstrated significantly lower mean body weights than the controls, beginning at week 1 and at week 3, respectively.
Male rats in low dose (100 mg/kg bw/day ; body weights in this group were not significantly affected.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences in feed and water intakes between the vehicle and water controls. when compared with respective control animals, sporadic differences in feed and water intakes were observed in female and male rats administered test item.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology and plasma clinical chemistry parameters recorded for rats exposed to test item are provided in the table under section " Any other information on results incl. tables"
Sporadic differences in the hematological and biochemisty parameters were found for silver acetate-treated groups when compared with their controls.
MCV (mean cells volumes) were significantly lower for mid and high dose treated female rats and for test item low and mid dose treatd male rats. Red Blood Cell (RBC) counts were significantly higher (p< 0.05 vs control) for the test item low and mid dose treated female rats and for the low dose male rats.
The significantly lower MCV values and high RBC counts by test item treated rats are suggestive of low iron status, however, minerals other than silver were not evaluated in blood samples.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematology and plasma clinical chemistry parameters recorded for rats exposed to test item are provided in the table under section " Any other information on results incl. tables"

Endocrine findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The effects of the test item on the absolute organ weights and relative organ weights (%; organ weight/necropsy body weight). Details of data presented in the table under section "Any other information on results, incl tables".
In male rats treated with test item (100 mg/kg bw/d), the absolute kidney weights were depressed (1.34g) and the relative weights were higher for the left epididymis (0.14%) and heart (0.36%); the relative liver weights was higher in the 100 and 200 mg/kg bw group (2.8% and 2.94% respectively).
In female rats exposed at 200 mg/kg bw/d, the absolute weights for the left kidney (0.85g) and thymus (0.22g) were lower when compared with controls (0.97 and 0.32 respectively).
At the high dose of test item (400 mg/kg bw/d), the absolute heart (0.88g) and thymus (0.13g) weights were lower when compared with the controls (1.09 and 0.32g respectively). The relative organ weights for liver were higher than control weights (2.55%) in the 200 mg/kg bw/d dose group (3.39%). Since the necropsy body weights were significantly lower in these same groups of rats, the significant changes in absolute and relative organ weights likely reflect mild dehydration due to the presence of gastrointestinal distress in these animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Non-neoplastic lesions were confined primarly to rats administered test item. with the exception of an increased dose-response in the incidence of histiocutic infiltrates in the lungs of female and male groups, there were no meaningful treatment-related differences from controls in the 100 and 200 mg/kg bw/d groups.

Increase incidences and severities of lesions were detected in rats exposed to highest dose of test item, and included mucosal hyperplasia in the small and large intestine, as well as thymic atrophy or necrosis - a stress-response to the gastrointestinal distrurbances experienced by these animals.

Dose descriptor:

LOAEL

Effect level:

<= 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Effect level:

>= 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Critical effects observed:

no

 Characterization of test item solutions and dose formulation:

The results of the characterisation for each of the individual lots of test item solutions. Stock of aqueous solution of test item (nominal concentration 10.4 mg/mL) were prepared in 18 megaohm sterilized water on a bi-weekly basis. The total silver and ioniic silver concentrations of the test materials were determined by ICP-MS, value presented the mean +/- SD of 22 shipments of 16 batch preparations of test item. 

The average total silver concentration of test item solution was 6.73 +/- 0.048 mg/mL and approximated the solubility (10.4 mg/mL) of test item in water; silver acetate is composed of 64.6% silver by weight.

Dose formulations were prepared on a weekly basis at 3 concetrations levels for silver acetate (1.62, 3.23 and 6.46 mg Ag/mL) and the total silver concetration of each formulation was determined by ICP-MS prior to insuance of the dose formulation to animals on the study.

 

Ratio of determined total silver concentration and targeted silver concentration:

	Target dose concetration (mg/mL)	Total silver concentration (mg/mL) +/- SD	Percentage of targeted dose +/- SD	Particle hydrodynamic size (nm) +/- SD
Silver acetate	1,62	1,684 (±0,05)	104 (±3,02)	225,14 (±14,45)
	3,23	3,327 (± 0,07)	103,20 (± 2,12)	225,77 (± 14,94)
	6,46	6,657 (±0,15)	103,10 (± 2,30)	226,03 (± 7,15)

 

Hematology and Clinical Chemistry of Sprague Dawley rats in the 13-weeks study of silver acetate:

	Female rats				Male rats
	H2O/MC	100 mg/kg bw/d	200 mg/kg bw/d	400 mg/kg bw/d	H2O/MC	100 mg/kg bw/d	200 mg/kg bw/d	400 mg/kg bw/d
Basophil (%)	0.2 ±0.0	0.4 ±0.1	0.2 ±0.0	0.2 ±0.1	0.2 ±0.0	0.2 ±0.0	0.2 ±0.0	n.a.
Eosinophil (%)	1.2 ±0.1	0.9 ±0.1	1.6 ±0.2	1.2 ±0.1	1.5 ±0.2	1.5 ±0.2	2.1 ±0.3	n.a.
Lymphocyte (%)	79.0 ± 1.0	80.3 ± 2.2	79.5 ± 1.5	75.7 ± 2.1	75.7 ± 0.9	76.9 ± 1.6	77.1 ± 2.5	n.a.
Monocyte (%)	5.1 ± 0.7	4.6 ± 0.6	5.2 ± 0.8	4.8 ± 1.3	6.8 ± 0.6	8.3 ± 0.5	6.8 ± 1.0	n.a.
Neutrophil (%)	14.5 ± 0.7	13.8 ± 1.8	13.6 ± 1.4	18.1 ± 1.9	15.8 ± 0.9	13.2 ± 1.2	13.9 ± 1.5	n.a.
Basophils (10^3/mL)	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	n.a.
Eosinophils (10^3/mL)	0.1 ± 0.0	0.1 ± 0.0	0.2 ± 0.0	0.1 ± 0.0	0.2 ± 0.0	0.2 ± 0.0	0.2 ± 0.0	n.a.
Lymphocytes (10^3/mL)	6.1 ± 0.5	7.3 ± 0.5	7.1 ± 0.7	7.3 ± 1.4	8.2 ± 0.7	8.4 ± 0.7	6.7 ± 0.6	n.a.
Monocytes (10^3/mL)	0.4 ± 0.1	0.4 ± 0.0	0.5 ± 0.1	0.5 ± 0.2	0.7 ± 0.1	0.9 ± 0.1	0.6 ± 0.1	n.a.
Neutrophils (10^3/mL)	1.1 ± 0.2	1.3 ± 0.2	1.2 ± 0.1	1.7 ± 0.4	1.7 ± 0.1	1.5 ± 0.2	1.2 ± 0.2	n.a.
Hematocrit (%)	45.5 ± 0.5	46.8 ± 0.5	44.9 ± 0.5	39.6 ± 1.5	47.3 ± 0.4	46.2 ± 0.5	44.9 ± 0.8	n.a.
MCHC (g/dL)	35.1 ± 0.2	35.1 ± 0.1	35.0 ± 0.2	33.4 ± 0.3	34.5 ± 0.2	34.0 ± 0.1	33.8 ± 0.2	n.a.
MCV (mm3)	55.2 ± 0.4	53.9 ± 0.6	51.6 ± 0.5	46.3 ± 0.9	51.3 ± 0.6	47.0 ± 0.3	46.6 ± 0.5	n.a.
PCV (mm3)	45.6 ± 0.5	46.8 ± 0.5	45.0 ± 0.5	39.7 ± 1.6	46.9 ± 0.4	46.1 ± 0.5	44.8 ± 0.8	n.a.
PLT (10^3/mL)	737 ± 34	764 ± 28	839 ± 37	775 ± 184	715 ± 20	805 ± 28	824 ± 31	n.a.
RBC (10^3/mL)	8.2 ± 0.1	8.7 ± 0.1	8.7 ± 0.1	8.6 ± 0.5	9.2 ± 0.1	9.8 ± 0.1	9.6 ± 0.2	n.a.
WBC (10^3/mL)	7.8 ± 0.7	9.1 ± 0.6	9.0 ± 0.9	9.7 ± 2.0	10.8 ± 0.8	11.0 ± 1.0	8.7 ± 0.8	n.a.
Albumin (g/dL)	4.2 ± 0.1	4.0 ± 0.1	4.1 ± 0.1	3.5 ± 0.0	3.6 ± 0.1	3.6 ± 0.1	3.6 ± 0.1	n.a.
Alkalin phosphatase (U/L)	57.4 ± 5.5	85.6 ± 6.1	106 ± 8	120 ± 28	100 ± 4	154 ±10	157 ± 13	n.a.
Alanine transferase (U/L)	33.1 ± 2.5	35.0 ± 2.3	41.8 ± 2.7	55.0 ± 8.3	35.8 ± 1.1	40.8 ± 2.0	45.7 ± 3.1	n.a.
Blood Urea nitrogen (mg/dL)	17.1 ± 0.6	19.9 ± 0.8	19.2 ± 0.3	20.7 ± 1.8	16.0 ± 0.6	16.9 ± 0.6	18.2 ± 1.0	n.a.
Cholesterol (mg/dL)	100 ± 6	122 ± 7	136 ± 5	118 ± 4	73.7 ± 5.1	110 ± 6.5	102 ± 5	n.a.
Creatine kinase (U/L)	190 ± 36	200 ± 28	212 ± 25	338 ± 146	201 ± 48	240 ± 46	232 ± 34	n.a.
Creatinine (mg/dL)	0.5 ± 0.0	0.4 ± 0.0	0.5 ± 0.0	0.3 ± 0.0	0.4 ± 0.0	0.3 ± 0.0	0.4 ± 0.0	n.a.
Glucose (mg/dL)	130 ± 6.2	115 ± 3.1	102 ± 3.6	136 ± 27	138 ± 4.6	127 ± 7.4	112 ± 4.1	n.a.
Sorbitol dehydrogenase (U/L)	8.1 ± 3.0	6.7 ± 2.4	8.8 ± 2.7	7.6 ± 6.9	7.5 ± 1.7	9.9 ± 1.7	9.4 ± 2.5	n.a.
Total bile acids (mmol/L)	42.4 ± 5.6	68.3 ± 13.1	76.2 ± 7.6	123 ± 37	39.1 ± 4.5	54.7 ± 9.0	92.6 ± 8.6	n.a.
Total protein (g/dL)	7.6 ± 0.2	7.0 ± 0.1	7.0 ± 0.1	6.0 ± 0.2	6.5 ± 0.1	6.3 ± 0.1	6.2 ± 0.1	n.a.
Triglycerides (mg/dL)	88.4 ± 10.6	70.3 ± 7.0	109 ± 14	54.0 ± 9.3	98 ± 8.7	103 ± 14	91.3 ± 9.6	n.a.

Absolute and relative organ weights of rats treated orally by daily gavage for 13-weeks with silver acetate

female	H2O/CM	100 mg/kg bw/day	200 mg/ kg bw/day	400 mg/kg bw/day
	Mean± SE	Mean± SE	Mean± SE	Mean± SE
Necropsy BW	312.9.6 ± 9.6	270.3 ±  4.3	269 ±  4.9	238 ±  5.9
Heart absolute	1.091 ±  0	1.034 ±  0	0.99 ±  0	0.877 ±  0
Heart relative	0.349 ±  0	0.383 ± 0	0.37 ±  0	0.367 ±  0
Kidney, left absolute	0.967 ±  0	0.979 ±  0	0.85 ±  0	0.9 ± 0
Kidney, left relative (%)	0.31 ± 0	0.325 ± 0	0.32 ± 0	0.377 ± 0
Kidney, right absolute	0.964 ± 0	0.884 ± 0	0.88 ± 0	0.887 ± 0
Kidsney, right relative	0.309 ± 0	0.327 ± 0	0.33 ± 0	0.371 ± 0
Liver absolute	7.855 ± 0.3	6.892 ± 0.2	7.15 ± 0.1	8.126 ± 0.9
Liver relative (%)	2.515 ± 0	2.548 ± 0	2.66 ± 0	3.387 ± 0.3
Lung absolute	1.309 ± 0	1.256 ± 0	1.25 ± 0	1.311 ± 0
Lung relative (%)	0.421 ± 0	0.466 ± 0	0.47 ± 0	0.55 ± 0
Ovary, left absolute	0.05 ± 0	0.046 ± 0	0.05 ± 0	0.045 ± 0
Ovary, left relative (%)	0.016 ± 0	0.017 ± 0	0.02 ± 0	0.019 ± 0
Ovary, right absolute	0.047 ± 0	0.039 ± 0	0.05 ± 0	0.041 ± 0
Ovary, right relative (%)	0.015 ± 0	0.015 ± 0	0.02 ± 0	0.017± 0
Thymus absolute	0.319 ± 0	0.259 ± 0	0.22 ± 0	0.131 ± 0
Thymus relative (%)	0.101 ± 0	0.096 ± 0	0.08 ± 0	0.055 ± 0
male	H2O/CM	100 mg/kg bw/day	200 mg/ kg bw/day	400 mg/kg bw/day
	Mean± SE	Mean± SE	Mean± SE	Mean± SE
Necropsy BW	530± 17.3	514.5 ±  18.8	466.6 ±  8.8	n.a.
Epididymis left absolute	0.59 ± 0	0.672 ± 0	0.060 ± 0	n.a.
Epididymis left relative	0.11 ± 0	0.144 ± 0	0.145 ± 0	n.a.
Epididymis right, absolute	0.6 ± 0	0.609 ± 0	0.607 ± 0	n.a.
Epididymis right, relative	0.12 ± 0	0.131 ± 0	0.146 ± 0	n.a.
Heart absolute	1.59 ± 0	1.663 ± 0.1	1.148 ± 0	n.a.
Heart relative	0.3± 0	0.356 ± 0	0.355± 0	n.a.
Kidney, left absolute	1.52 ± 0.1	1.337 ± 0.1	1.224 ± 0	n.a.
Kidney, left relative (%)	0.29 ± 0	0.287 ± 0	0.295 ± 0	n.a.
Kidney, right absolute	1.53 ± 0	1.381 ± 0.1	1.212± 0	n.a.
Kidsney, right relative	0.29 ± 0	0.296 ± 0	0.291± 0	n.a.
Liver absolute	13.3 ± 0.5	13.08 ± 0.5	12.24 ± 0.4	n.a.
Liver relative (%)	2.51 ± 0	2.799± 0.1	2.939± 0.1	n.a.
Lung absolute	1.74± 0	1.771± 0.1	1.529± 0.1	n.a.
Lung relative (%)	0.33± 0	0.381± 0	0.368 ± 0	n.a.
Testes, left absolute	1.75± 0.1	1.881 ± 0.1	1.749 ± 0.1	n.a.
Testes, left relative	0.33 ± 0	0.404 ± 0	0.421 ± 0	n.a.
Testes right absolute	1.68 ± 0.1	1.788 ± 0.1	1.76 ± 0	n.a.
Tested right relative	0.32 ± 0	0.385 ± 0	0.424 ± 0	n.a.
Thymus absolute	0.27 ± 0	0.277 ± 0	0.198 ± 0	n.a.
Thymus relative (%)	0.05 ± 0	0.059 ± 0	0.048 ± 0	n.a.

 

		Water/CM	100 mg/kg bw/d	200 mg/kg bw/d	400 mg/kg bw/d
Female	Blood Wk-1 (ng/mL)	53 ± 28*	1171 ± 91**	1631 ±188**	9047 ± 1883**
	blood Wk-2 (ng/mL)	18 ± 4*	792 ± 95**	1753 ± 212**	6960 ±
	Bone Marrow, femur (ng/mL)	3 ± 0*	800 ± 168**	3973 ± 1314**	13433 ± 2385**
	Heart (ng/g)	76 ± 69*	375244 ± 310684**	116150 ± 18153**	189000 ± 5686**
	Ileum (ng/g)	33 ± 21*	259000 ± 53550**	262000 ± 34697**	588667 ± 61167**
	Jejunum (ng/g)	12 ± 5*	336333 ± 52145**	574900 ± 49119**	1416667 ± 133708**
	Kidney (ng/g)	14 ± 9*	1481444 ± 205799**	1895000 ± 202640**	25256667 ± 188886**
	Liver lateral (ng/g)	4 ± 1*	759556 ± 69202***	1377600 ± 159674**	843667 ± 132640**
	Lymph node, mes. (ng/g)	10 ± 3*	830444 ± 107536**	2119000 ± 237821**	5556667 ± 788973**
	Colon (ng/g)	9 ± 2*	793778 ± 61053**	1175800 ± 87121**	117800 ± 232975**
	Spleen (ng/g)	24 ± 19*	560111 ± 85092**	2373000 ± 247557**	2843333 ± 101708**
	Uterus (ng/g)	67 ± 37*	56844 ± 6010**	76350 ± 5098**	126100 ± 40771**
Male		Water/MC	100mg/kg bw/d	200 mg/kg bw/d	400 mg/kg bw/d**
	Blood Wk-1 (ng/mL)	27  ± 8*	1121  ± 151**	2482  ± 546**	12270  ± 1846**
	blood Wk-2 (ng/mL)	34  ± 15*	595  ± 45**	1618  ± 227**	n.a.
	Bone Marrow, femur (ng/mL)	3  ± 0*	1020  ± 430**	6302  ± 1189**	n.a.
	Heart (ng/g)	2   ± 0*	58500  ± 5696**	77330  ± 8890**	n.a.
	Ileum (ng/g)	19  ± 10*	228920  ± 28619**	357100  ± 46308**	n.a.
	Jejunum (ng/g)	12  ± 5*	423800  ± 43898**	929100  ± 189193**	n.a.
	Kidney (ng/g)	8  ± 3*	440000  ± 60950**	768000  ± 96142**	n.a.
	Liver lateral (ng/g)	3  ± 0*	472620  ± 66058**	813300  ± 115528**	n.a.
	Lymph node, mes. (ng/g)	7  ± 0*	549200  ± 45321**	1661400  ± 215854**	n.a.
	Colon (ng/g)	18  ± 7*	310100  ± 44888**	658100  ± 52337**	n.a.
	Spleen (ng/g)	6  ± 1*	601000  ± 138471**	1207700  ± 214244**	n.a.

The limit of quantification for endpoint of interest is as follows:

blood: 20 ng/mL

bone marrow: 5ng/mL

heart: 4 ng/g

ileum and jejunum: 13 ng/g

kidney: 10 g/g

liver: 5ng/g

mesenteric lymph nodes and colon: 13g/g

spleen: 10 ng/g

uterus: 22ng/g

* In the control groups, signifies significant linear dose trend effects (P< 0.05) based on Bonferoni adjustments.

**Signifies values thar are significantly different (P<0.05) from the control group by Bonferoni adjustment

Disposition and accumulation of silver:

Tissue and organs from animals exposed to AgAc for 13 weeksn with exception of bone marrow, revealed statistically significant (p<0.05) dose-dependent increases in silver concentrations.

The silver content in blood and bone marriw averages 3-4 times lower than silver content of the heart, which had the lowest content of silver among the major organs analysed and indicated that the contribution of silver accumulation in the blood and bone marrow was minimal.

Blood levels of silver were not significantly higher after 12 weeks of dosing than levels found after only 1 week of dosing, these results suggest rapid clearance of silver from blood for all groups irrespective of silver form.
 
In preliminary studie, the half-time for elimination of silver from the blood was 24h regardless of sex of form of silver.

Silver concentrations were significantly higher in the blood and bone marrow of rats exposed to AgOAc when compared to AgNP. Results indicate that a much higher uptake of silver occurred following the administration of AgOAc, than could be accounted for by the differences in silver content of the dose.

In female and male rats exposed to AgAc, the kidney and spleen represented sites of significant silver accumulation. As observed in the intestinal tract, sex differences in silver concentrations were observed. In addition, the pattern of distribution in the kidneys and spleens of male rats differed from that of female rats.

The concentrations of silver in all organs appeared to be higher in females than males. The accumulation and pattern of silver distribution was similar in the kidney and spleen of female and male rats administered AgAc.

Localisation of silver in rat tissue

In rats exposed to AgAc (100 mg/kg bw), the accumulation of silver granules was detected primarily along the epithelial basement membrane of the jejunum ileum  and colon. The surface morphology of the accumulated electron dense silver granules in the intestine of rats exposed to AgAc was irregular and segmented. Mean diameters were larger for granules accumulated in the intestinal tract of rats exposed to AgAc.

The accumulation of silver was observed along the basement membranes of the glomeruli of kidneys from rats exposed to AgAc similarly found the accumulation of silver granules primarily along the basement membranes of the glomeruli in kidneys from rats exposed to AgAc.Those deposited in the kidneys of AgAc-exposed rats were large and irregular in shape, often with surface bulges.

AgAc appeared to have an affinity for extracellular membranes.

EDS was used to determine the compositional analysis of the granules within rat tissues. Strong energy signals were observed for silver, and the presence of selenium, sulfur, and chloride was noted in association with the silver
granules for AgAc.
Signals corresponding to copper and lead were also identified. These originated from the grids that were used in the visualization procedures; an osmium signal was observed and originated from the tissue fixation procedures. Whereas, only signals for lead, copper and osmonium were detected by EDS in tissues of the vehicle control and water control animals.

Conclusions:

In conclusion, male and female rats exposed to silver acetate showed significant effects on morbidity, mean body weight, organ weight, hematology and blood chemistry effect with the concentration tested 100, 200 and 400 mg/kg bw/d.

Executive summary:

In this study, Sprague Dawley rats of seven-week-old rats (10 rats per sex per group) were randomly assigned to treatments silver acetate (AgAc) at 100, 200, and 400 mg/kg bw; and controls (water). At termination, complete necropsies were conducted, histopathology, hematology, serum chemistry, micronuclei, and
reproductive system analyses were performed, and silver accumulations and distributions were determined.

Rats exposed to silver acetate at high dose (400 mg/kg bw/d) presented high morbidity with 70% of female and 100% of male rats being removed prior to the scheduled terminal sacrificed. Clinical findings suggested severe gastrointestinal symptoms, loss of body weight and unthrifty appearance among these animals, likely due to the bactericidal activity of silver ion on the intestinal microbiota.

 Significant lower mean body weight were observed in female rats administered AgAc of 100 and 400 mg/kg bw/d; the overall mean body weights were 88.5% and 74.4% of the control groups. Male rats administered with 400 and 200 mg/kg bw/d deminstrated significantly lower mean body weight than the controls, beginning at week 1 and week 3, respectively. body weight of male rats administered 100 mg/kg bw/d were not significantly affected. At the highest dose, the absolute heart and thymys weight were lower when compared with controls.

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Publication of a GLP guideline study reliable with restrictions Deviations from the guideline OECD 413 (1981): - no explicit statement is made in the publication if the actual concentration of the test substance was held as constant as practicable. No raw data of the actual concentration was presented, no indication was made if exposure started after a test atmosphere equilibration period. However, supporting publications by Jung, 2006 and Ji, 2007 specifically address and confirm the generation of a stable test atmosphere for this study in the same laboratory. - the temperature and humidity values are reported for the acclimatisation period only, but not explicitly for the test period - there was no indication that the animals were observed during the 6-hour exposure period. - animals were not observed every day, but on weekdays only - apparently, the animals were ophthalmologically examined, but it was not described when the examination took place and if all animals were examined. It seemed that at least the high dose group was examined, since it was stated that one animal from the high dose died during this examination, but no further information could be found regarding this examination. - it was not explicitly mentioned, if a full gross necropsy was performed. - baseline data was not given for haematological and biochemistry tests

Justification for type of information:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

, adopted 1981-05-12

Deviations:

yes

Remarks:

, please refer to "Rationale for reliability incl. deficiencies" above

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS - specific-pathogen-free
- Source: SLC (Tokyo, Japan)(originally derived from the Charles River SD in 1968)
- Age at study initiation: 8 week-old rats
- Weight at study initiation: males: approximately 253 g; females: approximately 162 g
- Fasting period before study: not stated
- Animals were not provided with food during the 6-hour exposure period.
- Housing: during the acclimation and experimental periods, the rats were housed in polycarbonate cages (five rats per cage).
- The animals were housed in individual wire cages during the exposure period.
- Diet (ad libitum, except during exposure): a rodent diet (Harlan Teklab, Plaster International Co., Seoul, South Korea)
- Water (ad libitum): filtered water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2°C
- Humidity: 55 ± 7%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: no data, please refer to "Any other information on materials and methods incl. tables" below.

Details on inhalation exposure:

Silver nanoparticles were generated as described in previous reports (Ji et al., 2007a,b; Jung et al., 2006)*.

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the rats were exposed to the silver nanoparticles in a whole-body-type exposure chamber (1.3 m^3, Dusturbo, Seoul, South Korea).

- System of generating particulates/aerosols: the generation consisted of a small ceramic heater connected to an alternating current power supply and housed within a quartz tube case. The heater dimensions were 50 X 5 X 1.5 mm^3, and a surface temperature of about 1500°C within local heating area of 5 X 10 mm^2 could be achieved within about 10 s. For long-term testing, the source material (about 160 mg) was positioned at the highest temperature point. The quartz tube case was 70 mm in diameter and 140 mm long. Clean (dry and filtered) air was used as the carrier gas, and the gas flow maintained at 30.0 L/min (Re = 572, laminar flow regime) using a mass flow controller (MFC, AERA, FC-7810CD-4V, Japan). This generator has been shown to generate nanoparticles from 2 to 65 nm in diameter which do not agglomerate in air. X-ray diffraction analysis using an X-ray diffractometer utilizing CuK2 radiation showed that particles generated are metallic silver, not silver oxide.
The system produced different concentrations of nanoparticles (high, middle, and low) in three separate chambers. For the high-concentration chamber, the nanoparticle generator was operated at 47 L/min and mixed with 200 L/min of clean ambient air. A portion of the high nanoparticle concentration was then diverted to the middle-concentration chamber using the MFC for the first dilution (6.76 ± 0.16 liter per minute (Lpm), mean ± SE), and a portion of the middle nanoparticle concentration then diverted to the low-concentration chamber using the second MFC (5.42 ± 0.18 Lpm).

- Method of particle size/particle concentration determination: the nanoparticle distribution with respect to size was measured directly in real-time using a differential mobility analysing system (DMAS); combining a differential mobility analyser (Short type-DMA, 4220, HCT Co., Ltd, Korea; range 5 - 150 nm) and condensation particle counter (CPC, 4312, HCT Co., Ltd, 0 - 10^8/cm^3 detection range). Nanoparticles were measured using sheath air at 5 L/min and polydispersed aerosol air at 1 L/min for DMA and CPC, respectively. The particle concentration in the fresh-air control chamber was measured using a particle sensor (4123, HCT Co., Ltd) that consisted of two channel; 300 - 1000 nm and over 1000 nm.
The filters used to sample the fume particles were coated with carbon, mounted on an electron microscope grid (200 mesh, Veco, Eerbeek, the Netherlands), and visualized under a transmission electron microscope (TEM, Hitachi 7100, Japan). The diameters of 800 randomly selected particles were measured at a magnification of 100,000, and the silver particles analysed using an energy-dispersive x-ray analyzer (EDX-200, Horiba, Japan) at an accelerating voltage of 75 kV.

* References:
Ji, J.H., Jung, J. H., Yu, I. J., and Kim, S.S. (2007a). Long-term stability characteristics of metal nanoparticle generator using small ceramic heater for inhalation toxicity studies. Inhal. Toxicol. 19, 745 - 51.
Ji, J. H., Jung, J. H., Kim, S. S., Yoon, J. U., Park, J. D., Choi, B. S., Chung, Y. H., Kwon, I. H., Jeong, J., Han, B.S., et al. (2007b). Twenty-eight days inhalation toxicity study of silver nanoparticles in Sprague-Dawley Rats. Inhal. Toxicol. 19, 857 - 871.
Jung, H. H., Oh, H. C., Noh, H.S., Ji, J.H., and Kim, S.S. (2006). Metal nanoparticle generation using a small-sized ceramic heater with a local heating area. J. Aerosol Sci. 37, 1662 - 1670.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Please refer to "Details on inhalation exposure" above.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
0 µg/m^3
Basis:
other: control

Remarks:

Doses / Concentrations:
49 µg/m^3
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
133 µg/m^3
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
515 µg/m^3
Basis:
analytical conc.

No. of animals per sex per dose:

10 males/10 females

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: as there is no current data on workplace air concentrations of silver nanoparticles or silver nanoparticle concentrations released from consumer products, the concentrations used in this study are difficult to relate human exposures. Concentrations used in this study were based on the current ACGIH silver dust threshold limit value (TLV) of 0.1 mg/m3 (ACGIH, 2001)*. As such, the low, middle, and high doses were 1/2, 1, and 5 times the ACGIH silver dust TLV, respectively, in terms of mass dose. In addition, the high dose used in this study is nearly 500-fold higher than the ACGIH silver dust TLV in terms of surface area.

Reference:
American Conference of Governmental Industrial Hygienists (ACGIH). (2001). Documentation of the TLVs and BEIs. ACGIH, Cincinnati, OH.

Positive control:

not used

Observations and examinations performed and frequency:

ANIMAL OBSERVATIONS: Yes
- Time schedule: daily on weekdays
- Observations checked: any evidence of exposure-related effects, including respiratory, dermal, behavioural, nasal, or genitourinary changes suggestive of irritation.

BODY WEIGHT: Yes
- Time schedule for examinations: at purchase, at the time of grouping, once a week during the inhalation exposure and before necropsy.

FOOD CONSUMPTION: Yes
- Time schedule: weekly

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: no data
- Dose groups that were examined: not explicitly stated, but it is assumed that all groups were checked

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 13-week experiment (before necropsy); blood was drawn from the abdominal aorta and collected in heparinized vacutainers.
- Anaesthetic used for blood collection: Yes; anesthetized with pentobarbital
- Animals fasted: Yes, food was withheld for 24 hours
- How many animals: all
- Parameters checked: white blood cell count, red blood cell count, haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red-cell distribution width, platelet count, mean platelet volume, number of neutrophils, percent of neutrophils, number of lymphocytes, percent of lymphocytes, number of monocytes, percent of monocytes, number of eosinophils, percent of eosinophils, number of basophils, and percent of basophils

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the 13-week experiment (before necropsy); blood was drawn from the abdominal aorta and collected in heparinized vacutainers.
- Anaesthetic used for blood collection: Yes; anesthetized with pentobarbital
- Animals fasted: Yes, food was withheld for 24 hours
- How many animals: all
- Parameters checked: albumin, alkaline phosphatase, calcium, cholesterol, creatinine, gamma-glutamyl transpeptidase, glucose, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, inorganic phosphorus, lactate dehydrogenase, magnesium, total protein, uric acid, blood urea nitrogen, total bilirubin, creatine phosphokinase, sodium, potassium, chloride, triglyceride, and ratio albumin to globulin

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

ORGAN WEIGHTS AND HISTOPATHOLOGY:
After collecting the blood, the rats were sacrificed by cervical dislocation, and the adrenal glands, bladder, testes, ovaries, uterus, epididymis, seminal vesicle, heart, thymus, thyroid gland, trachea, oesophagus, tongue, prostate, lungs, nasal cavity, kidneys, spleen, liver, pancreas, and brain all removed carefully. Organs were weighed and fixed in a 10% formalin solution containing neutral phosphate-buffered saline. The organs were embedded in paraffin, stained with hematoxylin and eosin. All organs from all animals were examined under light microscopy.

Other examinations:

DETERMINATION OF TISSUE SILVER:
After wet digestion using a flameless method, the tissue concentrations of silver were analysed using an atomic absorption spectrophotometer equipped with a Zeeman graphite furnace (Perkin Elmer 5100ZL, Zeeman Furnace Module, Waltham, MA) based on the NIOSH 7300 method (NIOSH (1999), NIOSH Manual of Analytical Methods. Method No. 7300, 7604. National Institute for Occupational Safety and Health, Cincinnati, OH).

Statistics:

All the results were expressed as the means ± SE. An ANOVA test and Duncan's multiple range tests were used to compare the body weights, bronchoalveolar lavage cell distributions, and parameters from the lung function test obtained for the three dose groups with those obtained for the control rats. Histopathological results were analysed by Chi-squared analysis. Level of significance was set at p < 0.05 and p < 0.01.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
No gross effects were observed during the 90-day exposure period. One animal from the high-dose group died during the opthalmological examination.

BODY WEIGHT AND WEIGHT GAIN
There were no significant changes in body weights of male rats. Although female rats showed a significant body weight difference between high and middle dose groups, there were no significant dose-related changes.

FOOD CONSUMPTION
No significant differences were observed in food consumption between the exposed rats and the control groups.

HAEMATOLOGY
There were no significant dose-related differences in the haematology values among groups.
To evaluate aggregation of red blood cells or blood coagulation attributable to silver nanoparticles, erythrocyte aggregation, activated partial thromboplastin time, and prothrombin time were tested. Only the percent of aggregation in the high-dose females showed a statistically significant difference compared with the controls.

CLINICAL CHEMISTRY
There were no significant dose-related differences in the blood biochemical parameters.

ORGAN WEIGHTS
No significant organ weight changes were observed in either the male or female rats after the 90 days of silver nanoparticle exposure.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver:
- minimal bile-duct hyperplasia was identified in 0/10, 0/10, 1/10, and 4/9 of the control, low, middle, and high dose males, respectively.
- one high-dose male had minimal bile-duct hyperplasia with minimal portal mineralization.
- the higher incidence of bile-duct hyperplasia in the high dose males, with or without mineralization suggested a minimal test article-related effect.
- minimal bile-duct hyperplasia was present in 3/10, 2/10, 4/10, and 8/10 of the control, low, middle, and high dose females, respectively.
- single-cell hepatocellular necrosis, characterised by increased cellular eosinophilia and shrunken condensed nucleo, was noted in 3/10 high dose females.
- one high dose female exhibited moderate bile-duct hyperplasia with concurrent moderate centrilobular fibrosis, minimal single-cell hepatocyte necrosis, mild pigment accumulation, and moderate multifocal necrosis.
- the higher incidence of bile-duct hyperplasia, with or without necrosis, fibrosis, and/or pigmentation, in high dose females suggested a test-article-related effecte, which was slightly mor obvious than in the males.
Please also refer to "Any other information on results incl. tables" below (Table 1 and 2).

Lung:
- examination revealed a high incidence of minimal alterations, including some chronic alveolar inflammation, a mixed cell perivascular infiltrate, and alveolar macrophage accumulation in high dose male and female animals when compared with the controls.
Please also refer to "Any other information on results incl. tables" below (Table 1 and 2).

Nasal pathways:
- no histopathologic findings in the nasal pathways were considered test article related.

Kidneys:
- incidence of minimal tubular basophilia was noted in all the groups, including the controls, and thus not considered to be test article exposure related.
- tubular basophilia was more prevalent in males compared with the females.
- although tubular dilatation, cast formation, mineralization, and inflammation were noted occasionally in the control and/or treated animals, they were considered to be within the range of expected background spontaneous change.

Heart:
- minimal degeneration/necrosis was observed in all the groups, including the controls, indicating that this alteration was not test article related.
- the change was more obvious in the males.
- the finding is a common spontaneous background change.

Harderian gland/prostate
- inflammation was noted occasionally, which is a common background spontaneous finding and thus not considered to be test article related.

OTHER FINDINGS
Silver distribution in tissue:
- silver concentration in lung tissue from groups exposed were statistically significant (p < 0.01) and increased with dose.
- a clear dose -dependent increase in silver concentration in the blood for both genders.
- 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).
- silver concentration in the kidneys showed a gender difference with the female kidneys containing two to three times more silver acculmulation than in male kidneys.
- because gender difference in silver accumulation was noted in kidneys, the kidney function was measured based on the N-acetylglutamate and protein in urine. No significant difference was noted among the dose groups and between genders, except for an increase of protein in the urine from the high-dose male rats (high-dose group: 2.57± 0.13 g/g creatinine; control group: 1.89 ± 0.11 g/g creatinine; p < 0.05).
Please also refer to "Any other information on results incl. tables" below (Table 3 and 4).

Dose descriptor:

NOAEC

Effect level:

133 other: µg/m^3

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

LOAEC

Effect level:

515 other: µg/m^3

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Histopathology

Table 1: Histopathological findings for male rats

Group				Control		Low		Middle		High
Number of animals				10		10		10		9
				N	%	N	%	N	%	N	%
Liver	No microscopic findings			10/10	100	10/10	100	9/10	90	5/9	55.6
	Abnormality*			0/10	0	0/10	0	1/10	10	4/9	44.4
	Necrosis	Multifocal	Minimum	0/10	0	0/10	0	0/10	0	1/9	11.1
	Hyperplasia*	Bile duct	Minimum	0/10	0	0/10	0	1/10	10	4/9	44.4
	Vacuolation	Hepatocellular	Minimum	0/10	0	0/10	0	0/10	0	1/9	11.1
	Mineralization	Portal	Minimum	0/10	0	0/10	0	0/10	0	1/9	11.1
Lungs	No microscopic findings			5/10	50	3/10	30	3/10	30	0/9	0
	Abnormality			5/10	50	7/10	70	7/10	70	9/9	100
	Accumulation	Macrophage, alveolar	Minimum	3/10	30	5/10	50	5/10	50	8/9	88.9
	Inflammation**	Chronic, alveolar	Minimum	2/10	20	3/10	30	2/10	20	8/9	88.9
	Infiltrate	Mixed cell perivascular	Minimum	3/10	30	4/10	40	6/10	60	7/9	77.8
	Hemorrhage	Alveolar	Minimum	1/10	10	0/10	0	0/10	0	0/9	0
	Osseous foreign body			0/10	0	0/10	0	0/10	0	1/9	11.1
	Hyperplasia	Respiratory epithelium level I		0/10	0	0/10	0	0/10	0	1/9	11.1

* p < 0.05, compared with control, ** p < 0.01, compared with control

Table 2: Histopathological findings for female rats

Group

Control

Low

Middle

High

Number of animals

10

10

10

10

N

%

N

%

N

%

N

%

Liver

No microscopic findings

7/10

70

5/10

50

5/10

50

1/10

10

Abnormality*

3/10

30

5/10

50

5/10

50

9/10

90

Necrosis

Multifocal

Minimum

2/10

20

0/10

0

0/10

0

0/10

0

Moderate

0/10

0

0/10

0

0/10

0

1/10

10

Focal

Minimum

0/10

0

0/10

0

1/10

10

0/10

0

Single-cell hepatocellular *

Minimum

0/10

0

0/10

0

0/10

0

3/10

30

Hyperplasia*

Bile duct

Minimum

3/10

30

2/10

20

4/10

40

8/10

80

Moderate

0/10

0

0/10

0

0/10

0

1/10

10

Granuloma

Multifocal

Minimum

0/10

0

2/10

20

0/10

0

0/10

0

Vacuolation

Hepatocellular

Minimum

0/10

0

1/10

10

0/10

0

0/10

0

Fibrosis

Centrilobular

Mild

0/10

0

0/10

0

0/10

0

1/10

10

Pigment

Centrilobular

Mild

0/10

0

0/10

0

0/10

0

1/10

10

Lungs

No microscopic findings

3/10

30

5/10

50

6/10

60

2/10

20

Abnormality

7/10

70

5/10

50

4/10

40

8/10

80

Accumulation

Macrophage, alveolar

Minimum

7/10

70

4/10

40

4/10

40

6/10

60

Inflammation**

Chronic, alveolar

Minimum

3/10

30

2/10

20

0/10

0

8/10

80

Infiltrate**

Mixed cell perivascular

Minimum

0/10

0

0/10

0

1/10

10

7/10

70

* p < 0.05, compared with control, ** p < 0.01, compared with control

Silver distribution in tissue

Table 3: Tissue content of silver in male rats (mean± SE) (Unit: ng/g tissue wet weight)

	Control	Low	Middle	High
Liver	0.70 ± 0.20 (3)	3.52 ± 0.98 (5)	13.75 ± 2.28 (5)	132.97 ± 22.87* (4)
Kidneys	0.85 ± 0.20 (5)	1.63 ± 0.33 (5)	3.58 ± 0.41** (5)	9.49 ± 0.86* (4)
Olfactory bulb	0.51 ± 0.38 (3)	6.44 ± 0.77 (5)	17.10 ± 1.61 (5)	30.48 ± 2.15*** (4)
Brain	1.12 ± 0.34 (2)	3.45 ± 0.73 (4)	7.89 ± 0.95** (5)	18.63 ± 1.24* (4)
Lungs	0.77 ± 0.25 (5)	613.57 ± 66.03† (5)	5450.29 ± 904.17** (5)	14645.42 ± 2630.24* (4)
Whole blood	0.09 ± 0.02 (7)	0.68 ± 0.08 (10)	1.82 ± 0.20** (10)	4.31 ± 0.37* (9)

Note. (): number of samples.

*p < 0.01, high-dose versus other groups.

** p < 0.01, middle-dose versus unexposed and low-dose groups.

*** p < 0.01, high-dose versus other groups (dose dependent).

†p < 0.05, male versus female in low-dose group.

Table 4: Tissue content of silver in female rats (mean± SE) (Unit: ng/g tissue wet weight)

	Control	Low	Middle	High
Liver	0.90±0.31 (5)	4.55±1.40 (5)	12.07±2.50 (5)	71.08±24.50* (5)
Kidneys	0.94±0.18 (4)	2.61±0.57 (5)	11.81±4.27 (4)	37.66±7.04*†(5)
Olfactory bulb	2.26±0.74 (4)	7.43±0.75 (5)	13.75±1.32 (5)	32.84±2.74*** (5)
Brain	0.66±0.26 (4)	4.09±0.46 (5)	10.22±1.19** (5)	19.97±2.41* (5)
Lungs	1.01±0.10 (3)	295.92±78.50 (5)	4241.17±641.10** (5)	20585.63±1880.31* (5)
Whole blood	0.05±0.01 (5)	0.85±0.14 (10)	2.10±0.22** (10)	6.86±0.60*,‡(10)

Note. (): number of samples.

*p < 0.01, high-dose versus other groups.

** p < 0.01, middle-dose versus unexposed and low-dose groups.

*** p < 0.01, high-dose versus other groups (dose dependent).

†p < 0.05, female versus male in low-dose group.

‡p < 0.01, female versus male in high-dose group.

Conclusions:

The results of this subchronic 90-day silver nanoparticle inhalation toxicity indicated that lungs and liver were the major target tissues for prolonged silver nanoparticle accumulation.
The NOAEC of 133 µg/m^3 is based on test article-related effects seen at higher (515 µg/m^3) exposure concentrations (minimal bile-duct hyperplasia in males and females, chronic alveolar inflammation and macrophage accumulation in the lungs of males and females, and erythrocyte aggregation in females) of nano-silver (test atmosphere characterised by a count median diameter of 18 nm).
This rat NOAEC was corrected to human equivalent concentrations (HEC) for typical, commercial non-nano silver metal powders (D50 in µm range) based on particle size considerations and modelled comparative fractional deposition in the respiratory tract (rats/humans). The resulting HECs under various considerations are all >> 3 mg/m³ (EBRC, 2012). In contrast, the current OEL set by SCOEL based on human data and taken forward as the DNEL is 0.1 mg/m^3. Since this is more than one order of magnitude lower than the HEC based on animal data with nano-silver, the more conservative approach based on human data as derived by SCOEL was adopted as the DNEL.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Introduction:

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

The key findings were:

• silver metal was substantially less absorbed than soluble silver salts and nanosilver. Based on matched dose assessments, the extent of systemic exposure was about 10 to 30-fold lower in the case of silver metal versus reference ionic silver salts.
• silver metal was considerably less distributed in tissues and organs than silver salts (ionic silver compounds). This links to predictions that silver metal (massive and powder) represents a correspondingly lower health hazard, i.e., is less likely to cause toxicity effects.

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

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

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

1. Oral exposure:

1.1. Problem formulation

No study is available for the repeated dose toxicity via oral route for silver metal (massive and powder). However, there are data available for nanosilver and soluble silver substances. Toxicity of silver is driven by released silver ions (Ag+) and the amount of Ag+ release is proportionate to the specific surface area of the particle. Therefore, the studies performed on nanosilver and silver compounds will be considered in a weight-of-evidence (WoE) approach for the repeated toxicity of silver metal (massive and powder) and the difference in bioavailability and systemic distribution between silver metal (massive and powder) vs nanosilver and soluble silver salts -demonstrated in a comparative in-vivo toxicokinetic assay- will be accounted for.

 

1.2. Collection - documentation and quality assessment of all information

The weight of evidence for repeated dose toxicity of silver metal (powder and massive) is built based on several piece of evidence provided by repeated dose toxicity studies via oral route with nanosilver and other silver compounds (like silver acetate). Since it is acknowledged that the silver ion is the relevant toxicophore for systemic toxicity of silver, the outcome of studies performed with nanosilver and soluble silver compounds (like silver acetate) can be considered as worst-case scenario to assess the potential effects of silver metal (massive and powder). Importantly, and based on the outcome of the comparative in-vivo toxicokinetics study (Melvin et al., 2021 and Charlton et al., 2021), the data of nanosilver and soluble silver salts (like silver acetate) cannot be directly read-across to silver metal (massive and powder) because of the demonstrated difference in bioavailability and systemic distribution. Therefore, the appropriate modification to address this difference in bioavailability needs to be accounted for to assess the effects of Ag metal (massive and powder).

1.3. Integration of data and weight of evidence

A Weight of Evidence (WoE) approach is considered relevant, reliable and justified to assess the repeated dose toxicity of silver metal (massive and powder). Repeated dose toxicity data obtained with nanosilver and silver compounds (as silver acetate and silver nitrate) in rats establish the evidence database.

The repeated dose toxicity assessment of silver metal (massive and powder) is based on repeated dose toxicity studies performed with nanosilver and soluble silver salts. It is assumed that systemic toxicity of silver substances is driven by Ag+ (silver ions) as the primary species relevant to tissues exposure, and hence hazard assessment. Thus, a low bioavailability, leading to a low internal concentration of Ag+ (toxicophore) leads to a correspondingly low level of biological interaction and hence correspondingly low adverse outcome in comparison with highly bioavailable silver compounds.

 

Therefore, the bioavailability (i.e. Ag+ release) is considered as key piece of evidence in the weight of evidence for the repeated dose toxicity of silver metal (massive and powder) by oral route.

In more detail: the bioavailability of AgAc (Silver acetate) via oral route is consistent between independent estimates presented in the scientific literature (Barraclough and Cotton 2017, Boudreau et al. 2012, Park et al. 2011 and Van der Zande et al. 2012) and the comparative in-vivo TK study – in the order of 3-5%. In comparative terms, silver metal powder (AgMP) is substantially less absorbed. Based on matched dose assessments performed in the in-vivo TK study (Melvin et al., 2021 and Charlton et al., 2021), the extent of systemic exposure was about 10 to 30-fold lower in the case of AgMP vs AgAc. Unlike of situation of AgAc, the degree of uptake is not linear as the amount of administered of AgMP was increased up to a limit dose, but instead there was evidence of absorption plateauing. As a generic observation, the following trend in bioavailability is being observed: AgAc / AgNO3 >> AgNP >>> AgMP.

Therefore, the toxicokinetic findings strongly suggest 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 respective toxicokinetic profiles, presented in figure 1).
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.
Therefore, the Toxicokinetic / bioavailability / Ag+ release parameter (Melvin et al., 2021; Charlton et al., 2021) is considered as key piece of evidence in the weight of evidence reasoning for the repeated dose toxicity of silver metal (massive and powder) by oral route.

Under these assumptions, the selection of the Lourens et al. 2022 (90-day study; OECD408; GLP compliant) study as key study is considered conservative and robust to extrapolate the repeated dose toxicity of Silver metal (massive and powder) via oral route. The No Adverse Effect Level (NOAEL) defined by Lourens et al. is 320 mg/kg bw/day for females and 120 mg/kg bw/day for males. Since the absorption of silver metal is 10 to 30-fold lower than AgAc it can be reasonable and confidently be extrapolated that that the corresponding NOAEL for silver metal (massive and powder) will be well above the limit dose (320*10 (most conservative %) = 3200 mg/kg bw/day for females and 120*10=1200 mg/kg bw/day for males).

1.4. Conclusion

In conclusion the evaluation of repeated dose toxicity of silver metal (massive and powder) via oral route was performed using a weight of evidence approach.

The approach was built on:

• confident and robust repeated dose toxicity studies via oral route using several silver test items (mainly Ag acetate and nanosilver).
• a correction to adjust for the difference in oral absorption/bioavailability between soluble silver salts & nanosilver vs silver metal (massive and powder).

A strong weight of evidence using the available data demonstrates that adverse effects via repeated exposure by oral route are not expected for silver metal (massive and powder) .

Please see in Section 13 the CSR Annex 11 - Weight of Evidence Justification for Silver Metal - human health endpoints, for further details. 

 

2. Inhalation Exposure:

2.1. Problem formulation

There is no study available for the repeated dose toxicity via inhalation route for silver metal massive and powder. However, studies performed on nanosilver via inhalation route are available and will be considered for repeated toxicity of silver metal (massive and powder) via inhalation route. Toxicity of silver is driven by released silver ions (Ag+) and the amount of Ag+ release is proportionate to the specific surface area of the particle. Therefore, the studies performed on nanosilver will be considered in a weight-of-evidence (WoE) approach for the repeated toxicity of silver metal (massive and powder). In addition, data from repeated dose toxicity studies via oral route with nanosilver and soluble silver compound will be considered as supporting evidence and the corresponding silver release will be considered according to their relative bioavailability (Ag+ released).

 

2.2. Collection - documentation and quality assessmet of all information

The WoE approach for repeated dose toxicity of silver metal (powder and massive) via inhalation route is built based on:

• Repeated dose toxicity studies via inhalation route for nanosilver supplemented by
• Repeated dose toxicity studies via oral route for silver salts (i.e. silver acetate) and nanosilver. 

Since it is acknowledged that silver ion is the toxicophore for systemic toxicity of silver, the outcome of studies performed with nanosilver can be considered as worst-case scenario to assess the potential effects of silver metal (massive and powder). Note that based on the outcome of the comparative in-vivo toxicokinetics data (Melvin et al., 2021 and Charlton et al., 2021), the data of nanosilver cannot be used in a direct read-across to silver metal (massive and powder) because of a demonstrated difference in bioavailability via oral route. Nevertheless, via inhalation, the potential absorption of silver metal (massive and powder) is based on dustiness, mass fractions and particle size distribution of airborne dusts.

2.3. Integration of data and weight of evidence

The WoE approach is considered relevant, reliable and justified to assess the repeated dose toxicity via inhalation route of silver metal (massive and powder). Repeated dose toxicity data for exposure via inhalation and oral route obtained with AgNP and silver compounds (silver acetate and silver nitrate) in rats establish the evidence database.

The inhalation potential of silver metal (massive and powder) is based on dustiness, mass fractions and particle size distribution of airborne dusts. Considering this evidence, for silver metal (massive and powder), the repeated dose inhalation toxicity studies performed with nanosilver are considered as worst-case as they are representing the smallest particle size of silver metal reaching the deeper lung areas at least at comparable and likely at higher quantities, plus they are dissolving quicker and at a greater extend (cfr. relationship between metal release/dissolution and surface area).

The inhalation studies with nanosilver are considered consistent, reliable, relevant and adequate to assess the inhalation repeated dose toxicity of silver metal (massive and powder). In these studies with nanoparticles, there was no evidence of systemic toxicity triggered via inhalation exposure. The main adverse effect observed remained local and limited to reversible or persistent lung inflammation, typically associated with nanosilver but not expected with microscale particles.

Furthermore, the repeated dose toxicity studies performed with nanosilver or soluble silver salts (like Ag acetate) via oral route are considered relevant and consistent to support the repeated dose toxicity of silver metal via inhalation route. It is assumed that systemic toxicity of silver substances is driven by silver ions (Ag+) as the primary species relevant to tissues exposure, and hence hazard assessment. Thus, the internal concentration of Ag+ (as toxicophore) drives biological interaction and hence the potential adverse outcome. Therefore, it is considered acceptable to use studies performed with highly bioavailable silver species (i.e. Ag acetate and nanosilver) via oral route as supporting evidence for the repeated dose toxicity of silver metal (massive and powder) via inhalation route. Importantly, and based on the outcome of the comparative in-vivo toxicokinetics study (Melvin et al., 2021 and Charlton et al., 2021), the data of nanosilver and soluble silver salts (like silver acetate) via repeated oral administration cannot be directly read-across to silver metal (massive and powder) because of the demonstrated difference in bioavailability and systemic distribution. Therefore, the appropriate modification to address this difference in bioavailability needs to be accounted for to assess the effects of Ag metal (massive and powder).

2.4. Conclusion:

In conclusion, the evaluation of repeated dose toxicity of silver metal (massive and powder) via inhalation route was performed using a weight of evidence approach.

The approach was built on:

• confident and robust repeated dose toxicity studies via inhalation route of nanosilver supported by
• the repeated dose toxicity studies via oral route performed with soluble silver salts (like Ag acetate) and nanosilver. For the latter, the proper modification needs to be performed to account for the demonstrated difference in bioavailability between Ag metal (massive and powder) vs nanosilver and silver acetate.
  

A strong weight of evidence using the available data demonstrates that adverse effects via repeated exposure by inhalation route are not expected for silver metal (massive and powder). The repeated dose inhalation toxicity studies performed with nanosilver only identify adverse local effects and inflammation, which in certain case is reversible but any case considered relevant for nanoparticles only. There is no systemic / local effect expected via repeated exposure to Ag metal (massive and powder) inhalation route.

Please see in Section 13 the CSR Annex 11 - Weight of Evidence Justification for Silver Metal - human health endpoints, for further details. 

Dermal Exposure:

No studies involving repeated dermal exposure to silver metal massive or powder is available

 

Justification for classification or non-classification

The available animal studies with silver compounds and nanosilver likewise do not indicate any specific target organ toxicity. in addition the new in-vivo comparative toxicokinetic study demonstrate that the direct read-across between sivler compounds and nanosilver to silver metal massive and powder is not supported. For these reasons, silver metal massive and powder do not meet any of the classification criteria for effects associated with repeated dosing.