Silver

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

bioaccessibility (or bioavailability)

tissue distribution

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Sub-micron sized powder-form (referenced as 'AgMP' throughout the study report; uncoated; >99% pure; d50 = ~0.3 μm; crystalline powder of highly uniform spheroidal shape) is the finest representative powder on the EU market and considered a worst-case type of silver metal powder in terms of expected bioavailability

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD IGS (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 8-10 weeks old at start of treatment
- Weight at study initiation: min. 170 g for females and 200 g for males
- Housing: up to four animals of the same sex per cag. Within each group, cages will be blocked together (by sex). The distribution of the groups within and between racks and the position of the racks within the room will be determined to equalize environmental influences across the study while minimizing the opportunity for inter-group contamination.
- Diet (e.g. ad libitum): SDS VRF-,1 pelleted diet, non-restricted availability (diet contains no added antibiotic or other chemotherapeutic or prophylactic agent)
- Water (e.g. ad libitum): potable water from the public supply, non-restricted availability via polycarbonate bottles with sipper tubes (except during urine collection)
- Acclimation period: at least 5 days before commencement of treatment
- Health status: visually inspected at least twice daily for evidence of ill-health

ENVIRONMENTAL CONDITIONS
- Temperature (°C): monitored and maintained within the range of 20-24ºC
- Humidity (%): monitored and maintained within the range of 40-70%
- Air changes (per hr): air filtered, not recirculated
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark
- Fasting period : none

IN-LIFE DATES: study performed in phases, with first phase started on 30 Sep 2020 and last (repeated) phase ended on 20 Aug 2021

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

1% w/v aqueous methylcellulose (400 cps)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Notes:
- Chloride and phosphate containing vehicles and reagents were avoided due to the potential for silver precipitation effects.
- Dissolved silver and silver particles adsorb onto material surfaces including glass and metals, and as such use of these was avoided.
- Formulations were protected from light during preparation (use of yellow-light instead).
- The formulation procedures are documented in the study data and will be included in the final report.
- The pH of the final formulations was measured and recorded in the data.
- A series of formulations at the required concentrations is prepared by dilution of individual weighing of the Test Item and dispensed in polypropylene screw top jars or similar

Method of preparation:
- 50% of the final volume of vehicle is added to an appropriate container. The required amount of Test Item is weighed and added to the vehicle whilst stirring at an appropriate speed to obtain a vortex that is half the depth of the suspension. The ‘weighing’ container is rinsed several times using 20% final volume of vehicle. After addition of the rinsing, the suspension is stirred for 60 seconds and then sonicated for approximately 30 minutes. This stir and sonicate step is then repeated, after which the remaining 30% final volume of vehicle is added and stirring continued at an appropriate speed to obtain a vortex that is half the depth of the final suspension for at least 60 minutes.
- The suspensions are sampled for formulation analysis (whilst under constant stirring, according to the previously described technique) prior to dispensing and aliquoting into suitable containers for dose administration.

Frequency of preparation:
- Repeat dose formulations were prepared weekly and split into daily aliquots.
- The suitability and homogeneity of formulations were confirmed as part of studies, undertaken separately by third parties designated by the Sponsor, and by the Principal Investigator for dose formulation analysis.

Storage of formulation: Silver AgMP suspensions: refrigerated (2 to 8°C), protected from light.

Duration and frequency of treatment / exposure:

Two treatments:
- single oral dose: single occasion on day 1
- repeat oral doses: 28 days; frequency: once daily at approximately the same time each day

Dose / conc.:

36 mg/kg bw/day (nominal)

Remarks:

single + repeat oral doses

Dose / conc.:

180 mg/kg bw/day (nominal)

Remarks:

repeat oral doses

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

single + repeat oral doses

No. of animals per sex per dose / concentration:

4 males + 4 females

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection and Route:
No reliable toxicokinetic information was previously available for micron-size silver metal powder (AgMP) and this study closes that data gap. Whilst very limited general toxicity data was available for Ag MP via the oral route, bulk silver forms are not known to produce significant systemic effects or to cause local tissue reactivity. Furthermore, in- chemico studies suggest that the bioavailability of this silver form will be more limited than the other Test Items. Hence a dose level of 1000 mg/kg bw/d via the oral route was selected, which corresponds to an OECD defined Limit Dose and should not produce any appreciable toxicities.

Details on dosing and sampling:

TOXICOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood, brain, bone marrow, small intestine, liver, spleen, ovaries, testes, uterus
- Time and frequency of sampling: blood: Day 15: PD, 6 hours post-dose, Day 28: PD, 3, 6, 9, 12, 24, 72, 96 hours post-dose. (10 time points) ; tissues: at termination (after 28 days treatment)

ANALYTICAL METHOD
Total Ag concentrations were determined in whole blood and tissues by ICP-MS after microwave digestion with nitric acid and hydrochloric acid. This method was successfully validated with a lower limit of quantification (LLOQ) of 10 ng/mL for silver in rat blood and 30 ng/g for silver in rat tissue.

Statistics:

- Ag analysis data were acquired and integrated using MassHunter (version C.01.05 Agilent). Peak area results were exported to Watson LIMS (Version 7.5 SP1, Thermo Scientific) in text file format. Watson LIMS was used to calculate standard curve parameters and concentration data for Silver. The concentration and statistical data were generated by computerized techniques.
- AUC(0-t): area under the concentration-time curve from hour 0 to the last measurable concentration, estimated by the linear trapezoidal rule.
- Dose normalized AUC(0-t): calculated as AUC(0-t) / dose level.

Preliminary studies:

Comprehensive enabling work was done to support the main study. The AgNP and AgMP test items were characterised to confirm suitability of the test items and to confirm stability in an appropriate aqueous-based vehicle. The AgMP test item formulated in the vehicle contained less than 0.01% of nanoparticles and ionic Ag (% of total Ag).

Type:

absorption

Results:

Based on matched dose assessments, the extent of systemic exposure was about 10 to 30-fold lower in the case of sub-micron Ag metal powder (‘AgMP’) versus a reference ionic Ag salt (Ag acetate).

Type:

distribution

Results:

Levels of Ag distributed into tissues and organs are considerably lower in the case of AgMP than an ionic Ag compound (Ag acetate).

Details on absorption:

- Comparisons via normalised administered doses (i.e. Ag equivalent dose) can be made between Ag acetate and Ag metal powder (AgMP). Based on these matched dose assessments, the extent of systemic exposure was about 10 to 30-fold lower in the case of Ag metal powder versus a reference ionic Ag salt
- Unlike the situation with ionic Ag, the degree of uptake of Ag metal powder was not linear when the amount of administered Ag metal powder was increased up to a limit dose. Instead, there was evidence of absorption plateauing.
- The maximum observed Ag concentration in blood occurs between 3-9h post-dosing for repeat dose groups.
- These data demonstrate that oral dosing of AgMP leads to appreciably lower Ag amplitude of systemic exposure in comparison to ionic Ag forms.

Details on distribution in tissues:

- When looking at differentials in silver in tissue concentrations following repeated dosing, for the majority of tissues, exposure to soluble silver compounds leads to tissue Ag levels which are 2 to 3 orders of magnitude higher than those evident for the silver powder.
- The tissues for which the difference is less than 2 to 3 orders of magnitude, are minor sites of distribution.
- The study shows that the testis and brain are minor sites of distribution. In contrast, the ovary has been shown to be an important site of distribution. However, ancillary investigations strongly suggest silver is present in the ovary as chemically stable silver sulphide/selenide complexes with very low local bioavailability.

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 82.4

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 36 mg/kg bw/d

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 145

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 36 mg/kg bw/d

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 36.1

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 180 mg/kg bw/d

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 50.9

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 180 mg/kg bw/d

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 8.85

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 1000 mg/kg bw/d

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 13.1

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 1000 mg/kg bw/d

Systemic exposure / Ag in blood

- Figure 1 (attached below) shows the extent of systemic exposure as area under the concentration time curve (AUC) values after repeated dose administration of Ag metal powder (AgMP) for 28 days.

- Comparisons via normalised administered doses (i.e. Ag equivalent dose) can be made between Ag acetate and Ag metal powder (AgMP). Based on these matched dose assessments, the extent of systemic exposure was about 10 to 30-fold lower in the case of Ag metal powder versus a reference ionic Ag salt.

- Unlike the situation with ionic Ag, the degree of uptake of Ag metal powder was not linear when the amount of administered Ag metal powder was increased up to a limit dose. Instead, there was evidence of absorption plateauing (Figure 1).

- Table 2 shows the blood Ag concentrations at predicted steady state (day 15) after repeated dose administration of AgMP. The maximum observed concentration occurs between 3-9h post-dosing for repeat dose groups, hence the 6h values for the respective test substances are shown as an indicator of peak exposure.

Table 2.  Blood Ag concentrations at day 15 after repeated oral dose administration of Ag metal powder (AgMP).

Dose level	Mean 6h blood [Ag]		Mean [Ag]6h/
(mg/kg Test Item bw/d)	ng/mL		Ag Equiv. Dose
			ng/mL
	♂	♀	♂	♀
Control	<LLOQ	<LLOQ	--	--
36	50	92	1.4	2.6
	± 16	± 16
180	74	138	0.41	0.77
	± 30	± 41
1000	138	182	0.14	0.18
	± 64	± 30

- Comparing these data to the data for other Ag test items, this demonstrates that oral dosing of AgMP leads to appreciably lower Ag amplitude of systemic exposure in comparison to ionic Ag forms.

 

Tissue exposure / Ag distribution

Table 3 shows the Ag tissue levels / distribution after repeated dose administration of AgMP.

Table 3. Terminal Ag tissue levels following repeated oral dose administration for 28 days of Ag metal powder (AgMP)

Dose (mg/kg bw/d) /Sex		Bone marrow	Brain	GI tract	Spleen	Testis	Liver
Control M	Mean	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
	SD	NC	NC	NC	NC	NC	NC
36 M	Mean	BLQ	76.0	261	77.5	76.8	BLQ
	SD	NC	17.7	134	31.4	36.0	NC
180 M	Mean	NC	117	1004	164	213	64.3
	SD	NC	47.0	1271	141	180	30.2
1000 M	Mean	NC	159	2620	516	296	48.7
	SD	NC	26.0	721	195	80.6	9.33

Dose (mg/kg bw/d) /Sex		Bone Marrow	Brain	GI Tract	Spleen	Ovaries	Uterus	Liver
Control F	Mean	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
	SD	NC	NC	NC	NC	NC	NC	NC
36 F	Mean	BLQ	75.6	310	89.8	81.9	BLQ	77.9
	SD	NC	16.1	162	58.7	31.8	NC	34.3
180 F	Mean	BLQ	166	3755	1006	1328	153	405
	SD	NC	34.9	1853	623	917	58.8	619
1000 F	Mean	243	187	5124	1448	2470	184	274
	SD	89.3	27.6	1645	830	1273	127	186

BLQ    <5.00 ng/mL
NC      Not calculated

- Comparing these data to the data for other Ag test items, this demonstrates that levels of Ag distributed into tissues and organs are considerably lower in the case of Ag metal powder than an ionic Ag compound. This links to predictions that silver powder represents a correspondingly lower health hazard, i.e., is less likely to cause toxic effects

Conclusions:

This in-vivo toxicokinetic study (in rats) by oral gavage comparing silver metal powder, nanosilver, silver nitrate and silver acetate shows a lower absorption/distribution and lower systemic exposure to tissues and organs for silver metal powder than for the other silver substances. The amount of Ag+ in blood and tissues is much higher for silver acetate/silver nitrate than for silver metal powder at comparable Ag dosing levels. Although all silver-containing substances have the ability to release Ag+, the extent of released silver ions is significantly different between substances. Therefore, a direct read-across of mammalian toxicity datasets for simple ionic silver salts like silver acetate or silver nitrate and nanosilver to silver metal powder/massive is not supported (from a scientifically valid toxicokinetic perspective).

Executive summary:

This in-vivo toxicokinetic study is an oral route investigation using adult CD Sprague Dawley rats. The design conformed to OECD TG 417 and was conducted in compliance to GLP. The study was designed as a comparative toxicokinetic assessment of the test items Silver Acetate (AgAc), Silver Nitrate (AgNO3), Micron-sized Silver (AgMP) and Nanoparticulate Silver (AgNP). This included assessment of bioavailability following single oral doses relative to single intravenous doses and systemic exposure following single and repeat oral doses. Groups of 4 male and 4 female CD Sprague Dawley rats were administered the test items, either as a single intravenous dose, a single oral dose or repeated daily oral doses for 4 weeks.

The silver metal powder (d50 ~0.3 µm) tested in the study is the finest representative powder on the EU market and considered a worst-case type of silver metal powder in terms of expected bioavailability.

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

The silver tissue levels after repeated dose administration are considerably lower in the case of silver metal powder (AgMP) than for silver acetate. This suggests that AgMP represents a correspondingly lower health hazard, i.e. AgMP (and by extrapolation also massive Ag with a tremendously lower particle size/reactive surface area) is expected to cause less effects at corresponding Ag dose levels than AgAc.

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

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

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2020-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

bioaccessibility (or bioavailability)

tissue distribution

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD IGS (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 8-10 weeks old at start of treatment
- Weight at study initiation: min. 170 g for females and 200 g for males
- Housing: up to four animals of the same sex per cag. Within each group, cages will be blocked together (by sex). The distribution of the groups within and between racks and the position of the racks within the room will be determined to equalize environmental influences across the study while minimizing the opportunity for inter-group contamination.
- Diet (e.g. ad libitum): SDS VRF-,1 pelleted diet, non-restricted availability (diet contains no added antibiotic or other chemotherapeutic or prophylactic agent)
- Water (e.g. ad libitum): potable water from the public supply, non-restricted availability via polycarbonate bottles with sipper tubes (except during urine collection)
- Acclimation period: at least 5 days before commencement of treatment
- Health status: visually inspected at least twice daily for evidence of ill-health

ENVIRONMENTAL CONDITIONS
- Temperature (°C): monitored and maintained within the range of 20-24ºC
- Humidity (%): monitored and maintained within the range of 40-70%
- Air changes (per hr): air filtered, not recirculated
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark
- Fasting period : none

IN-LIFE DATES: study performed in phases, with first phase started on 30 Sep 2020 and last (repeated) phase ended on 20 Aug 2021

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Notes:
- Chloride and phosphate containing vehicles and reagents were avoided due to the potential for silver precipitation effects.
- Dissolved silver and silver particles adsorb onto material surfaces including glass and metals, and as such use of these was avoided.
- Formulations were protected from light during preparation (use of yellow-light instead).
- The formulation procedures are documented in the study data and will be included in the final report.
- The pH of the final formulations was measured and recorded in the data.
- A series of formulations at the required concentrations is prepared by dilution of individual weighing of the Test Item and dispensed in polypropylene screw top jars or similar

Method of preparation:
The required amount of test item was weighed out, (Ag Acetate was ground in a pestle and mortar) and then added to ca 50% of the final volume of vehicle and magnetically stirred to mix/disperse. The ‘weighing’ vessel was rinsed, and the rinsing is added to the container before bringing to volume with the vehicle. The mixture was then transferred to a magnetic stirrer and stirred for a minimum of 20 minutes, recording the start and finish times in the raw data. Formulation analysis sampling will occur whilst under constant stirring, prior to dispensing and aliquoting into suitable containers for dose administration (stirrer bars will be included).

Frequency of preparation:
- Repeat dose formulations were prepared weekly and split into daily aliquots.
- The suitability and homogeneity of formulations were confirmed as part of studies, undertaken separately by third parties designated by the Sponsor, and by the Principal Investigator for dose formulation analysis.

Storage of formulation: Silver solutions – ambient temperature (15 to 25°C), protected from light.

Duration and frequency of treatment / exposure:

Two treatments:
- single oral dose: single occasion on day 1
- repeat oral doses: 28 days; frequency: once daily at approximately the same time each day

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

single + repeat oral doses

Dose / conc.:

55 mg/kg bw/day (nominal)

Remarks:

repeat oral doses

Dose / conc.:

175 mg/kg bw/day (nominal)

Remarks:

single + repeat oral doses

No. of animals per sex per dose / concentration:

4 males + 4 females

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection and Route:
The dose levels for silver acetate (AgAc) have been chosen to support a program of reproductive toxicology studies on this compound. Based on existing toxicity data for AgAc, from repeated administration studies in rodents the dose levels are expected to be well tolerated.

Details on dosing and sampling:

TOXICOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood, brain, bone marrow, small intestine, liver, spleen, ovaries, testes, uterus
- Time and frequency of sampling: blood: Day 15: PD, 6 hours post-dose, Day 28: PD, 3, 6, 9, 12, 24, 72, 96 hours post-dose. (10 time points) ; tissues: at termination (after 28 days treatment)

ANALYTICAL METHOD
Total Ag concentrations were determined in whole blood and tissues by ICP-MS after microwave digestion with nitric acid and hydrochloric acid. This method was successfully validated with a lower limit of quantification (LLOQ) of 10 ng/mL for silver in rat blood4 and 30 ng/g for silver in rat tissue.

Statistics:

- Ag analysis data were acquired and integrated using MassHunter (version C.01.05 Agilent). Peak area results were exported to Watson LIMS (Version 7.5 SP1, Thermo Scientific) in text file format. Watson LIMS was used to calculate standard curve parameters and concentration data for Silver. The concentration and statistical data were generated by computerized techniques.
- AUC(0-t): area under the concentration-time curve from hour 0 to the last measurable concentration, estimated by the linear trapezoidal rule.

Type:

absorption

Results:

Based on matched dose assessments, silver acetate (AgAc) and silver nitrate (AgNO3) exhibit very similar systemic exposure profiles while AgMP leads to appreciably lower Ag amplitude of systemic exposure.

Type:

distribution

Results:

Levels of Ag distributed into tissues and organs are similar for AgAc and AgNO3 and considerably lower in the case of AgMP .

Details on absorption:

- Comparisons via normalised administered doses (i.e. Ag equivalent dose) can be made between Ag acetate and bulk Ag (AgMP). Based on these matched dose assessments, the extent of systemic exposure was about 10 to 30-fold lower in the case of Ag metal powder versus AgAc
- The maximum observed Ag concentration in blood occurs between 3-9h post-dosing for repeat dose groups.

Details on distribution in tissues:

- Levels of Ag distributed into tissues and organs are similar for AgAc and AgNO3 (but considerably lower in the case of AgMP ).
- The study shows that the testis and brain are minor sites of distribution. In contrast, the ovary has been shown to be an important site of distribution. However, ancillary investigations strongly suggest silver is present in the ovary as chemically stable silver sulphide/selenide complexes with very low local bioavailability.

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 1770

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 5 mg/kg bw/d

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 2920

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 5 mg/kg bw/d

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 664

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 55 mg/kg bw/d

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 846

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 55 mg/kg bw/d

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 342

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 175 mg/kg bw/d

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 400

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 175 mg/kg bw/d

Systemic exposure / Ag in blood

- Figure 1 (attached below) shows the extent of systemic exposure as area under the concentration time curve (AUC) values after repeated dose administration of silver acetate (AgAc) for 28 days.

- Comparisons via normalised administered doses (i.e. Ag equivalent dose) can be made between different Ag test items. Based on these matched dose assessments, the extent of systemic exposure was similar for AgAc and AgNO3 but about 10 to 30-fold lower in the case of Ag metal powder versus a reference ionic Ag salt.

- Table 2 shows the blood Ag concentrations at predicted steady state (day 15) after repeated dose administration of AgAc. The maximum observed concentration occurs between 3-9h post-dosing for repeat dose groups, hence the 6h values for the respective test substances are shown as an indicator of peak exposure.

Table 2.  Blood Ag concentrations at day 15 after repeated oral dose administration of silver acetate (AgAc).

Dose level	Mean 6h blood [Ag]		Mean [Ag]6h/
(mg/kg Test Item bw/d)	ng/mL		Ag Equiv. Dose
			ng/mL
	♂	♀	♂	♀
Control	<LLOQ	<LLOQ	--	--
		(n=3)
5	113	204	34.8	62.8
	± 15	± 26
55	317	423	8.9	11.8
	± 93	± 72
175	652	918	5.7	8.1
	± 171	± 103
	(n=3)	(n=3)

- Comparing these data to the data for other Ag test items, this demonstrates that oral dosing of AgAc and AgNO3 lead to similar systemic exposure and AgMP leads to appreciably lower Ag amplitude of systemic exposure in comparison to ionic Ag forms.

 

Tissue exposure / Ag distribution

Table 3 shows the Ag tissue levels / distribution after repeated dose administration of AgAc.

Table 3. Terminal Ag tissue levels following repeated oral dose administration for 28 days of silver acetate (AgAc)

 

Dose (mg/kg bw/d) /Sex		Bone marrow	Brain	GI tract	Spleen	Testis	Liver
Control M	Mean	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
5 M	Mean	62.0	142	2770	283	168	55.4
	SD	13.5	36.0	1720	71.2	75.9	15.3
55 M	Mean	3500	637	19200	38700	1510	9470
	SD	1540	57.2	7680	11000	215	4120
175 M	Mean	21400	1460	83200	96400	1530	22700
	SD	15400	33.1	5270	55200	237	7630

Dose(mg/kg bw/d) /Sex		Bone Marrow	Brain	GI Tract	Spleen	Ovaries	Uterus	Liver
Control F	Mean	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
5 F	Mean	126	169	3800	990	2200	188	305
	SD	110	21.2	1490	455	1080	121	232
55 F	Mean	4500	805	50700	60800	24300	8000	16500
	SD	1420	35.9	11200	10400	2610	3300	3610
175 F	Mean	46800	1460	104000	142000	39700	11100	21900
	SD	19800	114	12600	93500	19800	2320	13100

BLQ    <5.00 ng/mL

- Comparing these data to the data for other Ag test items, this demonstrates that levels of Ag distributed into tissues and organs are similar for AgAc and AgNO3 but considerably lower in the case of Ag metal powder.

Conclusions:

This in-vivo toxicokinetic study (in rats) by oral gavage comparing silver metal powder, nanosilver, silver nitrate and silver acetate shows a lower absorption/distribution and lower systemic exposure to tissues and organs for silver metal powder than for the other silver substances. The amount of Ag+ in blood and tissues is much higher for silver acetate/silver nitrate than for silver metal powder at comparable Ag dosing levels. Although all silver-containing substances have the ability to release Ag+, the extent of released silver ions is significantly different between substances. Therefore, a direct read-across of mammalian toxicity datasets for simple ionic silver salts like silver acetate or silver nitrate and nanosilver to silver metal powder/massive is not supported (from a scientifically valid toxicokinetic perspective).

Executive summary:

This in-vivo toxicokinetic study is an oral route investigation using adult CD Sprague Dawley rats. The design conformed to OECD TG 417 and was conducted in compliance to GLP. The study was designed as a comparative toxicokinetic assessment of the test items Silver Acetate (AgAc), Silver Nitrate (AgNO3), Micron-sized Silver (AgMP) and Nanoparticulate Silver (AgNP). This included assessment of bioavailability following single oral doses relative to single intravenous doses and systemic exposure following single and repeat oral doses. Groups of 4 male and 4 female CD Sprague Dawley rats were administered the test items, either as a single intravenous dose, a single oral dose or repeated daily oral doses for 4 weeks.

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

The silver tissue levels after repeated dose administration are considerably lower in the case of silver metal powder (AgMP) than for silver acetate. This suggests that AgMP represents a correspondingly lower health hazard, i.e. AgMP (and by extrapolation also massive Ag with a tremendously lower particle size/reactive surface area) is expected to cause less effects at corresponding Ag dose levels than AgAc.

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

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

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2020-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

bioaccessibility (or bioavailability)

tissue distribution

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD IGS (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 8-10 weeks old at start of treatment
- Weight at study initiation: min. 170 g for females and 200 g for males
- Housing: up to four animals of the same sex per cag. Within each group, cages will be blocked together (by sex). The distribution of the groups within and between racks and the position of the racks within the room will be determined to equalize environmental influences across the study while minimizing the opportunity for inter-group contamination.
- Diet (e.g. ad libitum): SDS VRF-,1 pelleted diet, non-restricted availability (diet contains no added antibiotic or other chemotherapeutic or prophylactic agent)
- Water (e.g. ad libitum): potable water from the public supply, non-restricted availability via polycarbonate bottles with sipper tubes (except during urine collection)
- Acclimation period: at least 5 days before commencement of treatment
- Health status: visually inspected at least twice daily for evidence of ill-health

ENVIRONMENTAL CONDITIONS
- Temperature (°C): monitored and maintained within the range of 20-24ºC
- Humidity (%): monitored and maintained within the range of 40-70%
- Air changes (per hr): air filtered, not recirculated
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark
- Fasting period : none

IN-LIFE DATES: study performed in phases, with first phase started on 30 Sep 2020 and last (repeated) phase ended on 20 Aug 2021

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Notes:
- Chloride and phosphate containing vehicles and reagents were avoided due to the potential for silver precipitation effects.
- Dissolved silver and silver particles adsorb onto material surfaces including glass and metals, and as such use of these was avoided.
- Formulations were protected from light during preparation (use of yellow-light instead).
- The formulation procedures are documented in the study data and will be included in the final report.
- The pH of the final formulations was measured and recorded in the data.
- A series of formulations at the required concentrations is prepared by dilution of individual weighing of the Test Item and dispensed in polypropylene screw top jars or similar

Method of preparation:
The required amount of test item was weighed out, (Ag Acetate was ground in a pestle and mortar) and then added to ca 50% of the final volume of vehicle and magnetically stirred to mix/disperse. The ‘weighing’ vessel was rinsed, and the rinsing is added to the container before bringing to volume with the vehicle. The mixture was then transferred to a magnetic stirrer and stirred for a minimum of 20 minutes, recording the start and finish times in the raw data. Formulation analysis sampling will occur whilst under constant stirring, prior to dispensing and aliquoting into suitable containers for dose administration (stirrer bars will be included).

Frequency of preparation:
- Repeat dose formulations were prepared weekly and split into daily aliquots.
- The suitability and homogeneity of formulations were confirmed as part of studies, undertaken separately by third parties designated by the Sponsor, and by the Principal Investigator for dose formulation analysis.

Storage of formulation: Silver solutions – ambient temperature (15 to 25°C), protected from light.

Duration and frequency of treatment / exposure:

Two treatments:
- single oral dose: single occasion on day 1
- repeat oral doses: 28 days; frequency: once daily at approximately the same time each day

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

single + repeat oral doses

Dose / conc.:

55 mg/kg bw/day (nominal)

Remarks:

repeat oral doses

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

single + repeat oral doses

No. of animals per sex per dose / concentration:

4 males + 4 females

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection and Route:
The dose levels selected in the case of silver nitrate (AgNO3) extend existing but limited toxicokinetic data for this compound. A previous 28-day toxicity study demonstrated that up to 100 mg/kg bw/d of AgNO3 administered to rats via oral gavage produced minimal toxicity. The high-dose treatment level has been set with due regard for the local tissue irritancy profile of AgNO3.

Details on dosing and sampling:

TOXICOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood, brain, bone marrow, small intestine, liver, spleen, ovaries, testes, uterus
- Time and frequency of sampling: blood: Day 15: PD, 6 hours post-dose, Day 28: PD, 3, 6, 9, 12, 24, 72, 96 hours post-dose. (10 time points) ; tissues: at termination (after 28 days treatment)

ANALYTICAL METHOD
Total Ag concentrations were determined in whole blood and tissues by ICP-MS after microwave digestion with nitric acid and hydrochloric acid. This method was successfully validated with a lower limit of quantification (LLOQ) of 10 ng/mL for silver in rat blood4 and 30 ng/g for silver in rat tissue.

Statistics:

- Ag analysis data were acquired and integrated using MassHunter (version C.01.05 Agilent). Peak area results were exported to Watson LIMS (Version 7.5 SP1, Thermo Scientific) in text file format. Watson LIMS was used to calculate standard curve parameters and concentration data for Silver. The concentration and statistical data were generated by computerized techniques.
- AUC(0-t): area under the concentration-time curve from hour 0 to the last measurable concentration, estimated by the linear trapezoidal rule.

Type:

absorption

Results:

Based on matched dose assessments, silver acetate (AgAc) and silver nitrate (AgNO3) exhibit very similar systemic exposure profiles while AgMP leads to appreciably lower Ag amplitude of systemic exposure.

Type:

distribution

Results:

Levels of Ag distributed into tissues and organs are similar for AgAc and AgNO3 and considerably lower in the case of AgMP .

Details on absorption:

- Comparisons via normalised administered doses (i.e. Ag equivalent dose) can be made between the different Ag test items. Based on these matched dose assessments, the extent of systemic exposure was similar for AgAc and AgNO3 but about 10 to 30-fold lower in the case of Ag metal powder versus AgAc
- The maximum observed Ag concentration in blood occurs between 3-9h post-dosing for repeat dose groups.

Details on distribution in tissues:

- Levels of Ag distributed into tissues and organs are similar for AgAc and AgNO3 (but considerably lower in the case of AgMP ).
- The study shows that the testis and brain are minor sites of distribution. In contrast, the ovary has been shown to be an important site of distribution. However, ancillary investigations strongly suggest silver is present in the ovary as chemically stable silver sulphide/selenide complexes with very low local bioavailability.

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 1600

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 5 mg/kg bw/d

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 2240

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 5 mg/kg bw/d

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 581

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 55 mg/kg bw/d

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 601

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 55 mg/kg bw/d

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 476

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 125 mg/kg bw/d

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 601

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 125 mg/kg bw/d

Systemic exposure / Ag in blood

- Figure 1 (attached below) shows the extent of systemic exposure as area under the concentration time curve (AUC) values after repeated dose administration of silver nitrate (AgNO3) for 28 days.

- Comparisons via normalised administered doses (i.e. Ag equivalent dose) can be made between different Ag test items. Based on these matched dose assessments, the extent of systemic exposure was similar for AgAc and AgNO3 but about 10 to 30-fold lower in the case of Ag metal powder versus a reference ionic Ag salt.

- Table 2 shows the blood Ag concentrations at predicted steady state (day 15) after repeated dose administration of AgNO3. The maximum observed concentration occurs between 3-9h post-dosing for repeat dose groups, hence the 6h values for the respective test substances are shown as an indicator of peak exposure.

Table 2.  Blood Ag concentrations at day 15 after repeated oral dose administration of silver nitrate (AgNO3).

Dose level	Mean 6h blood [Ag]		Mean [Ag]6h/
(mg/kg Test Item bw/d)	ng/mL		Ag Equiv. Dose
			ng/mL
	♂	♀	♂	♀
Control	<LLOQ	<LLOQ	--	--
		(n=3)
5	131	199	26	40
	± 45	± 71
55	449	471	8.2	8.6
	± 204	± 48
		(n=3)
125	540	691	4.3	5.5
	± 111	± 133

- Comparing these data to the data for other Ag test items, this demonstrates that oral dosing of AgAc and AgNO3 lead to similar systemic exposure and AgMP leads to appreciably lower Ag amplitude of systemic exposure in comparison to ionic Ag forms.

 

Tissue exposure / Ag distribution

Table 3 shows the Ag tissue levels / distribution after repeated dose administration of AgNO3.

Table 3. Terminal Ag tissue levels following repeated oral dose administration for 28 days of silver nitrate (AgNO3)

Dose (mg/kg bw/d) /Sex		Bone marrow	Brain	GI tract	Spleen	Testis	Liver
Control M	Mean	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
	SD	NC	NC	NC	NC	NC	NC
5 M	Mean	73.1	142	1310	404	231	81.7
	SD	16.2	35.4	214	191	103	33.1
55 M	Mean	10739	632	5319	39789	1202	4668
	SD	2104	129	2412	5704	246	2919
125 M	Mean	46553	1091	10592	54070	1464	28859
	SD	26985	57.0	10669	21236	260	36648

Dose(mg/kg bw/d) /Sex		Bone Marrow	Brain	GI Tract	Spleen	Ovaries	Uterus	Liver
Control F	Mean	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
	SD	NC	NC	NC	NC	NC	NC	NC
5 F	Mean	87.0	177	2438	722	1484	145	130
	SD	36.8	41.7	425	335	561	81.7	39.8
55 F	Mean	NC	700	3624	41558	31357	8104	5295
	SD	NC	96.2	1449	5668	8793	3075	3561
125 F	Mean	25200	1019	14798	62335	44025	22466	16024
	SD	4630	209	7728	22794	16158	4327	6464

BLQ    <5.00 ng/mL
NC      Not calculated

- Comparing these data to the data for other Ag test items, this demonstrates that levels of Ag distributed into tissues and organs are similar for AgAc and AgNO3 but considerably lower in the case of Ag metal powder.

Conclusions:

This in-vivo toxicokinetic study (in rats) by oral gavage comparing silver metal powder, nanosilver, silver nitrate and silver acetate shows a lower absorption/distribution and lower systemic exposure to tissues and organs for silver metal powder than for the other silver substances. The amount of Ag+ in blood and tissues is much higher for silver acetate/silver nitrate than for silver metal powder at comparable Ag dosing levels. Although all silver-containing substances have the ability to release Ag+, the extent of released silver ions is significantly different between substances. Therefore, a direct read-across of mammalian toxicity datasets for simple ionic silver salts like silver acetate or silver nitrate and nanosilver to silver metal powder/massive is not supported (from a scientifically valid toxicokinetic perspective).

Executive summary:

This in-vivo toxicokinetic study is an oral route investigation using adult CD Sprague Dawley rats. The design conformed to OECD TG 417 and was conducted in compliance to GLP. The study was designed as a comparative toxicokinetic assessment of the test items Silver Acetate (AgAc), Silver Nitrate (AgNO3), Micron-sized Silver (AgMP) and Nanoparticulate Silver (AgNP). This included assessment of bioavailability following single oral doses relative to single intravenous doses and systemic exposure following single and repeat oral doses. Groups of 4 male and 4 female CD Sprague Dawley rats were administered the test items, either as a single intravenous dose, a single oral dose or repeated daily oral doses for 4 weeks.

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

The silver tissue levels after repeated dose administration are considerably lower in the case of silver metal powder (AgMP) than for silver acetate. This suggests that AgMP represents a correspondingly lower health hazard, i.e. AgMP (and by extrapolation also massive Ag with a tremendously lower particle size/reactive surface area) is expected to cause less effects at corresponding Ag dose levels than AgAc.

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

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

Cross-reference 4

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Objective of study:

other: investigation of copper status of rats exposed to silver powder

Qualifier:

no guideline available

Principles of method if other than guideline:

In the comparative in vivo TK study, at termination, analysis of serum for total copper concentration was undertaken in the case of the test items AgMP and AgNO3.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Sub-micron sized powder-form (referenced as 'AgMP' throughout the study report; uncoated; >99% pure; d50 = ~0.3 μm; crystalline powder of highly uniform spheroidal shape) is the finest representative powder on the EU market and considered a worst-case type of silver metal powder in terms of expected bioavailability

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD IGS (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River UK
- Age at study initiation: 8-10 weeks old at start of treatment
- Weight at study initiation: min. 170 g for females and 200 g for males
- Housing: up to four animals of the same sex per cage. Within each group, cages will be blocked together (by sex). The distribution of the groups within and between racks and the position of the racks within the room will be determined to equalize environmental influences across the study while minimizing the opportunity for inter-group contamination.
- Diet (e.g. ad libitum): SDS VRF-,1 pelleted diet, non-restricted availability (diet contains no added antibiotic or other chemotherapeutic or prophylactic agent)
- Water (e.g. ad libitum): potable water from the public supply, non-restricted availability via polycarbonate bottles with sipper tubes (except during urine collection)
- Acclimation period: at least 5 days before commencement of treatment
- Health status: visually inspected at least twice daily for evidence of ill-health

ENVIRONMENTAL CONDITIONS
- Temperature (°C): monitored and maintained within the range of 20-24ºC
- Humidity (%): monitored and maintained within the range of 40-70%
- Air changes (per hr): air filtered, not recirculated
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark
- Fasting period: none

IN-LIFE DATES: study performed in phases, with first phase started on 13 July 2021 and last (repeated) phase ended on 20 August 2021

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

1% w/v aqueous methylcellulose (400 cps)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Notes:
- Chloride and phosphate containing vehicles and reagents were avoided due to the potential for silver precipitation effects.
- Dissolved silver and silver particles adsorb onto material surfaces including glass and metals, and as such use of these was avoided.
- Formulations were protected from light during preparation (use of yellow-light instead).
- The formulation procedures are documented in the study data and will be included in the final report.
- The pH of the final formulations was measured and recorded in the data.
- A series of formulations at the required concentrations is prepared by dilution of individual weighing of the Test Item and dispensed in polypropylene screw top jars or similar

Method of preparation:
- 50% of the final volume of vehicle is added to an appropriate container. The required amount of Test Item is weighed and added to the vehicle whilst stirring at an appropriate speed to obtain a vortex that is half the depth of the suspension. The ‘weighing’ container is rinsed several times using 20% final volume of vehicle. After addition of the rinsing, the suspension is stirred for 60 seconds and then sonicated for approximately 30 minutes. This stir and sonicate step is then repeated, after which the remaining 30% final volume of vehicle is added and stirring continued at an appropriate speed to obtain a vortex that is half the depth of the final suspension for at least 60 minutes.
- The suspensions are sampled for formulation analysis (whilst under constant stirring, according to the previously described technique) prior to dispensing and aliquoting into suitable containers for dose administration.

Frequency of preparation:
- Repeat dose formulations were prepared weekly and split into daily aliquots.
- The suitability and homogeneity of formulations were confirmed as part of studies, undertaken separately by third parties designated by the Sponsor, and by the Principal Investigator for dose formulation analysis.

Storage of formulation: Silver AgMP suspensions: refrigerated (2 to 8°C), protected from light.

Duration and frequency of treatment / exposure:

28 days; frequency: once daily at approximately the same time each day

Dose / conc.:

36 mg/kg bw/day (nominal)

Dose / conc.:

180 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

4 males + 4 females

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

silver nitrate; doses: 5, 55 and 125 mg/kg bw/day (nominal)

Details on dosing and sampling:

TOXICOKINETIC STUDY (Absorption, distribution, excretion)
- Body fluids sampled: blood
- Sample treatment: Whole blood was left to clot according to standard operating procedures. Serum was collected and frozen upright on dry ice following centrifugation, pending transfer to frozen storage.
- Time and frequency of sampling: at termination (after 28 days treatment)

ANALYTICAL METHOD
Serum samples were analysed for copper using ICP-MS method. The method was successfully validated with a lower limit of quantification (LLOQ) of 30 ng/mL.

Statistics:

Summary statistics (e.g. means and standard deviations) presented in the report were calculated from computer-stored individual raw data.

Type:

other: investigation of Cu status of rats exposed to AgMP

Results:

After AgMP dosing, Cu conc. at all 3 dose levels were similar to those of controls; no dose response relationship. After AgNO3 dosing, Cu conc. at LD were similar to those of controls, at MD (55 mg/kg/day) 20% lower and at HD (125 mg/kg/day) 26-38% lower.

Conclusions:

There was no treatment-related effect on the concentrations of copper in serum following repeated oral doses of AgMP, however, after repeated oral doses of AgNO3, the serum concentrations of copper decreased with increasing dose.

Executive summary:

The copper status of rats exposed to micron-sized silver (AgMP) has been evaluated and compared to results for an ionic Ag reference (AgNO3). For AgMP, there was no indication of any depletion in circulating Cu levels after 28 days treatment via the oral route, even with treatment corresponding to a limit dose (1000 mg/kg bw/d). This equates to a Ag equivalent dose level which was 12 times higher than that employed as the AgNO3 high dose comparator. In contrast, a depression in circulating Cu levels was apparent from a quite low exposure in the case of the ionic reference compound (being evident from 35 mg Ag/kg bw/d).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Objective of study:

absorption

bioaccessibility (or bioavailability)

tissue distribution

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Silver

EC Number:

231-131-3

EC Name:

Silver

Cas Number:

7440-22-4

Molecular formula:

Ag

IUPAC Name:

Silver

Test material form:

solid: nanoform

Details on test material:

- Surfactant stabilized aqueous dispersion of nanoparticles containing 4% (w/w) polyoxyethylene glycerol (25) trioleate [TAGAT™ TO] and 4% (w/w) polyoxyethylene (20) sorbitan mono-laurate [Tween 20™] together with 7.5% (w/w) ammonium nitrate
- Particle size distribution: mean: 15 nm; d99: 20 nm
- Density: 1.1 kg/dm3

Specific details on test material used for the study:

Well-characterised Ag nanoparticle reference material (referenced as 'AgNP' throughout the study report). Surfactant stabilized aqueous dispersion of nanoparticles containing 4% (w/w) polyoxyethylene glycerol (25) trioleate [TAGAT™ TO] and 4% (w/w) polyoxyethylene (20) sorbitan mono-laurate [Tween 20™] together with 7.5% (w/w) ammonium nitrate

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD IGS (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 8-10 weeks old at start of treatment
- Weight at study initiation: min. 170 g for females and 200 g for males
- Housing: up to four animals of the same sex per cag. Within each group, cages will be blocked together (by sex). The distribution of the groups within and between racks and the position of the racks within the room will be determined to equalize environmental influences across the study while minimizing the opportunity for inter-group contamination.
- Diet (e.g. ad libitum): SDS VRF-,1 pelleted diet, non-restricted availability (diet contains no added antibiotic or other chemotherapeutic or prophylactic agent)
- Water (e.g. ad libitum): potable water from the public supply, non-restricted availability via polycarbonate bottles with sipper tubes (except during urine collection)
- Acclimation period: at least 5 days before commencement of treatment
- Health status: visually inspected at least twice daily for evidence of ill-health

ENVIRONMENTAL CONDITIONS
- Temperature (°C): monitored and maintained within the range of 20-24ºC
- Humidity (%): monitored and maintained within the range of 40-70%
- Air changes (per hr): air filtered, not recirculated
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark
- Fasting period : none

IN-LIFE DATES: study performed in phases, with first phase started on 30 Sep 2020 and last (repeated) phase ended on 20 Aug 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

1% w/v aqueous methylcellulose (400 cps)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Notes:
- Chloride and phosphate containing vehicles and reagents were avoided due to the potential for silver precipitation effects.
- Dissolved silver and silver particles adsorb onto material surfaces including glass and metals, and as such use of these was avoided.
- Formulations were protected from light during preparation (use of yellow-light instead).
- The formulation procedures are documented in the study data and will be included in the final report.
- The pH of the final formulations was measured and recorded in the data.
- A series of formulations at the required concentrations is prepared by dilution of individual volumes of the Test Item and dispensed in polypropylene screw top jars or similar

Method of preparation:
- 50% of the final volume of vehicle is added to an appropriate container. The required amount of Test Item is weighed and added to the vehicle whilst stirring at an appropriate speed to obtain a vortex that is half the depth of the suspension. The ‘weighing’ container is rinsed several times using 20% final volume of vehicle. After addition of the rinsing, the suspension is stirred for 60 seconds and then sonicated for approximately 30 minutes. This stir and sonicate step is then repeated, after which the remaining 30% final volume of vehicle is added and stirring continued at an appropriate speed to obtain a vortex that is half the depth of the final suspension for at least 60 minutes.
- The suspensions are sampled for formulation analysis (whilst under constant stirring, according to the previously described technique) prior to dispensing and aliquoting into suitable containers for dose administration.

Frequency of preparation:
- Repeat dose formulations were prepared weekly and split into daily aliquots.
- The suitability and homogeneity of formulations were confirmed as part of studies, undertaken separately by third parties designated by the Sponsor, and by the Principal Investigator for dose formulation analysis.

Storage of formulation: Silver AgNP suspensions: refrigerated (2 to 8°C), protected from light.

Duration and frequency of treatment / exposure:

Two treatments:
- single oral dose: single occasion on day 1
- repeat oral doses: 28 days; frequency: once daily at approximately the same time each day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3.6 mg/kg bw/day (nominal)

Remarks:

Silver equivalent value; single + repeat oral doses

Doses / concentrations 2

Dose / conc.:

36 mg/kg bw/day (nominal)

Remarks:

Silver equivalent value; repeat oral doses

Doses / concentrations 3

Dose / conc.:

360 mg/kg bw/day (nominal)

Remarks:

Silver equivalent value; single + repeat oral doses

No. of animals per sex per dose / concentration:

4 males + 4 females

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection and Route:
The dose levels selected in the case of nanoparticulate silver (AgNP) extend existing but limited toxicokinetic information which is available for this form of silver. Previous repeat dose studies in rats via the oral route for durations up to 13 weeks have shown that no marked systemic toxicity or local tissue reactivity has occurred with Ag NP dose levels of up to 500 mg/kg bw/d. Therefore, it is expected that the high-dose group level selected for this study (360 mg/kg bw/d) should be well tolerated.

Details on dosing and sampling:

TOXICOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: blood, brain, bone marrow, small intestine, liver, spleen, ovaries, testes, uterus
- Time and frequency of sampling: blood: Day 15: PD, 6 hours post-dose, Day 28: PD, 3, 6, 9, 12, 24, 72, 96 hours post-dose. (10 time points) ; tissues: at termination (after 28 days treatment)

ANALYTICAL METHOD
Total Ag concentrations were determined in whole blood and tissues by ICP-MS after microwave digestion with nitric acid and hydrochloric acid. This method was successfully validated with a lower limit of quantification (LLOQ) of 10 ng/mL for silver in rat blood4 and 30 ng/g for silver in rat tissue.

Statistics:

- Ag analysis data were acquired and integrated using MassHunter (version C.01.05 Agilent). Peak area results were exported to Watson LIMS (Version 7.5 SP1, Thermo Scientific) in text file format. Watson LIMS was used to calculate standard curve parameters and concentration data for Silver. The concentration and statistical data were generated by computerized techniques.
- AUC(0-t): area under the concentration-time curve from hour 0 to the last measurable concentration, estimated by the linear trapezoidal rule.

Results and discussion

Preliminary studies:

Comprehensive enabling work was done to support the main study. The AgNP and AgMP test items were characterised to confirm suitability of the test items and to confirm stability in an appropriate aqueous-based vehicle. For the AgNP suspension, the ionic Ag was ~5% of the total Ag concentration.

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Based on matched dose assessments, nanosilver (AgNP) exhibits a systemic exposure profile between sub-micron Ag metal powder (‘AgMP’) and a reference ionic Ag salt.

Main ADME results 2

Type:

distribution

Results:

Levels of Ag distributed into tissues and organs after AgNP exposure are between those after AgMP exposure and an those after exposure to an ionic Ag compound

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Comparisons via normalised administered doses (i.e. Ag equivalent dose) can be made between different Ag test items. Based on these matched dose assessments, it can be concluded that oral dosing of AgMP leads to appreciably lower Ag amplitude of systemic exposure in comparison to ionic Ag forms, while AgNP exhibits an intermediate systemic exposure profile.

Details on distribution in tissues:

- Levels of Ag distributed into tissues and organs are considerably lower in the case of AgMP than an ionic Ag compound. AgNP exhibits an intermediate TK profile with achieved tissue levels more closely resembling those of the ionic Ag test items rather than those evident for Ag metal powder (AgMP).
- The study shows that the testis and brain are minor sites of distribution. In contrast, the ovary has been shown to be an important site of distribution. However, ancillary investigations strongly suggest silver is present in the ovary as chemically stable silver sulphide/selenide complexes with very low local bioavailability.

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 686

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 3.6 mg/kg bw/d

Toxicokinetic parameters 2

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 1080

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 3.6 mg/kg bw/d

Toxicokinetic parameters 3

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 186

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 36 mg/kg bw/d

Toxicokinetic parameters 4

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 276

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 36 mg/kg bw/d

Toxicokinetic parameters 5

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 35.3

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for male rats at dose level of 360 mg/kg bw/d

Toxicokinetic parameters 6

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 67.3

Remarks:

Dose normalised AUC(0-t) after 28d dosing; for female rats at dose level of 360 mg/kg bw/d

Any other information on results incl. tables

Systemic exposure / Ag in blood

- Figure 1 (attached below) shows the extent of systemic exposure as area under the concentration time curve (AUC) values after repeated dose administration of nanosilver (AgNP) for 28 days.

- Comparisons via normalised administered doses (i.e. Ag equivalent dose) can be made between different Ag test items. Based on these matched dose assessments, it can be concluded that oral dosing of AgMP leads to appreciably lower Ag amplitude of systemic exposure in comparison to ionic Ag forms, while AgNP exhibits an intermediate systemic exposure profile.

- Table 2 shows the blood Ag concentrations at predicted steady state (day 15) after repeated dose administration of AgNP. The maximum observed concentration occurs between 3-9h post-dosing for repeat dose groups, hence the 6h values for the respective test substances are shown as an indicator of peak exposure.

Table 2.  Blood Ag concentrations at day 15 after repeated oral dose administration of nanosilver (AgNP).

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

- Comparing these data to the data for other Ag test items, this demonstrates that oral dosing of AgMP leads to appreciably lower Ag amplitude of systemic exposure in comparison to ionic Ag forms, while AgNP exhibits an intermediate systemic exposure profile.

Tissue exposure / Ag distribution

Table 3 shows the Ag tissue levels / distribution after repeated dose administration of nanosilver (AgNP).

Table 3. Terminal Ag tissue levels following repeated oral dose administration for 28 days of nanosilver (AgNP)

Dose (mg/kg bw/d) /Sex		Bone marrow	Brain	GI tract	Spleen	Testis	Liver
Control M	Mean	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
	SD	NC	NC	NC	NC	NC	NC
3.6 M	Mean	BLQ	77.5	156	77.0	66.1	BLQ
	SD	NC	13.9	42.8	38.6	26.6	NC
36 M	Mean	181	226	3888	938	452	154
	SD	107	46.5	1799	920	173	114
360 M	Mean	NC	575	11466	14928	1310	1949
	SD	NC	136.9	8252	5257	152	900

Dose(mg/kg bw/d) /Sex		Bone Marrow	Brain	GI Tract	Spleen	Ovaries	Uterus	Liver
Control F	Mean	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
	SD	NC	NC	NC	NC	NC	NC	NC
3.6 F	Mean	BLQ	64.8	415	53.2	116	BLQ	77.2
	SD	NC	12.5	196	21.8	93.7	NC	50.4
36 F	Mean	NC	221	5784	2233	5957	225	382
	SD	NC	13.4	726	1069	2255	133	233
360 F	Mean	NC	604	9630	28081	19170	3865	4325
	SD	NC	36.8	3721	7067	6142	1915	1544

BLQ    <5.00 ng/mL
NC      Not calculated

- Comparing these data to the data for other Ag test items, this demonstrates that levels of Ag distributed into tissues and organs are considerably lower in the case of Ag metal powder than an ionic Ag compound. The silver nanoparticle reference material (AgNP) exhibits an intermediate TK profile with achieved tissue levels more closely resembling those of the ionic Ag test items rather than those evident for Ag metal powder (AgMP)

Applicant's summary and conclusion

Conclusions:

This in-vivo toxicokinetic study (in rats) by oral gavage comparing silver metal powder, nanosilver, silver nitrate and silver acetate shows a lower absorption/distribution and lower systemic exposure to tissues and organs for silver metal powder than for the other silver substances. The amount of Ag+ in blood and tissues is much higher for silver acetate/silver nitrate than for silver metal powder at comparable Ag dosing levels. Although all silver-containing substances have the ability to release Ag+, the extent of released silver ions is significantly different between substances. Therefore, a direct read-across of mammalian toxicity datasets for simple ionic silver salts like silver acetate or silver nitrate and nanosilver to silver metal powder/massive is not supported (from a scientifically valid toxicokinetic perspective).

Executive summary:

This in-vivo toxicokinetic study is an oral route investigation using adult CD Sprague Dawley rats. The design conformed to OECD TG 417 and was conducted in compliance to GLP.cThe study was designedcas a comparative toxicokinetic assessment of the test items Silver Acetate (AgAc), Silver Nitratec(AgNO3), Micron-sized Silver (AgMP) and Nanoparticulate Silver (AgNP). This included assessment of

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

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

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

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

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