Silver bromide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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

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)

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:

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:
- 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.:

5 mg/kg bw/day (nominal)

Dose / conc.:

55 mg/kg bw/day (nominal)

Dose / conc.:

125 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

4 males + 4 females

Control animals:

yes, concurrent vehicle

Details on study design:

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 tissues irritancy profile of AgNO3

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 AgNO3

Results:

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.

Details on absorption:

n.a.

Details on distribution in tissues:

n.a.

Details on excretion:

n.a.

Metabolites identified:

no

Details on metabolites:

n.a.

Conclusions:

There was a treatment-related effect on the concentration of copper in serum following repeated oral dose of AgNO3, the serum concentration of copper decreased with increasing dose.

Executive summary:

The copper status of rats exposed to an ionic Ag (AgNO3) was investigated in this study. a treatment-related effects on concentration of Cu in serum was observed after 28 days treatment via the oral route. This depression in circulating Cu levels was apparent from a quite low exposure in the case of the ionic reference compound (AgNO3) (being evident from 35 mg Ag/kg bw/day).

Reference 2

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

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.

Details on excretion:

not investigated

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

Metabolites identified:

no

Remarks:

n.a.

Details on metabolites:

n.a.

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).

Reference 3

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

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.

Details on excretion:

not investigated

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

Metabolites identified:

no

Remarks:

n.a.

Details on metabolites:

n.a.

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).

Reference 4

Endpoint:

dermal absorption in vivo

Remarks:

percutaneous absorption in guiena pigs

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1965

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Reasonably well-documented publication, despite major methodological deficiencies.

Qualifier:

no guideline followed

GLP compliance:

no

Species:

guinea pig

Details on test animals or test system and environmental conditions:

Guinea pig with the average weight of 374-380 g

Type of coverage:

occlusive

Vehicle:

not specified

Remarks:

An amount of 2.0mL of the aqueous solution of the silver nitrate was applied to a skin depot (exposure area 3.1cm2), which was subsequently covered.

Duration of exposure:

5 hours

Doses:

0.239 moles in 2.0 mL of aqueous solution

No. of animals per group:

20 animals

Details on study design:

Guinea pigs were observed for symptoms, food and water intake, and number of deaths. They were weighted once a week.

Total recovery:

After 3 weeks no animals died after exposure to silver nitrate via dermal route.

The weight of animals treated with silver nitrate increased after 1 week 

Conclusions:

Less than 1 % of an applied dose of silver nitrate was absorbed across intact skin of
guinea pigs within 5 hours of exposure. Test system not according to current standards
(i.e., no mass balance, no analysis of actual percutaneous transfer or residual Ag in skin
layers).

Reference 5

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1964

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Reasonably well-documented publication, despite major methodological deficiencies.

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study investigated the absorption of radioactive silver nitrate through the skin of the living guinea pig by following the disappearance of the radioactivity compounds from the cutaneous surface, so called "disappearance measurements".

GLP compliance:

no

Specific details on test material used for the study:

Silver nitrate were obtained from J.T. Baker's Chemical Co (USA) 110m Ag, half-life 253 days.

Radiolabelling:

yes

Remarks:

110mAg (Half-life = 253 days)

Species:

guinea pig

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

weight 300-500 g

Type of coverage:

not specified

Vehicle:

water

Remarks:

distilled water

Duration of exposure:

5 hours

Doses:

The concentrations were chosen which permitted direct comparison with previous investigations.
The lowest concentration was chosen on account of the specific activity of the perspective isotope.
The highest concentration was chosen by limiting solubility in distilled water at room temperature

No. of animals per group:

10-15 animals/dose

Total recovery:

The absorption of silver nitrate is relatively small, most of the value given are <1.0% after 5 hours.

Disappearance constant (k) and disappearance percentage during 5 hours for silver nitrate for different concentration
k.10^5 min-1			< 3.4	3.4-6.6	6.7 -10.1	10.2-13.3
%/ 5 hours			< 1.0	1.0-1.9	2.0-2.9	3.0-3.9
Conc. M	pH	No.	No.	No.	No.	No.
0.00048	6.0	10	9	-	1	-
0.005	5.7	10	6	1	2	1
0.080	5.5	15	9	4	2	-
0.118	5.2	10	9	-	1	-
0.239	4.7	14	12	2	-	-
0.398	4.6	10	8	2	-	-
0.753	4.4	11	8	3	-	-
4.870	3.3	10	10	-	-	-

Disappearance constants and the disappearance percentage per 5-hours periods for silver nitrate for various concentration is presented in the table above.

 

Conclusions:

Less than 1 % of an applied dose of silver nitrate was absorbed across intact skin of
guinea pigs within 5 hours of exposure. Test system not according to current standards
(i.e., no mass balance, no analysis of actual percutaneous transfer or residual Ag in skin
layers).

Description of key information

Introduction:

Most recently, a comparative in-vivo toxicokinetic study (Melvin et al. 2021 and Charlton et al. 2021) with several test substances (silver acetate, silver nitrate, silver metal powder and nanosilver) was performed by the European Precious Metals Federation (EPMF) to supplement the Ag mammalian toxicity database and to strengthen the basis of the read-across approach. This in-vivo oral gavage toxicokinetic study (using rat as test species) shows similar systemic exposure and absorption/distribution to tissues and organs for silver acetate and silver nitrate, notably the amount of silver in blood and tissues is similar after exposure to both silver compounds. Since it is assumed that the systemic toxicity of silver is due to the potential release of Ag⁺, a direct read-across of mammalian toxicity datasets between simple ionic silver compounds (like silver acetate (AgAc) and silver nitrate(AgNO₃)) is considered robust and scientifically valid.

No reliable and consistent in-vivo data are available to demonstrate the toxicokinetic of silver bromide. Nevertheless, in the absence of data measuring the amount of silver ions released from silver bromide, a worst-case approach was adopted to extrapolate the absorption via the different route of exposure. The oral and dermal absorption were extrapolated from silver acetate/silver nitrate and from Ag⁺. While the inhalation absorption has been extrapolated from the MPPD model performed with disilver oxide.

Note: in-vivo TK data have been generated since in-vitro bioaccessibility testing (‘bio elution testing’) is not applicable to assess the bioavailability of silver substances because of the strong binding of Ag⁺ with chloride (Cl^-) resulting in an immediate and excessive AgCl precipitate.

Soluble silver salts, like silver nitrate or silver acetate, rapidly dissociate to Ag⁺ and the relevant counter-ion in aqueous solution:

AgL ⇌ Ag⁺ + L^- with L = NO₃, acetate...

Upon dissolution, the Ag⁺ ions are highly reactive to inorganic and organic ligands, especially sulfidic/thiosulfidic moieties or ammines with formation constants (‘K_f’) of logK_f(AgHS) = 13.6, logK_f(Ag₂S) = 23.7, logK_f(Ag-2-mercaptoethanol) = 13.2, logK_f(Ag-cysteine) = 11.9 or logK_f(Ag-thiourea) = 7.1, logK_f values for Ag-NH₃ complexes or Ag-amines = 3-6 and logK_f(Ag-EDTA) = 7 (Bell and Kramer, 1999).

Upon ingestion and in reasonably expected conditions, Cl is excessively present in the stomach compared to the above-mentioned ligands. Considering this excess of Cl in relation to other potential ligands but also compared to Ag⁺ concentrations, a considerable reaction between Ag⁺ and Cl^- will take place. AgCl (LogK_f(AgCl)) = 3) or AgCl_n-complexes will be formed.

At first, a majority of the released silver ions will be precipitated as:

Ag⁺(aq) + Cl⁻(aq) ⇌ AgCl(s)                                                                   {1}

However, a minor part of AgCl(aq) will remain in solution, and soluble AgCl_n-complexes might be formed at increasing Cl^- concentrations as:

Ag⁺(aq) + Cl⁻(aq) ⇌ AgCl(aq)                             logK=3.70                     {2}

AgCl(aq) + Cl⁻(aq) ⇌ AgCl₂^-(aq)                          logK=1.92                     {3}

AgCl₂^-(aq) + Cl⁻(aq) ⇌ AgCl₃²⁻(aq)                      logK=0.78                     {4}

Artificial stomach fluid (typically applied in bioelution testing) consists of a 0.07 N HCl solution and has an approximate pH 1.2-1.5. This is considered a good approximation of pH and Cl concentrations in human stomach. Looking at the above figure and at the relevant range of Cl-concentrations, the concentration of dissolved silver species is at its minimum and the majority of silver precipitates as AgCl(s). This is typically observed in bioelution tests as an immediate and excessive white precipitate. A minor fraction that remains in solution and does not precipitate as AgCl(s), is present as Ag⁺ and soluble AgCl(aq) or AgCl₂^–-complexes. Only these soluble Ag ionic species are considered available for systemic uptake and circulation.

Oral absorption

Melvin et al. 2021 and Charlton et al. 2021 performed a comparative in-vivo toxicokinetic study via oral route using adult CD Sprague Dawley rats. The design conformed to OECD and EU guidelines (OECD 417 (Toxicokinetics), and was conducted in compliance to GLP.

Four test items were used: two ionic Ag salts (silver acetate and silver nitrate; both > 99.5% pure); a well-characterised and small-size Ag nanoparticles reference material (surfactant stabilized aqueous dispersion with 10% Ag; d50= 15 nm); and a sub-micron sized powder form (AgMP; uncoated; >99% pure; d50~0.3 µm; crystalline powder of highly uniform spheroidal shape) of bulk elemental silver (registered under REACh). Simple soluble salts (both silver nitrate and silver acetate) do undergo some absorption when orally administered. Bioavailability via oral route is estimated at 3-5%, a value that appears consistent with other estimates presented in the scientific literature.

Boudreau et al. 2016 studied the pharmacokinetics of silver. The blood concentration-time curves of silver following a single oral gavage administration of 10 mg/kg bw of AgNP (10, 75 and 110 nm) or AgAc are shown in Figure 3 of orginial paper. The calculated half-times for distribution (4-5 h) and elimination (~24 h), and the time to reach maximum concentration levels (12 h) were similar for both male and female rats. In contrast, the analysis of the AUC (Area Under the Curve) revealed differences due to sex, form of silver, and particle size. The AUC profiles were similar for female (A) and male (B) rats; however, they were ~2-fold greater for female rats, irrespective of silver form or particle size. The AUCs were greater for AgAc than for AgNP, and among AgNP, the AUCs showed an inverse relationship with particle size (i.e. smaller particles had larger AUCs than larger particles – see CSR section).

In a well-documented comparative investigation assessing the bioavailability of radiosilver ^110mAg (as silver nitrate) in mice, rats, monkeys and dogs via oral, intravenous and intraperitoneal administration, less than 10% was absorbed for all routes and more than 90% of silver was excreted in the faeces. About 1% or less of the oral dose was absorbed in all animals with the exception of dogs (<10%). The higher absorption rate in dogs can be explained by the related gut passage time which is in the order of 24 hours for dogs and a bit more than 8 hours in rats (Furchner et al. 1968).

Dermal absorption

Several publications report information on percutaneous absorption of silver.

In a well-documented study with guinea pigs less than 1% of the applied dose of silver nitrate was absorbed through the skin. However, this study has major methodical deficiencies, and is therefore considered only as supporting data (Wahlberg et al., 1965). A review article refers to earlier publications in which an in-vivo percutaneous absorption study in guinea pigs with ¹¹⁰Ag as tracer is described. Despite this study having considerable methodological shortcomings compared to current standards, the authors conclude on a dermal absorption rate of <1% (Skog & Wahlberg, 1964). Other similarly outdated data relate to either non-standard test systems or absorption through wounded or burned skin and is therefore not considered relevant for the assessment of percutaneous absorption through intact skin, as required for risk assessment purposes (e.g. Nguyen et al., 1999).

Overall, the available data for soluble silver substances indicate dermal absorption rates well below 1% of the applied dose.

These conclusions are consistent with the methodology proposed in HERAG guidance for metals (HERAG fact sheet – assessment of occupational dermal exposure and dermal absorption for metals and inorganic metal compounds; EBRC Consulting GmbH / Hannover /Germany; August 2007). The default dermal absorption factors (reflective of full-shift exposure, i.e. 8 hours) are considered adequately conservative for silver substances, i.e. 1% from exposure to liquid/wet media, and 0.1% from exposure to dry (dust).

Inhalation absorption

Disilver oxide is taken as a data source to extrapolate the inhaled absorption of silver bromide. Even though the likelihood of inhalation absorption of silver bromide is very limited due to the manufacturing process, an extrapolation from disilver oxide was performed in a worst-case scenario approach. This extrapolation is considered reasonable and coherent due to their similar very low water solubility (1.6 mg/L for Ag₂O and 0.13 mg/L for AgBr).

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

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

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

	Disilver oxide Batch PMC1	Disilver oxide Batch PMC2
D50	ca. 4 µm	ca. 7-18 µm
Total dustiness	149 mg/g	126 mg/g
Deposition (total)	46.1 %	49.1 %
Deposition (head), fH	45.3 %	47.9 %
Deposition (TB), fTB	0.3 %	0.5 %
Deposition (pulmonary) fPU	0.6 %	0.8 %

 

Based on the fractional deposition, an inhalation absorption factor can be calculated under the following assumption: the material deposited in the head and tracheobronchial regions would be translocated to the gastrointestinal tract without any relevant dissolution, where it would be subject to an assigned default gastrointestinal uptake at a ratio of 3 to 5% (see above).

The material that is deposited in the pulmonary region may conservatively be assumed by default to be absorbed to 100%. This absorption value is chosen in the absence of relevant scientific data regarding alveolar absorption although knowing that this is a conservative choice specifically for poorly soluble substances.

Formula (f_H+ f_TB) * abs_oral+ f_PU* abs_pul= inhalation absorption factor.

Calculation for batch PMC1: (45.3% + 0.3%) * 5 % + 0.6 % * 100 % = 2.88%

Calculation for batch PMC2: (47.9% + 0.5%) * 5 % + 0.8 % * 100 % = 3.22%

 

Estimated inhalation absorption factor of disilver oxide (PMC1)	2.88%
Estimated inhalation absorption factor of disilver oxide (PMC2)	3.22%

Given the inherently conservation assumption of 100% absorption for pulmonary deposition and oral absorption of disilver oxide (extrapolated from silver acetate) at worst-case at 5%, it is considered adequate and conservative to use a value of 3% for inhalation absorption of disilver oxide and by extrapolation of silver bromide.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

5

Absorption rate - dermal (%):

1

Absorption rate - inhalation (%):

3

Additional information

For details, please refer to separate report "CSR Annex 7_Read Across justification Human Health_Nov 2022" which is attached to the CSR in section 13.