Silver bromide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in-vitro: in-vitro bacterial mutagenicity (Ames test)

Waiver: This study has been waived due to the well-known antimicrobial activity of silver substances. Therefore, in-vitro gene mutation tests in bacteria are irrelevant for assessing the genotoxicity potential of silver compounds. In addition, bacterial mutagenesis assays of inorganic metal compounds are frequently negative due to limited capacity for uptake of metal ions and/or the induction of large DNA deletions by metals in bacteria potentially leading to an increased death rate in mutants. The high prevalence of false negatives for metal compounds might suggest that mutagenesis assays with mammalian cells, as opposed to bacterial cells, would be the preferred starting point for testing for this class of Annex VII substances (ECHA Guidance - Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017 (p. 565).

 

Genetic toxicity in-vitro: induction of micronuclei (MN) in mammalian cells

Key study - Lloyd et al. 2010 performed an in-vitro micronucleus test according to the OECD 487 (draft guideline) and following the GLP principles on disilver sulphate. The in vitro micronucleus tests was performed in human peripheral blood lymphocytes (PBL’s) from whole blood cultures. Under all treatments testing was performed up to and above (in the case of the 24 hour treatment) the limits of toxicity. At all tested concentrations there were no cultures that resulted in micronucleus frequencies that lay outside the historical control range in use at the time. As such, it was concluded that disilver sulfate was negative for induction of micronuclei and neither clastogenic nor aneugenic in human PBL’s.

Genetic toxicity in-vitro: Gene mutations in mammalian cells (thymidine kinase)

Key study - Lloyd et al. 2010 performed a GLP and OECD guideline compliant MLA assay in L5178Y cells on disilver sulfateusing the microplate method comprising a range finder and two main experiments in the presence and absence of Araclor 1254 induced rat liver S-9.The range finding experiment was performed to define the concentrations tested in the main experiments. The highest concentration under all conditions were limited by toxicity, measured as a decrease in relative total growth (RTG). In the main experiments treatments were for 3 hours in the presence and absence of S-9 (Experiment 1) and 24 hours in the absence of S-9 (Experiment 2).

• In experiment 1 (3 hour treatments): in the presence of S-9, the mutation frequencies did not show any significant changes across the concentration range tested. In the absence of S-9, a significant increase in mutation frequency was noted at the highest concentration only (1.5μg/mL) at 12% RTG which only marginally exceeded the GEF (MF of 200 compared to GEF of 197). When the individual cultures were examined, only one exceeded the GEF.


• In experiment 2 (3 hour treatments): in the presence of S-9  no significant increases in mutation frequency. in the absence of S-9  a larger increase in mutation frequency with the highest two concentrations (1.2 and 1.3μg/mL) clearly exceeding the GEF (188x106) alongside a positive linear trend test confirming a dose related increase in mutant frequency.


• 24 hours in absence of S-9 there were slight increases in mutation frequency that resulted in a positive linear trend test however, all values were well within the GEF (182x106) and as such this timepoint was considered negative for induction of mutants.

In the case of Disilver sulfate, there were increases in both small and large colonies. As such the study concludes that silver sulfate is positive for induction of point mutations. It should be noted that interpretation of positive effects seen only under conditions of high toxicity (10-20% RTG) should be interpreted with care. In this case the only concentrations resulting in mutation frequencies that exceeded the GEF were found within the 10-20% RTG level and as such while this study fulfils the criteria for a positive response it is only under conditions that are unlikely to occur in vivo.

Other data:

Butler et al. 2015 evaluated nanoparticle genotoxicity, used silver nitrate as a control in Ames, in vitro MN and the comet assay in Jurkat and THP-1 cell lines. It was reported that AgNO3 did not induce mutation in salmonella strains TA98, TA100 and TA102 as well as E.coli strains WP2 PKM101 and WP2uvraPKM101. Revertant numbers were reduced at higher concentrations indicative of a bacteriotoxic effect that would be expected from treatment with silver ions. Silver nitrate induced elevated levels of MN and comets in both cell lines. However it is noted that the present study used a measure of toxicity that does not include a proliferative marker, as such testing could easily have proceeded into the toxic range (as measured by OECD 487recommended procedures). The cell lines THP-1 and Jurkat are not commonly used in genotoxicity studies. As such this data is considered unreliable for estimation of chromosome damaging potential of silver salts.

Jiang et al. in 2013 used silver nitrate as a control in a study evaluating the genotoxic potential of silver nanoparticles in the CHO-K1 cell line. The mitochondrial activity was used to evaluate toxicity, 8-oxodG adducts were also measured to evaluate the contribution of reactive oxygen. AgNO3 induced both increases in MN frequency as well as 8-oxodG adducts implicating reactive oxygen in the genotoxic potential of silver ions. It should be noted that whilst OECD 487 does not preclude the use of rodent derived cell lines the use of p53 competent cells should be used in preference to reduce “misleading positives” that these cell lies are susceptible to Fowler et al. 2012.

Guo et al. published in 2015 a study evaluating predominantly silver nanoforms but also tested silver nitrate and silver acetate in an Ames test with two strains, TA100 and TA98, both were negative but noted bacterial toxicity from treatments with silver acetate and silver nitrate. Both silver salts gave positive responses in the mouse lymphoma test performed within this study although it should be noted that the GEF was not used in interpretation and the raw data not given to accurately determine toxicity (chart only). The same study also evaluated micronuclei, positive responses are only generally seen as cell survival decreases.

Whilst these additional sources of data find positive responses with exposure to silver ions, they have critical defects in study design and methodology that makes them difficult to interpret. None compare endpoint increases against historical ranges or use adequate statistical analyses (i.e. data is not normally distributed). These studies may add to the pattern already seen i.e. Ames negative, scattered MLA positive (under high toxicity) but conflict other data with difficult to interpret in vitro clastogenicity effects.

As high quality in vitro and in vivo clastogenicity test data are negative and considering the limitations of these additional data, little weight should be given to these studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-07-27 till 2009-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: the current version of draft OECD guideline 487 (2008)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: from human donors

Details on mammalian cell type (if applicable):

Blood from two healthy, non-smoking male volunteers was used for each experiment in this study. The measured cell cycle time of the donors used at
Covance falls within the range 13 +/- 1.5 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes on the day of culture initiation.
- Type and identity of media: HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 50 μg/mL gentamycin. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37°C±1°C for 48 hours and rocked continuously.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Range-Finder:
- without S9 mix, 3 hours treatment: 3.265, 5.442, 9.07, 15.12, 25.19, 41.99, 69.98, 116.6, 194.4, 324.0, 540.0 and 900.0 µg/mL
- without S9 mix, 24 hours treatment: 3.265, 5.442, 9.07, 15.12, 25.19, 41.99, 69.98, 116.6, 194.4, 324.0, 540.0 and 900.0 µg/mL
- with S9 mix, 3 hours treatment: 3.265, 5.442, 9.07, 15.12, 25.19, 41.99, 69.98, 116.6, 194.4, 324.0, 540.0 and 900.0 µg/mL
Concentrations for the Main Experiment were selected based on the results of this cytotoxicity Range-Finder Experiment.
Main Experiment:
- without S9 mix, 3 hours treatment: 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 40.0, 60.0, 80.0, 100.0 and 120.0 µg/mL
- without S9 mix, 24 hours treatment: 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, 20.0, 30.0, 40.0, 60.0 and 80.0 µg/mL
- with S9 mix, 3 hours treatment: 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 60.0, 80.0, 100.0 and 120.0 µg/mL
For more details see table in the field "Any other information on material and methods incl. tables" in the technical dossier.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Disilver(I)sulfate was soluble in purified water at a concentration of approximately 9.590 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

0.6 and 0.8 µg/mL, dissolved in purified water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

6.25 and 12.5 µg/mL, dissolved in anhydrous analytical grade dimethyl sulphoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: vinblastine; 0.03 and 0.04 µg/mL, dissolved in purified water

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

Treatments were conducted 48 hours following mitogen stimulation by Phytohaemagglutinin (PHA).

DURATION
- Exposure duration: 3 hours (+21 hours recovery) or 24 hours; For removal of the test article, cells were pelleted, washed twice with sterile saline and resuspended in fresh pre-warmed medium containing foetal calf serum and gentamycin.
- Inhibition of cell division: Cytochalasin B, formulated in DMSO, was added to post wash-off culture medium to inhibite cytokinesis (cell division).
- Fixation time (start of exposure up to fixation or harvest of cells): Cells were harvested 72 hours after culture initiation.

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): After fixation and preparation of the slides, the cells were stained for 5 minutes in filtered 4% (v/v) Giemsa in pH 6.8 buffer.

NUMBER OF REPLICATIONS: 2 replicates were cultured

NUMBER OF CELLS EVALUATED: Where possible, 1000 binucleate cells from each culture (2000 per concentration) were analysed for micronuclei.

DETERMINATION OF CYTOTOXICITY
- Method: replication index (RI):
A cytotoxicity Range-Finder test was performed. S-9 mix or KCl (0.5 mL/culture) was added appropriately immediately prior to treatment to the cultures. Cultures were treated with the test article or vehicle control (1 mL/culture). Cytochalasin B, formulated in DMSO, was added directly to all continuous (24+0 hour –S-9) cultures at the time of treatment. Cultures were incubated at 37°C ± 1°C for the designated exposure time.
Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined.

RI, which indicates the relative number of nuclei compared to controls was determined using the formulae below:
RI = number binucleate cells + 2(number multinucleate cells)/total number of cells in treated cultures.
Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows:
Relative RI (%) = RI of treated cultures/RI of vehicle controls x 100.
Cytotoxicity (%) is expressed as (100 – Relative RI).

OTHER EXAMINATIONS:
Binucleate cells were only included in the analysis if all of the following criteria were met:
1) the cytoplasm remained essentially intact, and
2) the daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1) the micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2) any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3) micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result.

Statistics:

After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportion of MNBN cells for each treatment condition were compared with the proportion in negative controls by using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell were obtained and recorded.

Species / strain:

lymphocytes: from humans

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Treatment resulted in frequencies of micronucleated binucleate (MNBN) cells that were generally similar to (and not significantly different from) those observed in concurrent vehicle controls for all concentrations analysed.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The highest tested concentration in the Main Experiment (120.0 μg/mL) was limited by toxicity (determined in a preliminary cytotoxicity Range-Finder Experiment).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality an pH were measured in the cytotoxicity Range-Finder Experiment.
- Effects of pH: No marked changes in pH were observed at the highest concentration tested (900.0 μg/mL) as compared to the concurrent vehicle controls.
- Effects of osmolality: No marked changes in osmolality were observed at the highest concentration tested (900.0 μg/mL) as compared to the concurrent vehicle controls.
- Water solubility: Preliminary solubility data indicated that Disilver(I)sulfate was soluble in purified water at a concentration of approximately 9.590 mg/mL.
- Precipitation: The solubility limit in culture medium was less than 479.5 μg/mL, as indicated by precipitation at this concentration which persisted for approximately 20 hours after test article addition. A maximum concentration of 900.0 μg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to a precipitating concentration.

RANGE-FINDING/SCREENING STUDIES: no further data

COMPARISON WITH HISTORICAL CONTROL DATA: The MNBN cell frequency of all Disilver(I)sulfate treated cultures fell within the observed normal historical ranges.

ADDITIONAL INFORMATION: For the 3+21 hour treatment in the presence of S9 mix, the MNBN cell frequencies in single cultures at 15.0 and 80.0 μg/mL marginally exceeded the 95th percentile of the normal range but both fell within the observed normal range. These observations were not considered biologically relevant.

Conclusions:

Interpretation of results: negative

It is concluded that Disilver(I)sulfate did not induce micronuclei in cultured human peripheral blood lymphocytes in the absence and presence of S-9 when tested up to the limit of cytotoxicity.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-07-24 till 2009-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells were stored as frozen stocks in liquid nitrogen.
- Type and identity of media: RPMI 1640 media supplemented with heat inactivated horse serum (RPMI A = 0% v/v; RPMI 10 = 10% v/v and RPMI 20 = 20% v/v), penicillin (100 units/mL) and steptomycin (100 μg/mL), Amphotericin B (2.5 μg/mL), Pyruvic acid (0.2 mg/mL) and Pluronic (0.5 mg/mL for RPMI A and RPMI 10).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Range-Finder:
- with and without metabolic activation, 3 and 24 hours treatment: 0.03906, 0.07813, 0.1563, 0.3125, 0.625, 1.25, 2.5, 5.0 and 10.0 µg/mL
Concentrations selected for Experiment I and II were based on the results of the second cytotoxicity Range-Finder Experiment.
Experiment I:
- without metabolic activation, 3 hours treatment: 0.0, 0.1, 0.2, 0.4, 0.7, 1.0, 1.2, 1.3, 1.5, 1.75 and 2.0 µg/mL
- with metabolic activation, 3 hours treatment: 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 µg/mL
Experiment II:
- without metabolic activation, 3 hours treatment: 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.3, 1.4, 1.5 and 1.75 µg/mL
- with metabolic activation, 3 hours treatment: 0.0, 0.5, 1.0, 1.5, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0 and 3.5 µg/mL
- without metabolic activation, 24 hours treatment: 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, 0.9, 1.0, 1.1, 1.2 and 1.5 µg/mL
For more details see table in the filed "Any other information on material and methods incl. tables" in the technical dossier.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Disilver(I)sulfate was soluble in water for irrigation (purified water) with vortex mixing, extensive ultrasonication and warming at 37°C at a concentration of approximately 9.590 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation
15.0 and 20.0 µg/mL (Exp.I) and 5.0 and 7.5 µg/mL (Exp.II), dissolved in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

2.0 and 3.0 µg/mL, dissolved in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours (Exp. I and II) and 24 hours (Exp. II):
After 3 hours incubation at 37±1°C with gentle agitation, cultures were centrifuged, washed and resuspended in fresh RPMI 10 medium.
After static incubation for 24 hours at 37±1°C, cultures were centrifuged washed and resuspended in fresh RPMI 10 medium.
- Expression time (cells in growth medium): Cultures were maintained in flasks for a period of 2 days during which the tk-/- mutation would be expressed. During the expression period, subculturing was performed as required with the aim of not exceeding 1 x 10^6 cells/mL.
- Selection time (if incubation with a selection agent): At the end of the expression period, TFT (300 μg/mL) was added to the cell cultures in a final concentration of 3 μg/mL. Plates were incubated at 37±1°C in a humidified incubator gassed with 5% v/v CO2 in air until scoreable (12 days).

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Each treatment, in the absence or presence of S9 mix, was in duplicate (single cultures only used for positive control treatments).

NUMBER OF CELLS EVALUATED: not applicable; Two types of TFT-resistant mutant colonies are selected and these are designated large colonies and small (slow-growing) colonies.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
In the absence of S9 mix, 3 and 24 hours treatment incubation periods were used and in the presence of S9 mix a 3 hour treatment incubation was used for the cytotoxicity Range-Finder Experiment.
A maximum concentration of 900 μg/mL was originally selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed in excess of the limit of solubility in culture medium. However, extreme toxicity was observed under all treatment conditions in this experiment, therefore a second Range-Finder Experiment was performed at concentrations up to a maximum of 10 μg/mL. Concentrations selected for Experiment 1 were based on the results of the second cytotoxicity Range-Finder Experiment.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The mutant frquency (MF) of any test concentration exceeded the sum of the mean control mutant frequency plus GEF
2. The linear trend test was positive.
The test article was considered as positive in this assay if both of the above criteria were met.
The test article was considered as negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RTG lower than 10%.

Statistics:

The significance of increases in mutant frequencies (total wells with clones), by comparison with concurrent controls and the global evaluation factor (GEF), was assessed according to the recommendations of the Mouse Lymphoma Workgroup, Aberdeen, 2003. The control mutant frequency was compared with each test article treatment and the data were checked for a linear trend in mutant frequency with treatment concentration using weighted regression. The test for linear trend is one-tailed, therefore negative trend was not considered significant.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

An increase in mutant frequency was observed at 1.5 μg/mL, the highest concentration analysed.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2 days after treatment the highest 2 concentrations tested in the absence of S9 mix and the highest 4 concentrations tested in the presence of S9 mix were considered too toxic for selection to determine viability and TFT resistance.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

In the presence of S9 mix, no marked increases in mutant frequency were observed at any concentration analysed. A weak linear trend was observed, but this observation was not considered biologically relevant.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2 days after treatment the highest 2 concentrations tested in the absence of S9 mix and the highest 4 concentrations tested in the presence of S9 mix were considered too toxic for selection to determine viability and TFT resistance.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Increases in mutant frequency were observed at 1.2 and 1.3 μg/mL, respectively.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2 days after treatment the highest 2 concentrations tested in the absence of S9 mix and the highest 5 concentrations in the presence of S9 mix were considered too toxic for selection to determine viability and TFT resistance.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

In the presence of S9 mix, no marked increases in mutant frequency were observed at any concentration analysed.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2 days after treatment the highest 2 concentrations tested in the absence of S9 mix and the highest 5 concentrations in the presence of S9 mix were considered too toxic for selection to determine viability and TFT resistance.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No marked increases in mutant frequency were observed at any concentration analysed.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2 days after treatment the highest 3 concentrations tested were considered too toxic for selection to determine viability and TFT resistance.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH measurements on post-treatment incubation medium were taken in the cytotoxicity Range-Finder Experiment.
- Effects of pH: No marked changes in pH were observed in the 3 and 24 hour Range-Finder Experiment at the highest concentration tested (10.0 μg/mL), compared to the concurrent vehicle controls.
- Effects of osmolality: No marked changes in osmolality were observed in the 3 and 24 hour Range-Finder Experiment at the highest concentration tested (10.0 μg/mL), compared to the concurrent vehicle controls.
- Water solubility: Preliminary solubility data indicated that Disilver(I)sulfate was soluble in water at a concentration of approximately 9.590 mg/mL.
- Precipitation: The solubility limit in culture medium was less than 479.5 μg/mL, as indicated by precipitation at this concentration which was present approximately 24 hours after test article addition.

RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity Range-Finder Experiment, 24 hour treatment, 9 concentrations were tested in the absence of S9 mix, ranging from 0.03906 to 10.00 μg/mL. The highest concentration to provide >10% RTG was 0.625 μg/mL, which gave 45% RTG.

COMPARISON WITH HISTORICAL CONTROL DATA: no further data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: The highest concentrations selected were 1.5 μg/mL in the absence of S9 mix and 3 μg/mL in the presence of S9 mix, which gave 12% and 5% Relative Total Growth (RTG), respectively. Steep concentration-related toxicity was observed in the presence of S9 mix, such that a concentration of 2.5 μg/mL gave 61% RTG, therefore it was considered prudent to analyse cultures at 2.5 and 3 μg/mL for mutation.
- Experiment 2 (3 hour treatment): The highest concentrations selected in the absence and presence of S9 mix (1.4 and 2.2 μg/mL, respectively) were later rejected from analysis due to extreme toxicity (<10% RTG). The highest concentrations analysed were 1.3 μg/mL in the absence of S9 mix and 2 μg/mL in the presence of S9 mix, which gave 11% and 20% RTG, respectively.
- Experiment 2 (24 hour treatment): The highest two concentrations selected (0.9 and 1 μg/mL) were later rejected from analysis due to extreme toxicity (<10% RTG). The highest concentration analysed was 0.8 μg/mL, which gave 18% RTG.

Conclusions:

Interpretation of results:
positive without metabolic activation
negative with metabolic activation

It is concluded that Disilver(I)sulfate was mutagenic in this test system when tested up to toxic concentrations for 3 hours in the absence of S-9, but was not mutagenic when tested up to toxic concentrations for 3 hours in the presence of S-9 and for 24 hours in the absence of S-9.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Genetic Toxicity in-vivo: Induction of micronuclei (MN) in mammalian cells

Boudreau et al (2016) studied the genotoxicity effects of silver acetate in male and female rats by an in-vivo micronucleus assay using peripheral blood cells. Animals were treated during 90 days with 100, 200 or 400 mg/kg bw/day of silver acetate by oral gavage. For the micronucleus endpoint blood was taken via the tail vein at weeks 1, 4 and 12 weeks, fixed in ice cold methanol and stored frozen (-80ºC) until analysis. Micronuclei were evaluated from thawed frozen samples using a litron MicroFlow™ kit, a well validated and established method. A total of 20000 reticulocytes were analysed for the presence of micronuclei. 

The authors concluded that silver acetate did not induce micronuclei in the peripheral blood of SD rats after 13 week treatments. There was an increase in micronucleated reticulocytes at 400mg/kg at week 4 however the authors report that at the 400mg/kg dose animals suffered severe gastric distress and few made it to 12 weeks. At the 12 week point animal numbers were reduced to single animals in some cases therefore 400 mg/kg could be considered too high a dose to analyse for micronuclei.

The results of the micronucleus assay of peripheral blood concluded that silver acetate does not induce micronuclei in reticulocytes of female and male rats.

Based on the above study on silver acetate and read-across, it is assumed that other silver compounds do not induce micronuclei in-vivo and therefore, might not induce chromosome breaks or losses.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

According to OECD guideline for testing chemicals in toxicity studies in rodents (Guidelines for the Testing of Chemicals, Section.Health Effects. Test No. 408: Repeated dose 90-day oral toxicity study in rodents.) and NTP specification for the conduct of studies in laboratory animals NTP, 2011)

Principles of method if other than guideline:

The genotoxic effects of silver acetate exposure in mal and female rats were examined with a micronucleus assay, using flow cytometric analysis of peripheral blood (Witt et al., 2008). Blood obtained from the rat at week 1, 4 and 12 of the study were fixed in ultracold methanol and stored at -80°C until analysis for the frequency of micronucleated cells in 20,000 reticulocytes per samples.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

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

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

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

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

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

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

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

Route of administration:

oral: gavage

Vehicle:

Water/0.1% methyl cellulose, methylcellulose was used in this study as bulking agents to inhibit somewhat the gastroinstestinal passage of the mostly aqueous dose formulation. The compounds is not toxic and do not promote allergic reactions in rodents.

Details on exposure:

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

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

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

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

Duration of treatment / exposure:

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

Frequency of treatment:

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

Post exposure period:

none

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

List of tissues examined:
- jejunum, ileum, colon, kidney, liver, spleen,

Details of tissue and slide preparation:

At necropsy, organs and tissues were examined for grossly visible lesions, and protocol designated tissues and organs were weighed and preserved in 10% neutral buffered formalin (NBF), with the exception that modified Davidson’s fixative was used for the right testes and eyes. Femur bone marrow was collected for histopathology (left) and ICP-MS (right). The testes and epididymides were collected for sperm analysis (left) and histopathology (right). A section of the ileum and scrapping of the ileal mucosa were collected for intestinal microbiota and immune response evaluations (Williams et al., 2015). Sections
of the proximal ileum, jejunum, proximal colon, and mesenteric lymph nodes, the right kidney, median and left lateral liver lobes, and spleen were preserved for 2 weeks at 4°C in Karnovsky’s fixative and stored in 10% NBF at 4°C for TEM evaluations.
Additional sections of these organs and tissues, along with the heart and uterine horn, were collected, flash frozen in liquid nitrogen, and stored at 80°C for total silver analysis by ICP-MS.

Statistics:

Micronuclei analysis was conducted for each sex using a 2-way fixed effect ANOVA to determine the effect of treatment using the arcsin square root transformation for percent data. Pairwise comparisons of the 2 control groups and each of the treatment groups to the appropriate control group were performed with Bonferroni adjustments. All tests were conducted as 2-sided with significance at the .05 probability level.

Sex:

male/female

Genotoxicity:

ambiguous

Remarks:

high toxicity level

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks on result:

other: 400 mg/kg bw/d small but significant increase in the frequency of micronucleated reticulocytes at week-4, but not subsequent time point. Those animals had severe gastroinestinal distress and only 1 female and no male rats survived to week 12

Conclusions:

Male and female rats administered with 400 mg/kg bw/day (highest dose) had a small but significant increase in frequency of micronucleated reticulocytes at week-4 only, but not at subsequent time points. In addition, these animals had severe gastrointestinal distress and only 1 female and no male rats survived to week 12.

In conclusion, under the study conditions, silver acetate do not induce micronuclei in reticulocytes of female and male rats.

Executive summary:

The genotoxic effects of silver acetate exposure in mal and female rats were examined with a micronucleus assay, using flow cytometric analysis of peripheral blood (Witt et al., 2008). Blood obtained from the rat at week 1, 4 and 12 of the study were fixed in ultracold methanol and stored at -80°C until analysis for the frequency of micronucleated cells in 20,000 reticulocytes per samples.

Male and female rats administered with 400 mg/kg bw/day (highest dose) had a small but significant increase in frequency of micronucleated reticulocytes at week-4 only, but not at subsequent time points. In addition, these animals had severe gastrointestinal distress and only 1 female and no male rats survived to week 12.

In conclusion, under the study conditions, silver acetate do not induce micronuclei in reticulocytes of female and male rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The genotoxicity database consists of in-vitro and in-vivo studies performed with disilver sulphate (in-vitro) and silver acetate (in-vivo). Following a rigorous assessment of the in-vitro and in-vivo micronucleus assays, it can be concluded, by a read-across approach, that silver compounds do not show clastogenic or aneugenic potential.

However, the in-vitro cytogenicity assay in mammalian cells test concluded that disilver sulphate was mutagenic, in this test system, up to toxic concentrations for 3 hours in the absence of S-9 (metabolic activation). While it was not mutagenic under the other conditions tested (up to toxic concentrations for 3 hours in the presence of S-9 and for 24 hours in the absence of S-9).

Overall, there is no evidence that disilver sulphate have toxicity in chromosome (clastogen or aneugen), however, with only in-vitro positive results (in absence of S-9), it cannot be demonstrated that disilver sulphate, and by read-across other silver compounds, is not a mutagen in-vivo.

Therefore, according to ECHA guidance R.7a to identify evidence of gene mutation a TGR (OECD 489) or a Comet Assay test (OECD 488) would be the appropriate follow up test. Nevertheless, according to the column 2 of Annex VIII of REACh regulation, an in-vivo mutagenicity study is not required if the substance is classified as Carcinogen Cat. 1. Since silver acetate is undergoing an in-vivo carcinogenicity testing (OECD 453) and that the study report will be available in September 2024; it was decided to consider at first the outcome of ongoing carcinogenicity study prior intiating a new in-vivo test for the assessment of mutagenicity on silver compounds, in order to avoid unnecessary use of additional animals. Hence, depending on the outcome of the carcinogenicity study, further evaluation of this specific endpoint will be conducted, if necessary.