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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
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

Test material

Constituent 1
Reference substance name:
disilver(I)sulfate
IUPAC Name:
disilver(I)sulfate
Constituent 2
Reference substance name:
Ag2SO4
IUPAC Name:
Ag2SO4
Constituent 3
Chemical structure
Reference substance name:
Disilver(1+) sulphate
EC Number:
233-653-7
EC Name:
Disilver(1+) sulphate
Cas Number:
10294-26-5
Molecular formula:
Ag.1/2H2O4S
IUPAC Name:
disilver(1+) sulfate
Details on test material:
- Name of test material (as cited in study report): Disilver(I)sulfate, also known as Silver Sulphate
- Physical state: solid, white crystalline powder
- Storage condition of test material: stored at 15 to 25°C in the dark
- It is important to note that Disilver(I)sulfate has most probably formed precipitates with halide anions, i.e. silver chloride (water sol. 1.86mg/L at 25°C). Silver halides are unstable to extended exposure to light (formation of elemental silver and chloride). Therefore exposure to light was kept to the necessary minimum.

Method

Target gene:
thymidine kinase locus
Species / strain
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.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation

Migrated to IUCLID6: 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

Migrated to IUCLID6: 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.

Results and discussion

Test resultsopen allclose all
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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Experiment I, 3 hours treatment

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
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.