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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
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:
other: the current version of draft OECD guideline 487 (2008)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
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 10-30°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:
not applicable
Species / strain
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.
Controlsopen allclose all
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

Migrated to IUCLID6: 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

Migrated to IUCLID6: 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.

Results and discussion

Test results
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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
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.