Calcium thiosulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Experimental work started on 21 July 2010 and was completed on 20 September 2010.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP and 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

Test material information

Constituent 1

Reference substance name:

Ammonium thiosulphate

EC Number:

231-982-0

EC Name:

Ammonium thiosulphate

Cas Number:

7783-18-8

Molecular formula:

H3N.1/2H2O3S2

Constituent 2

Reference substance name:

ammonium thiosulfate

IUPAC Name:

ammonium thiosulfate

Details on test material:

- Name of test material (as cited in study report): ammonium thiosulfate
- Molecular formula (if other than submission substance): (NH4)2S2O3
- Molecular weight: 148.2 g/mol
- Physical state: solid, white powder
- Storage condition of test material: stored at 15-25°C in the dark

Method

Target gene:

hprt

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 media containing 2.5 µg/mL Amphotericin B, 100 µg/mL Streptomycin, 100 units/mL Penicillin, 0.5 µg/mL Pluronic (ecxept for RPMI 20) and 0%, 10% or 20% (v/v) heat inactivatet horse serum for RPMI A, RPMI 10 and RPMI 20, respectively.

The master stock of L5178Y tk+/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; each batch of frozen cells was confirmed to be mycoplasma free.
- Periodically checked for karyotype stability: yes; each batch of frozen cells was purged of mutants.
- Periodically "cleansed" against high spontaneous background: yes
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

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 experiment:
- with and without S9-mix: 46.31, 92.63, 185.3, 370.5, 741 and 1482 µg/mL
Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range-Finder Experiment.

Experiment I:
- with and without S9-mix: 200, 400, 600, 800, 1000, 1200, 1350 and 1482 µg/mL
Experiment II:
- with and without S9-mix: 100, 300, 600, 900, 1100, 1300 and 1482 µg/mL

Cultures selected for mutation assessment:
Experiment I:
- with and without S9-mix: 200, 400, 600, 800, 1000, 1200, 1350 and 1482 µg/mL
Experiment II:
- with and without S9-mix: 300, 600, 900, 1100, 1300 and 1482 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that ammonium thiosulfate was soluble in water for irrigation (purified water) at concentrations up to at least 34.99 mg/mL.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

treatment with the vehicle (purified water) diluted 10 fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide; 0.1 and 0.15 µg/mL (dissolved in DMSO)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

treatment with the vehicle (purified water) diluted 10 fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Migrated to IUCLID6: 2 and 3 µg/mL (dissolved in DMSO)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours at 37°C
- Expression time (cells in growth medium): 7 days during which the hprt mutation would be expressed: After the incubation period, cultures were centrifuged (200 g), washed and resuspended in RPMI 10. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival (scoreable after 7 days).
- Selection time (if incubation with a selection agent): 12 to 14 days: At the end of the expression period, aliquots of cell suspension were placed into each well of 4 x 96 well microtitre plates (384 wells at 2 x 104 cells/well). Plates were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air and wells containing clones were identified and counted.

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: 2

EVALUATION: wells containing clones were identified and counted

DETERMINATION OF CYTOTOXICITY
- Method: relative survival:
Treatment and post treatment dilution of cell cultures for the cytotoxicity Range Finder Experiment was as described for the Mutation Experiments. However, single cultures only were used and positive controls were not included.
Following treatment, cells were centrifuged (200 g) washed with tissue culture medium and resuspended. Cells were plated into each well of a 96 well microtitre plate for determination of relative survival. The plates were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air for 7 days. Wells containing viable clones were identified by eye using background illumination and counted.

OTHER:
- Probable number of clones/well (P) = -ln (empty wells (without clones) /total of TW),
- Plating efficiency (PE) = P/No of cells plated per well,
- Percentage relative survival (% RS) = % RS = [PE (test)/PE (control)] x 100,
- Mutant frequency (MF) = [PE (mutant)/PE (viable)] x 10^6.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p<0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p<0.05).
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No statistically significant increases in mutant frequency were observed following treatment with ammonium thiosulfate at any concentration tested and there were no significant linear trends.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 media were taken in the cytotoxicity Range-Finder Experiment.
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations analysed (1482 µg/mL) as compared to the concurrent vehicle controls (individual data not reported).

RANGE-FINDING/SCREENING STUDIES: 6 concentrations were tested in the absence and presence of S9 ranging from 46.31 to 1482 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration analysed was 1482 µg/mL, which gave 103% and 65% RS in the absence and presence of S9, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: yes; comparison of controls with historical means of the positive control substances.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I, 8 concentrations, ranging from 200 to 1482 g/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration analysed gave 107% and 95% RS in the absence and presence of S9 respectively.
In Experiment II, 7 concentrations, ranging from 100 to 1482 µg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration analysed gave 68% and 107% RS, respectively.

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 ammonium thiosulfate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study in the absence and presence of a rat liver metabolic activation system (S9).

Executive summary:

Ammonium thiosulfate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells. The study consisted of a cytotoxicity Range-Finder experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation (S9).

The test article was formulated in water for irrigation (purified water). A 3 -hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, 6 concentrations were tested in the absence and presence of S9, ranging from 46.31 to 1482 mg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration analysed was 1482 mg/mL, which gave 103% and 65% relative survival (RS) in the absence and presence of S‑9, respectively.

In Experiment I, concentrations, ranging from 200 to 1482 mg/mL, were tested in the absence and presence of S9. 7 days after treatment all concentrations in the absence and presence of S9 were selected to determine viability and 6TG resistance. The highest concentration analysed was 1482 mg/mL, which gave 107% and 95% RS in the absence and presence of S9 respectively.

In Experiment II, concentrations, ranging from 100 to 1482 mg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration analysed to determine viability and 6TG resistance was 1482 mg/mL, which gave 68% and 107% RS in the absence and presence of S9, respectively.

Vehicle and positive control treatments were included in each Mutation Experiment.

In Experiments I and II no statistically significant increases in mutant frequency were observed following treatment with ammonium thiosulfate at any concentration tested in the absence and presence of S9 and there were no significant linear trends.