Magnesium thiosulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

from 2001-04-26 till 2001-05-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Justification for type of information:

The basis for the read-across concept for this project is the equilibrium between sulfites, hydrogensulfites, and metabisulfites in aqueous solutions depending on pHvalue which is clearly described in published literature and summarised in the following equations:[1],[2]
           SO2+ H2O <->`H2SO3´         H2SO3<->H++ HSO3-<->2H++SO32-    2HSO3-<->H2O +S2O52-
As the nature of the cation should make no significant difference in this case concerning toxicity and solubility (all substances are very soluble in water), only the chemical and biological properties of the anion are considered relevant. Based on the described equilibrium correlations, we propose unrestricted read-across between the groups of sulfites, hydrogensulfites and metabisulfites.

Additionally, it is known that sodium dithionite disproportionates in water to form sodium hydrogen sulfite and sodium thiosulfate (equation II) so that this substance can also be added to the read-across concept.[2],[1]It is expected for this case that the substance is not stable enough under physiological conditions to fulfil the requirements of study guidelines and so the products of decomposition have to be considered.
       2 S2O42-+ H2O→2HSO3-+ S2O32 -
 
Not completely included in this read-across concept is the substance class of thiosulfates. Although thiosulfates may also disproportionate in aqueous solution to form polythionic acids and SO2(HSO3-), the required conditions are somewhat different (more acidic) and are therefore not strictly comparable with physiological conditions, except for the case of oral application where read-across should be considered unrestricted due to the strongly acidic conditions in the stomach:
       HS2O3-+ H2S2O3→HS3O3- + SO2+ H2O
Nevertheless, read-across for all other routes (dermal, inhalation) should also be considered.
The proposed read-across concept only applies to toxicological and ecotoxicological/environmental fate endpoints.
[1]Hollemann Wiberg, Lehrbuch der Anorganischen Chemie, 101.Auflage
[2]Handbook of Chemistry and Physics, Ed. Lide, DR, 88thedition, CRC Press

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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

IUPAC Name:

diammonium thiosulfate

Constituent 2

Reference substance name:

Ammonium thiosulfate

IUPAC Name:

Ammonium thiosulfate

Constituent 3

Reference substance name:

ammonium thiosulfate

IUPAC Name:

ammonium thiosulfate

Details on test material:

- Name of test material (as cited in study report): Ammonium thiosulfate
- Substance type: pure active substance
- Physical state: liquid, clear and colourless
- Storage condition of test material: at room temperature, protected from exposure to light

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. This cell line has an average call cycle time of 10-14 hours with a modal chromosome number of 20.
- Type and identity of media: complete medium (McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS), 100 units penicillin and 100 µg streptomycin/mL, and 2 mM L-glutamine)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; The freeze lot of cells was tested using the Höchst staining procedure and found to be free of mycoplasma contamination.
- Periodically checked for karyotype stability: yes; In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 20.
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

185, 370, 740 and 1480 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: A solubility test was perfomed.

Controls

Controls 1

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: stock concentration of 1 and 2 µg/mL (dissolved in distilled water)

Controls 2

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: stock concentration of 100 and 200 µg/mL (dissolved in distilled water)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 16-24 hours at 37°C in a humidified atmosphere of 5% CO2.
- Exposure duration: a) 4 hours and 20 hours (continuously treatment) without metabolic activation at 37°C in a humidified atmosphere of 5% CO2 and b) 4 hours at 37°C in a humidified atmosphere of 5% CO2 with metabolic activation.
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours after study initiation: Two hours after addition of Colcemid, metaphase cells were harvested by trypsinisation. To prepare slides, fixed cells (fiexed in Carnoy's fixative) were centrifuged, the supernatant was aspirated and 1 mL fresh fixative was added. After removal of the fixative by centrifugation, a sufficient amount of cell suspension was dropped onto a glass slide and was air dried.

SPINDLE INHIBITOR (cytogenetic assays): Two hours pior to the scheduled cell harvest, Colcemid was added at a final concentration of 0.1 µg/mL and the flasks returned to the incubator until cell collection.
STAIN (for cytogenetic assays): Dried slides were stained with 5% Giemsa, air dried and permanently mounted.

NUMBER OF REPLICATIONS: duplicate flasks

NUMBER OF CELLS EVALUATED: A minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth:
The preliminary toxicity assay was performed for the purpose of selecting dose levels for the chromosome aberration (CA) assay. CHO cells were seeded for each treatment condition at approximately 5 x 10^5 cells/25 cm² flask and were incubated at 37°C in a humidified atmosphere of 5% Co2 in air for 16-24 hours. Treatment was carried out for the non-activated study and for the activated study. The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9. At completion of the 4 hours exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline, refed with 5 mL complete medium and returned to the incubator for a total of 20 hours from the initiation of treatment. At 20 hours after initiation of treatment the cells were harvested by trypsinisation and counted. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control.

OTHER: mitotic index:
To ensure, that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group.

Evaluation criteria:

All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test item was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p<0.05). However, values that are statistically significant but do not exceed the range of historic solvent controls may be judged as not biologically significant. Test item not demonstrating a statistically significant increase in aberrations will be concluded to be negative. Negative results with metabolic activation may need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not necessary, justification will be provided.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test item dose level, the Cochran-Armitage test was used to measure dose-responsivness.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

The percentage of cells with structural and numeric aberrations in the test item treated groups was not significantly increased above that of the solvent control.

Cytotoxicity / choice of top concentrations:

other: absence of substantial toxicity: Toxicity in CHO cells was 5% at 1480 µg/mL. The mitotic index at the highest dose level evaluated was 4% reduced relative to the solvent control.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

The percentage of cells with aberrations was significantly increased at dose level 1480 µg/mL. However, the percentage was within the historical control range; therefore it is not considered to be biologically significant.

Cytotoxicity / choice of top concentrations:

other: absence of substantial toxicity: Toxicity in CHO cells was 7% at 1480 µg/mL. The mitotic index at the highest dose level evaluated was 33% reduced relative to the solvent control.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

The percentage of cells with structural and numeric aberrations in the test item treated groups was not significantly increased above that of the solvent control.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxicity was observed when treated for 4 hours

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest concentration of test item in treatment medium was approximately 7.5.
- Effects of osmolality: The osmolarity in treatment medium of the highest concentration tested, 1480 µg/mL, was 280 mmol/kg. The osmolarity of the solvent in treatment medium was 270 mmol/kg.
- Water solubility: Water was determined to be the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article was soluble in water at a concentration of 50 mg/mL, the maximum concentration tested.

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity assay, the maximum dose tested was 1480 µg/mL (10mM). The test item was soluble in treatment medium at all dose levels tested. Substantial toxicity, i.e., at least 50% cell growth inhibition (relative to the solvent control), was not observed at any dose level in the 4 and 20 hour non-activated exposure groups or in the S9 activated 4 hour exposure groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 185 to 1480 µg/mL for both the non-activated exposure groups and the S9 activated exposure group.
In the absence of both test item precipitation in the treatment medium and at least 50% toxicity, the highest dose level evaluated was 10 mM. The next two lower dose levels were included in the evaluation.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical control values for structural and numerical aberrations (1997-1999) in non-activated test system and activated systems with solvent and positive controls are available.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No further details.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: ; 4 hours treatment

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the findings of this study, Ammonium thiosulfate was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.

Executive summary:

The test item, Ammonium thiosulfat, was tested in the chromosome aberration (CA) assay using Chinese hamster ovary (CHO) cells in both the absence and presence of metabolic activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The CA assay was used to evaluate the clastogenic potential of the test article. Based on the findings, the doses chosen for the chromosome aberration assay ranged from 185 to 1480 µg/mL for both the non-activated exposure groups and the S9 activated exposure group.

In the CA assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9 activated test system, and all cells were harvested at 20 hours after treatment initiation. In the absence of both substantial toxicity and test item precipitation in the treatment medium, 1480 µg/mL was chosen as the high dose for microscopic analysis for CA in all hervests. The next two lower doses were also included in the analysis.

Based on the findings of this study, Ammonium thiosulfate was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.