Reaction mass of disodium sulphide and sodium carbonate and sodium hydrogensulphide

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:

Study was started on 17 December 2009 and was completed on 23 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same 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:

Disodium sulphide

EC Number:

215-211-5

EC Name:

Disodium sulphide

Cas Number:

1313-82-2

Molecular formula:

Na2S

IUPAC Name:

disodium sulfide

Constituent 2

Reference substance name:

sodium sulfide, anhydrous

IUPAC Name:

sodium sulfide, anhydrous

Details on test material:

- Name of test material (as cited in study report): Sodium sulfide, anhydrous
- Molecular formula (if other than submission substance): 78.04 g/mol
- Substance type: technical product
- Physical state: solid, slightly yellow granules
- Storage condition of test material: stored at 2 to 8°C in the dark, with desiccant and under nitrogen

Method

Target gene:

hprt locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk +/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co.
- Type and identity of media: RPMI 1640 media supplemented with heat inactivated horse serum (0%, 10% and 20%, respectively), 100 units/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL Amphotericin B and 0.5 mg/mL pluronic (except for RPMI 20%) were used.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; Each batch of cells was checked for that it was mycoplasma free.
- Periodically "cleansed" against high spontaneous background: yes; Each batch of cells was purged of TK- mutants, checked for spontaneous mutant frequency.
The cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range-Finder Experiment. Values are based on test material (Na2S, anhydrous).

Range-Finder:
- with metabolic activation: 24.41, 48.81, 97.63, 195.3, 390.5 and 781.0 µg/mL
- without metabolic activation: 24.41, 48.81, 97.63, 195.3, 390.5 and 781.0 µg/mL

Experiment I:
- with metabolic activation: 50, 100, 150, 200, 300, 400*, 500*, 600*, 700* and 781 µg/mL
- without metabolic activation: 50, 100*, 150*, 200, 300*, 400*, 500*, 600*, 700 and 781 µg/mL

Experiment II:
- with metabolic activation: 100, 200, 400*, 600*, 650*, 700*, 720*, 740*, 760* and 781 µg/mL
- without metabolic activation: 50*, 100*, 200*, 400*, 500*, 600*, 625*, 650*, 700 and 781 µg/mL

* = Concentrations selected for mutation assessment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Treatments with the vehicle purified water diluted 10 fold in the treatment medium.

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation

Migrated to IUCLID6: 0.1 and 0.15 µg/mL; dissolved in DMSO

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Treatments 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: 2.0 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 at 37 +/- 1°C
After incubation, the cells washed and resuspended in RPMI 10 medium. Cells were transferred to flasks for growth through the expression period or were diluted to be plated for survival (scoring after 7 days).
- Expression time (cells in growth medium): Cultures were maintained for a period of 7 days during which the hprt- mutation would be expressed.
Thereafter cultures were selected to be plated for viability and 6TG resistance.
- Selection time (if incubation with a selection agent): 12 to 13 days; At the end of the expression period, the cell suspension was placed into each well of 4 x 96 well microtitre plates (384 wells at 2 x 10^4 cells/well). Plates were incubated at 37 +/-1ºC in a humidified incubator gassed with 5% v/v CO2 in air until scoreable and wells containing clones were identified and counted.

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

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

EVALUATION: Wells containing viable clones were identified by eye using background illumination and counted.

DETERMINATION OF CYTOTOXICITY
- Method: relative survival:
Single cultures only were used and positive controls were not included. Following treatment, cells were washed with tissue culture medium and resuspended in tissue culture medium. Cells were plated into each well of a 96 well microtitre plate for determination of relative survival. The plates were incubated at 37 +/- 1ºC 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: Plating efficiency (PE), percentage relative survival (% RS) and mutant frequency (MF) were determined.

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. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly 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:

In both experiments there were no significant increases in MF at any concentration analysed.

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A weak but statistically significant linear trend was observed in the presence of S9 mix in Experiment II but, in the absence of any marked increases in mutant frequency at any test article concentration analysed in this experiment, this observation was not considered biologically relevant.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: At the highest concentration tested in the cytotoxicity Range-Finder Experiment (781 µg/mL), no marked changes in osmolality, compared to the concurrent vehicle controls, were observed but a marked increase in pH (of ≥1 unit, compared to the concurrent vehicle controls) was observed at 781 µg/mL in the absence and presence of S9 mix. Further pH measurements were therefore taken in Experiments I and II.
In Experiment I, marked increases in pH (of ≥1 unit, compared to the concurrent vehicle controls) were observed at 500 µg/mL in the absence and presence of S9 mix. In Experiment II, marked increases in pH (of ≥1 unit, compared to the concurrent vehicle controls) were observed at 400 µg/mL and above in the absence of S9 mix and at 600 µg/mL and above in the presence of S9 mix.
However, the marked increases in pH observed in the absence and presence of S-9 did not affect the interpretation of the data, as there were no significant increases in mutant frequency.
- Water solubility: Preliminary solubility data indicated that Sodium sulfide, anhydrous was soluble in water for irrigation (purified water) at a concentration of to at least 12.88 mg/mL. The solubility limit in culture medium was in excess of 1288 µg/mL (no precipitation was present at this concentration 3 hours after test article addition).

RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity Range-Finder Experiment, 6 concentrations were tested in the absence and presence of S9 mix ranging from 24.41 to 781 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide >10% RS was 390.5 µg/mL, which gave 68% and 53% RS in the absence and presence of S9 mix, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA: no

ADDITIONAL INFORMATION ON CYTOTOXICITY: no further data

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 Sodium sulfide, anhydrous did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study.

It is concluded that Sodium sulfide, anhydrous did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9 mix).

Executive summary:

Sodium sulfide, anhydrous was assayed for mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6 -thioguanine [6TG] resistance) in mouse lymphoma cells.

In the cytotoxicity Range-Finder Experiment, 6 concentrations were tested in the absence and presence of S9 mix, ranging from 24.41 to 781 µg/mL (equivalent to 10 mM at the highest concentration tested).

According to the results from the Range-Finder test, in Experiment I 10 concentrations, ranging from 50 to 781 µg/mL, were tested in the absence and presence of S9 mix.

In Experiment II 10 concentrations, ranging from 50 to 781 µg/mL in the absence of S9 mix and from 100 to 781 µg/mL in the presence of S9 mix, were tested.

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

When tested up to toxic concentrations in the absence and presence of S9 mix in Experiments I and II, there were no significant increases in mutant frequency at any concentration analysed. A weak but statistically significant linear trend was observed in the presence of S9 mix in Experiment II but, in the absence of any marked increases in mutant frequency at any test article concentration analysed in this experiment, this observation was not considered biologically relevant.

Read-across from Na₂S to NaHS:

Given that sodium sulfide and sodium hydrogensulfide dissociate in aqueous media, it can safely be assumed that under most physiologically relevant conditions ( i.e., neutral pH) sulfide and hydrogen sulfide anions are present at almost equimolar concentrations, thus facilitating unrestricted read-across between both species. Only under extreme conditions such as gastric juice (pH << 2), sulfides will be present predominantly in the form of the non-dissociated hydrogen sulfide. In turn, hydrogen sulfide (H₂S) may be formed from both soluble sulfides, according to the following equilibria:

                           Na₂S + H₂O   →  NaOH + NaHS (2Na⁺/ OH^-/ HS^-)

                           NaHS + H₂O  →  NaOH + H₂S (Na⁺/ OH^-/ H₂S)

Similarly, hydrogen sulfide dissociates in aqueous solution to form two dissociation states involving the hydrogen sulfide anion and the sulfide anion, according to the following equilibrium:

                           H₂S  ↔  H⁺  +  HS^-  ↔  2 H⁺  +  S^2-

In conclusion, under physiological conditions, inorganic sulfides or hydrogensulfides as well as H2S will dissociate to the respective species relevant to the pH of the physiological medium, irrespective the nature of the “sulfide”, which is why read-across between these substances and H2S is considered to be feasible without any restrictions.