Strontium

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

Study started on 16 December 2009 and was completed on 1 March 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Justification for type of information:

Strontium metal is highly reactive and instantly oxidizes upon contact with water. It decomposes completely. During the redox-reaction with water, a strong evolution of hydrogen gas and an immediate precipitation of a white, crystalline solid (i.e. Sr(OH)2) is observed (Sr2+ + 2OH- + H2 (g). The amount of dissolved Sr cations is determined by the solubility of the Sr(OH)2 precipitate. According to OECD guideline 105 (1995) and EU method A.6 (2006), the water solubility of strontium was determined to be 6.74 ± 0.14 g/L under the conditions of the test (flask method under protective gas atmosphere; loading of 41 g Sr/L, at 20.0 ± 1.0 °C, pH >13).
Strontium ions are highly mobile, occur only in one valence state (2+), i.e. are not oxidized or reduced, and do not form strong complexes with most inorganic and organic ligands (Krupka et al. 1999. EPA 402-R-99-004B; Salminen et al. 2015; Carbonaro and Di Toro. 2007. Geochim Cosmochim Acta 71 3958–3968; Carbonaro et al. 2011. Geochim Cosmochim Acta 75: 2499-2511 and references therein). Thus, it may be assumed that systemic toxicological effects (not local) are related to the strontium ion. Therefore, the assessment of the systemic toxicity of strontium is based on elemental strontium concentrations. Read-across of systemic toxicity data available for soluble strontium substances is applied since the strontium ions determine the toxicological potential of strontium.

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 (incl. QA statement)

Remarks:

signed by The Department of Health of the Government of the United Kingdom (2010-06-23)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 002

Cas Number:

10042-76-9

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Strontium nitrate - Molecular formula (if other than submission substance): Sr(NO3)2 - Molecular weight (if other than submission substance): 211.63 g/mol - Physical state: solid, white powder - Storage condition of test material: stored at 15 - 25°C in the dark

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 (except for RPMI 20%) were used.- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes; Each batch of cells was checked 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.Range-Finder test (with and without S9 mix): 66.13, 132.3, 264.5, 529, 1058 and 2116 µg/mL Experiment I: - without S9 mix: 250*, 500*, 750*, 1000*, 1200*, 1400*, 1600*, 1800*, 2000 and 2116 µg/mL - with S9 mix: 250*, 500*, 750*, 1000*, 1200*, 1400*, 1600*, 1800, 2000 and 2116 µg/mL Experiment II: - without S9 mix: 200, 400*, 700*, 1000*, 1200*, 1400*, 1600*, 1800*, 2000 and 2116 µg/mL- with S9 mix: 200, 400*, 700*, 1000*, 1200*, 1400*, 1600*, 1800, 2000 and 2116 µg/mL* = Concentration were selected to be plated for viability and 6TG resistance.

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°CAfter cells were washed and resuspended in RPMI 10 medium, they were transferred to flasks for growth through the expression period or were diluted to be plated for survival (scoring after 7-10 days).- Expression time (cells in growth medium): for a period of 7 days during which the hprt- mutation would be expressed - Selection time (if incubation with a selection agent): 12 to 14 days; At the end of the expression period, the cell were plated 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 20 mL tissue culture medium. Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was 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) in each test culture was 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:

No statistically significant increases in mutant frequency were observed following treatment with Strontium nitrate at any concentration tested in Experiments I and II and there were no statistically significant linear trends.

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:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH and osmolality: No marked changes in osmolality or pH were observed at the highest concentration tested in the cytotoxicity Range-Finder (2116 µg/mL), compared to the concurrent vehicle controls (individual data not reported).- Water solubility: Preliminary solubility data indicated that Strontium nitrate was soluble in sterile water for irrigation (purified water) at concentrations up to at least 58.53 mg/mL. The solubility limit in culture medium was less than 5853 µg/mL, as indicated by precipitation which was observed at this concentration at least 3 hours after test article addition (no lower concentrations were assessed). - Precipitation: Precipitation was observed in the Range-Finder test following treatment incubation period at a concentration of 2116 µg/mL. In Experiment I and II, after the 3 hour treatment incubation period, precipitate was observed at the highest 3 concentrations in the absence of S9 mix (1800 to 2116 µg/mL) and at the highest 4 concentrations in the presence of S9 mix (1600 to 2116 µg/mL). RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity Range-Finder Experiment, 6 concentrations were tested in the absence and presence of S9 mix, ranging from 66.13 to 2116 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide >10% RS was 1058 µg/mL, which gave 63% and 71% RS in the absence and presence of S 9, respectively. COMPARISON WITH HISTORICAL CONTROL DATA: yes; results for positive controls were compared with historical means.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):negativeIt is concluded that Strontium nitrate 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 precipitating concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9 mix).

Executive summary:

Strontium nitrate was assayed for mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6 -thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. 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 mix).

According to the results from a Range-Finder test, for Experiment I 10 concentrations, ranging from 250 to 2116 µg/mL were tested in the absence and presence of S9 mix. 7 days after treatment, the highest concentrations selected to determine viability and 6TG resistance were 1800 µg/mL in the absence of S9 mix and 1600 µg/mL in the presence of S9 mix (both limited by the appearance of post-treatment precipitate), which gave 56% and 70%  relative survival (RS), respectively.

In Experiment II 10 concentrations, ranging from 200 to 2116 µg/mL were tested in the absence and presence of S9 mix. 7 days after treatment, the highest concentrations selected to determine viability and 6TG resistance were 1800 µg/mL in the absence of S9 mix and 1600 µg/mL in the presence of S9 mix (both limited by the appearance of post-treatment precipitate), which gave 32% and 36% RS, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment.

No statistically significant increases in mutant frequency were observed following treatment with Strontium nitrateat any concentration tested in the absence or presence of S9 in Experiments I and II and there were no statistically significant linear trends.