Strontium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read-across:

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.

 

Soluble strontium substances have been tested in bacterial reverse mutation assay, in vitro gene mutation and chromosome aberration test. Read across from soluble strontium substances is applied since potential effects may be regarded to as strontium-ion related effects. A negative response was observed in all three tests as follows:

Tests on the mutagenic potential of strontium compounds in bacteria are considered dispensable for principal considerations, since inorganic metal compounds are frequently negative in this assay due to limited capacity for uptake of metal ions (Guidance on information requirements and chemical safety assessment, Chapter R.7a, p. 387; HERAG facts sheet mutagenicity, Chapter 2.1). However, strontium chloride hexaydrate was considered to be non-mutagenic either with or without metabolic activation at any concentration tested.

 

Strontium nitrate did not induce micronuclei in cultured human peripheral blood lymphocytes following treatments in the absence and presence of an Aroclor induced rat liver metabolic activation system (S-9 mix). Concentrations were tested and analysed up to 2116 µg/mL.

 

Strontium nitrate did not induce mutation at the tk 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 (S-9 mix).

 

 

Thus, a negative response was observed in all three tests; soluble strontium substances are not genotoxic or mutagenic. Further testing of in vivo genetic toxicity tests is not considered necessary.

Link to relevant study records

Reference

Reference 1

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.

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

Target gene:

hprt locus

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

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

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.

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'.

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

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:

Study started on 15 December 2009 and was completed on 11 February 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.

Qualifier:

according to guideline

Guideline:

other: OECD (2008) ‘Genetic Toxicology: OECD Guideline for the testing of chemicals. Draft proposal for a new Guideline 487: In vitro micronucleus 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:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: from female volunteers

Details on mammalian cell type (if applicable):

Blood from two healthy, non-smoking female 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.- Type and identity of media: Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 8.1 mL HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum and 50 µg/mL gentamycin, so that the final volume following addition of S9 mix/KCl and the test article in its chosen vehicle is 10 mL. The mitogen Phytohaemagglutinin (PHA) 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.No further details are given.

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 Main Experiment were based on the results of this cytotoxicity Range-Finder Experiment.Range-Finder:- 3+21 hours, without and with metabolic activation; as well as 24+0 hours, without metabolic activation: 7.677, 12.79, 21.32, 35.54, 59.23, 98.72, 164.5, 274.2, 457.1, 761.8, 1270 and 2116 µg/mLMain Experiment:- 3+21 hours, without metabolic activation: 200, 400, 800, 1200*, 1500*, 1800* and 2116 µg/mL- 3+21 hours, with metabolic activation: 200, 400, 800*, 1200*, 1500*, 1800 and 2116 µg/mL- 24+0 hours, without metabolic activation: 200, 400, 800, 1200, 1500*, 1800* and 2116* µg/mL* = Concentrations selected for analysis.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sterile purified water was added to cultures.

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation Migrated to IUCLID6: 0.8 and 0.8 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sterile purified water was added to cultures.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation Migrated to IUCLID6: 6.25 and 15.5 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Sterile purified water was added to cultures.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: vinblastine; 0.02, 0.03 and 0.04 µg/mL

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium DURATION- Exposure duration: 3 hours (+ 21 hours recovery) or 24 hoursCytochalasin B, formulated in DMSO, was added directly (0.1 mL/culture) to all 24-hour cultures at the time of treatment. Cultures were incubated at 37°C +/- 1°C for the designated exposure time.- Addition of Cytochalasin B: Cytochalasin B (formulated in DMSO) was added to post wash-off culture medium after approximately 51 hours for the cultures exposed 3 hours to the test substance or after 48 hours for cultures exposed continuously (test substance was removed at time of harvest). - Fixation time (start of exposure up to fixation or harvest of cells): Cells were harvested after 72 hours. Cells were fixed by dropping the suspension into fresh, cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation and resuspension. This procedure was repeated until the cell pellets were clean.SLIDE PREPARATION: Several drops of fixed cell suspension were gently spread onto multiple clean, dry microscope slides. STAIN (for cytogenetic assays): After the slides had dried the cells were stained for 5 minutes in filtered 4% (v/v) Giemsa in pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.NUMBER OF REPLICATIONS: Cultures exposed to the test article and the positive controls were tested in duplicate; for the negative controls 4 cultures were tested for each exposure. NUMBER OF CELLS EVALUATED: Where possible, a minimum of 1000 binucleate cells from each culture (at least 2000 per concentration) were analysed for micronuclei. The number of cells containing micronuclei and the number of micronuclei per cell on each slide was noted. DETERMINATION OF CYTOTOXICITY- Method: other:Single cultures were treated with the test article for 3 hours (+ 21 hours recovery; in the absence and presence of metabolic activation) or continuously for 24 hours (without S9 mix). Vehicle controls (1 mL/culture) were tested in duplicates. Positive control treatments were not included. Cytochalasin B, formulated in DMSO was added directly (0.1 mL/culture) to all continuous cultures at the time of treatment. Cultures were incubated at 37°C +/- 1°C for the designated exposure time.OTHER EXAMINATIONS:Slides from the cytotoxicity Range-Finder Experiment were examined, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) and the relative RI were determined. Cytotoxicity (%) is expressed as (100 – Relative RI).

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:

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.

Species / strain:

lymphocytes: from humans

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Treatment of cells with Strontium nitrate (+/- S9) resulted in frequencies of MNBN cells that were similar to (and not significantly different from) those observed in concurrent vehicle controls at all concentrations analysed. For details see table below.

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 purified water at concentrations up to at least 58.53 mg/mL. The solubility limit in culture medium was in excess of 5853 µg/mL. RANGE-FINDING/SCREENING STUDIES: The results of the cytotoxicity Range-Finder Experiment were used to select suitable maximum concentrations for the Main Experiment.COMPARISON WITH HISTORICAL CONTROL DATA: yes; The MNBN cell frequency of all Strontium nitrate treated cultures fell within normal ranges.ADDITIONAL INFORMATION ON CYTOTOXICITY: no further data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Main experiment (48 hours PHA) - Summary of results

Treatment	Concentration (mg/mL)	Cytotoxicity (%)	Mean MNBN cell frequency (%)	Historical(%) #	Statistical significance
3+21 hour -S-9	Vehicle^a	-	0.62	0.1-1.2	-
Trial 2	1200	0	0.32		NS
	1500	0	0.34		NS
	1800	0	0.79		NS
	*MMC, 0.80	ND	11.02		p<0.001
	*VIN, 0.02♦	ND	4.86		p<0.001
3+21 hour +S-9	Vehicle^a	-	0.32	0.0-1.2	-
Trial 2	800.0	0	0.39		NS
	1200	0	0.42		NS
	1500	0	0.34		NS
	*CPA, 12.5	ND	2.03		p<0.001
24+0 hour -S-9	Vehicle^a	-	0.25	0.1-1.2	-
Trial 1	1500	5	0.20		NS
	1800	19	0.20		NS
	2116	10	0.25		NS
	*VIN, 0.02	ND	4.40		p<0.001
	*MMC, 0.80♥	ND	8.45		p<0.001
♦ 24+0 hour –S-9 treatment ♥ 3+21 hour –S-9 treatment ^aVehicle control waspurified water * Positive control ^#95^thpercentile of the observed range NS = Not significant          ND = Not determined

Conclusions:

Interpretation of results (migrated information):negativeIt is concluded that Strontium nitrate did not induce micronuclei in cultured human peripheral blood lymphocytes when tested in excess of the limit of solubility in both the absence and presence of S-9.

Executive summary:

Strontium nitrate was tested in an in vitro micronucleus assay using human lymphocytes, both in the absence and presence of metabolic activation.

Treatments covering a broad range of concentrations, the highest concentration used in the Main Experiment, 2116 mg/mL, (equivalent to 10 mM) was determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatments were conducted 48 hours following mitogen stimulation by Phytohaemagglutinin (PHA). In the Main Experiment, micronuclei were analysed at 3 concentrations. Appropriate vehicle control cultures were included in the test system under each treatment condition.

Treatment of cells with Strontium nitrate in the absence and presence of S9 mix resulted in frequencies of MNBN cells that were similar to (and not significantly different from) those observed in concurrent vehicle controls at all concentrations analysed under all treatment conditions. The MNBN cell frequency of all Strontium nitrate treated cultures fell within normal ranges.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

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:

The study was performed between 10 December 1996 and 03 February 1997.

Reliability:

2 (reliable with restrictions)

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

; signed by the Depertment of Health of the Government of the UK (21.02.1996)

Type of assay:

bacterial reverse mutation assay

Target gene:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

not applicable (S. typhimurium strains are no mammalian cells); but prior to use, the strains were checked for characteristics, viability and spontaneous reversion rate and were all found to be satisfactory.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

not applicable (S. typhimurium strains are no mammalian cells); but prior to use, the strain was checked for characteristics, viability and spontaneous reversion rate and was found to be satisfactory.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary toxicity study: 0, 50, 150, 500, 1500 and 5000 µg/plateMain study: 0, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

A solvent treatment group was used as the vehicle control.

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

with metabolic activation; strains TA100 and TA1535 Migrated to IUCLID6: 3.0 and 5.0 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

A solvent treatment group was used as the vehicle control.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

with metabolic activation; strain TA1537 Migrated to IUCLID6: 80 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

A solvent treatment group was used as the vehicle control.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine; 5 µg/plate

Remarks:

with metabolic activation; strain TA1538

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

A solvent treatment group was used as the vehicle control.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide; 0.2 µg/plate

Remarks:

with metabolic activation; strain TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

A solvent treatment group was used as the vehicle control.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene; 1.0, 2.0 and 0.5 µg/plate

Remarks:

without metabolic activation; all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) DURATION- Exposure duration: 48 hours at 37°C NUMBER OF REPLICATIONS: The test material and the positive controls were assayed in triplicate, respectively. NUMBER OF CELLS EVALUATED: not applicable; The frequency of revertant colonies was assessed using a Domino colony counter. DETERMINATION OF CYTOTOXICITY- Method: relative total growth:A mixture of 0.1 mL of bacterial suspension (TA100), 2 mL of molten, trace histidine supplemented media (histidine/biotin and top agar), 0.1 mL of test material and 0.5 mL phosphate buffer was overlaid onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). 5 doses of test material and a vehicle control were tested in duplicate. In addition, the sterility of the test material was tested. After 48 hours incubation at 37°C the plates were assessed for revertant colonies using a Domino colony counter and examined for a thinning og the background lawn. OTHER: The plate incorporation assay was repeated in an second experiment using fresh bacterial cultures, test material and control solutions.

Evaluation criteria:

For the substance to be considered positive in this test system, it schould have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of S9 microsomal enzymes in both experiments at sub-toxic dose levels. In the event of the two experiments giving conflicting or equivocal results, then a third experiment my be performed to confirm the correct response. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between two and five fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.

Statistics:

All data are statistically analysed using the methods recommended by the UKEMS (Kirkland, D.J. (Ed), 1989, Statistical Evaluation of Mutagenicity Test Data.) and normally Dunnett's method of linear regression is used to evaluate the results.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No significant increase in the frequency of revertant colonies of bacteria were recorded for any of the strains of S. typhimurium used, at any dose level.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxicity was exhibited to any of the strains tested.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No significant increase in the frequency of revertant colonies of bacteria were recorded for any of the strains of S. typhimurium used, at any dose level.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxicity was exhibited to any of the strains tested.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORSNo details are reported.RANGE-FINDING/SCREENING STUDIES: The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500, 1500 and 5000 µg/plate. The test material was non-toxic to the strain of S. typhimurium TA100. COMPARISON WITH HISTORICAL CONTROL DATA: no dataADDITIONAL INFORMATION ON CYTOTOXICITY: No further details are reported.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):negativeIn conclusion, under the test conditions described the test material, strontium chloride-6-hydrate extra pure, was considered to be non-mutagenic either with or without metabolic activation at any concentration tested.

Executive summary:

S. typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test material using the Ames plate incorporation method at 5 dose levels, both with and without metabolic activation. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1.

Positive and vehicle controls were included in the test.

The test material caused no visible reduction in the growth of the bacterial lawn at any of the dose levels to any of the strains of Salmonella tested.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any of the concentrations tested, either with or without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Mutagenic / genotoxic effects were not observed in relevant in vitro tests with soluble strontium substances (bacterial reverse mutation assay, in vitro gene mutation and chromosome aberration test). A similar conclusion is derived for strontium metal since potential effects would be regarded to as strontium-ion related effects. Hence, classification and labelling of strontium metal is not required.