Strontium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Strontium nitrate

EC Number:

233-131-9

EC Name:

Strontium nitrate

Cas Number:

10042-76-9

IUPAC Name:

strontium dinitrate

Constituent 2

Reference substance name:

233-13-9

IUPAC Name:

233-13-9

Test material form:

solid: particulate/powder

Remarks:

migrated information: 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:

not applicable

Species / strain

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/mL

Main 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

Controls

Controls 1

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

Controls 2

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

Controls 3

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 hours
Cytochalasin 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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It 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.