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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Strontium neodecanoate was assessed in three in vitro test systems for its ability to induce gene, chromosome and genome mutations. None of the test systems retuned a positive result when tested up to the maximum concentrations.Therefore, strontium neodecanoate is considered to be non-mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-06-22 to 2015-07-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997-07-21
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
, signed 2013-04-11
Type of assay:
bacterial reverse mutation assay
Target gene:
TA98: his D 3052
TA100: his G 46
TA1535: his G 46
TA1537: his C 3076
E. coli WP2 uvr A: trp-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9: phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system (The S9 was prepared and stored according to the currently valid version of the Harlan CCR SOP for rat liver S9 preparation)
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate (with and without metabolic activation)
Experiment II: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF; purity > 99 %)
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control without metabolic activation: concentration: 10 µg/plate; vehicle: deionised water; strains: TA1535 & TA100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Positive control without metabolic activation: concentration: 10 μg/plate in strain TA 98, 50 μg/plate in strain TA 1537; vehicle: DMSO (purity >99 %)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Positive control without metabolic activation: concentration: 2.0 μL/plate; vehicle: deionised water; strain: E. coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Positive control with metabolic activation: concentration: 2.5 μg/plate in strains TA 1535, TA 1537, TA 98, TA 100, 10.0 μg/plate in WP2 uvrA; vehicle: DMSO (purity >99 %)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation; Pre- experiment/Experiment I) & preincubation (Experiment II)

Pre-Experiment/Experiment I:
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Experimental Performance:
For each strain and dose level, including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
Experiment I (Plate Incorporation)
25 μL Test solution at each dose level or solvent or 100 μL reference mutagen solution (positive control),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 μL Overlay agar
Experiment II (Pre-Incubation)
In the pre-incubation assay 25 μL test solution or solvent or 100 μL reference mutagen solution (positive control), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

In parallel to each test a sterile control of the test item was performed. Therefore, 25 μL of the stock solution, 500 μl S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager (v.1.21).

ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
A statistical analysis of the data is not mandatory.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
please refer to the field "Additional information on results" below
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
please refer to the field "Additional information on results" below
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test item in the overlay agar in the test tubes occurred up to the highest investigated dose with and without metabolic activation in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 μg/plate with and without metabolic activation. The undissolved particles had no influence on the data recording.

DOSE SELECTION:
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since toxic effects were observed in experiment I, eight concentrations were tested in experiment II. 5000 μg/plate were chosen as maximal concentration.

RESULTS:
The plates incubated with the test item showed reduced background growth in experiment I in strain TA 1535 and TA 1537 with S9 mix and strain TA98 with and without S9 mix at 5000 μg/plate.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at the following concentrations (µg/plate):
- Experiment I: 2500 - 5000 µg/plate (strain TA1537; without metabolic activation); 5000 µg/plate (strain TA100; with and without metabolic activation)
- Experiment II: 2500- 5000 µg/plate (strains TA1535, TA98, TA100; with and without metabolic activation); 2500 - 5000 µg/plate (strain: TA1537; without metabolic activation); 5000 µg/plate (strain 1537; with metabolic activation); 5000 µg/plate (strain E. coli WP2 uvrA; without metabolic activation); 2500 - 5000 µg/plate (strain E. coli WP2 uvrA; with metabolic activation)
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Strontium Neodecanoate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Strontium neodecanoate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay. It did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-06-10 to 2015-07-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 487 "In vitro Mammalian Cell Micronucleus Test", adopted 26 September, 2014
Deviations:
yes
Remarks:
please refer to the field "Prinicples of method if other than guideline" below
Principles of method if other than guideline:
The following alterations from the guidelines were performed:
A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2013-04-11
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: isolated from healthy female human blood samples.
Details on mammalian cell type (if applicable):
- Type and identity of media: culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
Pre-test/ Experiment I: 17.2, 30.2, 52.8, 92.4, 161.7, 283.0, 495.2, 886.6, 1516.6, 2654.0 µg/mL (with and without metabolic activation; exposure period: 4 hours)
Experiment II: 17.2, 30.2, 52.8, 92.4, 161.7, 283.0, 495.2, 886.6, 1516.6, 2654.0 µg/mL (without metabolic activation; exposure period: 20 hours)
Experiment II: 92.4, 161.7, 283.0, 495.2, 886.6, 1516.6, 2654.0 µg/mL (with metabolic activation; exposure period: 4 hours)
Vehicle / solvent:
- Vehicle/solvent used: THF: tetrahydrofuran, purity: 99.85 %, final concentration in the culture medium: 0.5 %.
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Positive control (without metabolic activation): dissolved in deionised water; concentration: 1.0 µg/mL; pulse treatment
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Demecolcin
Remarks:
Positive control (without metabolic activation): dissolved in deionised water; concentration: 150.0 ng/mL; continuous treatment
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Positive control (with metabolic activation): dissolved in saline; concentration: 17.5 µg/mL (Experiment I & II)
Details on test system and experimental conditions:
NOTE: in the experiments the following concentrations were evaluated:
Experiment I (without metabilic activation; exposure period: 4 hours): 495.2, 886.6, 1516.6 µg/mL
Experiment I (with metabilic activation; exposure period: 4 hours): 283.0, 495.2, 886.6 µg/mL
Experiment II (without metabilic activation; exposure period: 20 hours): 161.7, 283.0, 495.2 µg/mL
Experiment II (with metabilic activation; exposure period: 4 hours): 495.2, 886.6, 1516.6 µg/mL

PRE-EXPERIMENT/EXPERIMENT I
- a preliminary cytotoxicity test (concentrations: 17.2 to 2654.0 µg/mL; with and without metabolic activation; exposure period: 4 hours) was performed to determine the concentrations to be used in the main experiment.
- solvent and positive control were tested.
- cytotoxicity is characterized by the percentages of reduction in the cytokinesis-block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate.
- experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
- all cell cultures were set up in duplicate.
- preparation interval was 40 hours after start of the exposure.
- since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

CYTOGENETIC EXPERIMENT
1) puls exposure:
- 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration.
- culture medium was replaced with serum-free medium containing the test item.
- for the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added.
- after 4 hours the cells were spun down by centrifugation.
- supernatant was discarded
- cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose •H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4), which was done twice .
- cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period.
- after the recovery period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

2) continuous exposure (without S9 mix)
- 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration.
- culture medium was replaced with complete medium (with 10 % FBS) containing the test item.
- after 20 hours the cells were spun down by centrifugation.
- supernatant was discarded
- cells were re-suspended in and washed with "saline G", which was done twice.
- cells were re-suspended in complete culture medium containing 10 % FBS (v/v).
- cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

PREPARATION OF CELLS
- cultures were harvested by centrifugation 40 hours after beginning of treatment.
- supernatant was discarded
- cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation.
- cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes.
- 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended.
- after removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold.
- slides were prepared by dropping the cell suspension in fresh fixative onto a microscope slide.
- cells were stained with Giemsa.

EVALUATION OF CYTOTOXICITY AND CYTOGENETIC DAMAGE
- evaluation of the slides was performed using NIKON microscopes with 40 x objectives.
- micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976)*.
- micronuclei have to be stained in the same way as the main nucleus.
- area of the micronucleus should not extend the third part of the area of the main nucleus.
- at least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
- frequency of micronucleated cells was reported as % micronucleated cells.
- to describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis.
- CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

CBPI = ((MONC x 1) + (BINC x 2) + (MUNC x 3))/n

CBPI = cytokinesis-block proliferation index
n = total number of cells
MONC = mononucleate cells
BINC = binucleate cells
MUNC = multinucleate cells

Cytostasis % = 100 – 100 [(CBPI T – 1) / (CBPI C – 1)]

T = test item
C = solvent control

ACCEPTABILITY CRITERIA
The micronucleus assay will be considered acceptable if it meets the following criteria:
− the concurrent solvent control will normally be within the laboratory historical solvent control data range.
− the concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase.
− cell proliferation criteria in the solvent control are considered to be acceptable.
− all experimental conditions described were tested unless one exposure condition resulted in a clearly positive result.
− the quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
− the criteria for the selection of top concentration are consistent with those described.

*Reference:
- Countryman P.I. and Heddle J.A. (1976) The production on micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.
Evaluation criteria:
A test item can be classified as non-clastogenic and non-aneugenic if:
− the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
− no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent control.

A test item can be classified as clastogenic and aneugenic if:
− the number of micronucleated cells is not in the range of the historical laboratory control data and
− either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.

If the above mentioned criteria for the test item are not clearly met, the test item will be classified as equivocal or a confirmatory experiment may be performed.

An increase in the number of micronucleated mononucleate cells may indicate that the test item has aneugenic potential.
Statistics:
Chi squared test (α < 0.05)
Species / strain:
lymphocytes: human (females)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
please refer to the field "Additional information on results" below
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
1) Effects of pH: no relevant influence on pH value was observed.
2) Effects of osmolarity: no relevant influence on osmolarity was observed.
3) Precipitation:
- Experiment I without S9 mix at concentration ≥ 1516.6 μg/mL and with S9 mix at concentrations ≥ 866.,6 µg/m Lat the end of treatment.
- Experiment II with and without S9 mix at concentrations ≥ 1516.6 μg/mL at the end of treatment.

RESULTS:
CYTOTOXICITY:
- Experiment I (with and without S9 mix; up to the highest evaluated concentration): no cytotoxicicty
- Experiment II (without S9 mix; at the highest evaluated concentration): clear cytotoxicity (67.9 %)
- Experiment II (with S9 mix; at the highest evaluated concentration): moderate cytotoxicity (44.0 %)

CYTOGENETIC:
In Experiment I & II, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary of results

Exp.

Preparation interval

Test item
concentration
in μg/mL

Proliferation index
CBPI

Cytostasis
in %*

Micro-nucleated cells
in %**

Exposure period 4 hrs without S9 mix

I

40 hrs

Solvent control1

1.87

 

0.75

 

 

Positive control2

1.34

61.2

18.65S

 

 

495.2

1.84

2.9

0.85

 

 

866.6

1.76

12.0

0.65

 

 

1516.6P

1.84

3.3

0.65

Exposure period 20 hrs without S9 mix

II

40 hrs

Solvent control1

1.91

 

0.65

 

 

Positive control3

1.45

50.8

3.90S

 

 

161.7

1.70

23.6

0.35

 

 

283.0

1.46

49.9

0.40

 

 

495.2

1.29

67.9

0.50

Exposure period 4 hrs with S9 mix

I

40 hrs

Solvent control1

1.90

 

0.65

 

 

Positive control4

1.52

42.5

10.85S

 

 

283.0

1.99

n.c.

0.75

 

 

495.2

1.80

11.2

0.70

 

 

866.6P

1.89

0.3

0.60

II

40 hrs

Solvent control1

1.90

 

0.85

 

 

Positive control4

1.50

44.5

9.45S

 

 

495.2

1.75

17.4

1.05

 

 

866.6

1.69

23.1

0.55

 

 

1516.6P

1.51

44.0

1.00

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

P Precipitation occurred at the end of treatment

S The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1 THF 0.5 % (v/v)

2 MMC 1.0 μg/mL

3 Demecolcin 150.0 ng/mL

4 CPA 17.5 μg/mL

Conclusions:
Interpretation of results (migrated information):
negative

Strontium neodecanoate is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations. The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-06-09 to 2015-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 1997-07-21
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2013-04-11
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, Darmstadt, Germany) are stored in liquid nitrogen. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is checked for spontaneous mutant frequency.
- Checked for mycoplasma contamination: yes
- Checked for karyotype stability: yes

Thawed stock cultures are propagated at 37 °C in 75 cm² plastic flasks. About 5x10^5 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS, neomycin (5 µg/mL) and amphotericin B (1 %). The cells were sub-cultured twice weekly. The cell cultures were incubated at 37°C in a 1.5 % carbon dioxide atmosphere (98.5 % air).
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9 (protein concentration: 35.0 mg/mL (pre-experiment & experiment 1) or 32.3 mg/mL (experiment 2)
Test concentrations with justification for top dose:
Pre-test: 30.3, 40.6, 81.1, 162.5, 325.0, 650.0, 1300.0, and 2600.0 µg/mL (4 hour treatment with and without metabolic activation; 24 hours without metabolic activation)
Experiment 1: 125, 250.0, 500.0, 1000.0, 2000.0, and 4000.0 µg/mL (4 hour treatment with and without metabolic activation)
Experiment 2: 250.0, 500.0, 750.0, 1000.0, 1500.0, and 2000.0 µg/mL (24 hour treatment without metabolic activation)
Experiment 2: 250.0, 500.0, 750.0, 1000.0, 1500.0, and 2000.0 µg/mL (4 hour treatment with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF; pre-experiment) / MEM culture medium /Experiment 1 & Experiment 2)
On the day of the pre-experiment (immediately before treatment), the test item was dissolved in THF. The final concentration of THF in the culture medium was 0.5% (v/v). In experiment 1 the test item was suspended in culture medium. In experiment 2 the test item was dissolved in culture medium.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF (Pre-experiment) and MEM culture medium (Experiment 1 & Experiment 2)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Positive control without metabolic acitvation: purity: 99 %; vehicle: nutrient medium; final concentration: 0.15 mg/mL = 1.2 mM
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF (Pre-experiment) and MEM culture medium (Experiment 1 & Experiment 2)
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Positive control with metabolic acitvation: purity: ≥ 95 %; vehicle: dimethylsulfoxide (final concentration in nutrient medium 0.5%); final concentration: 2.2 µg/mL = 8.6 µM
Details on test system and experimental conditions:
PRE-TEST ON TOXICITY:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency.
The highest concentration used in the pre-test was chosen with regard to the solubility of the test item in tetrahydrofuran (THF). Test item concentrations between 20.3 and 2600 μg/mL were used to evaluate toxicity in the presence (4 hour treatment) and absence (4 hour and 24 hour treatment) of metabolic activation.
No relevant toxic effects were observed after 4 hours treatment up to the maximum concentration with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. No precipitation or phase separation occurred up to the maximum concentration after 4 and 24 hours treatment with and without metabolic activation.
Since neither precipitation nor toxicity was noted in the pre-experiment after 4 hour treatment up to the maximum concentration of 2600 μg/mL, culture medium was used as solvent in the main experiments. The maximum concentration in the first main experiment was based on the solubility properties of the test item in culture medium (4000 μg/mL). The concentration range of second main experiment was based on the results of the pre-experiment (without metabolic activation) and the first main experiment (with metabolic activation).

MAIN EXPERIMENT (Experiment 1 & Experiment 2):
NOTE: following the expression phase the cultures at the two highest concentrations in experiment 1 without metabolic activation (concentrations: 2000.0 and 4000.0 µg/mL) and the cultures at the highest concentration with metabolic activation (concentration: 4000.0 µg/mL) were not continued due to exceedingly severe cytotoxic effects. In experiment 2 the cultures at the highest concentration without metabolic activation (concentration: 2000.0 µg/mL) were not continued for the same reason. The cultures at the lowest concentration in experiment 2 with metabolic activation (concentration: 250.0 µg/mL) were not continued since a minimum of only four concentrations is required by the guidelines.

-Culture medium:
For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, 10 % FBS (except during 4 hour treatment), neomycin (5 μg/mL) and amphotericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
- Seeding:
Two to three days after sub-cultivation stock cultures were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in PBS.
Prior to the trypsin treatment the cells were rinsed with PBS buffer containing 200 mg/L EDTA (ethylene diamine tetraacetic acid). Approximately 1.5x10^6 (single culture) and 5x10^2 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment.
- Treatment:
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.
The pH was adjusted to 7.2.
The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below.
Three or four days after treatment 1.5x10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5x10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked witha preparation microscope.

OTHER EXAMINATIONS:
- pH-value: pH was determined in culture medium of the solvent control and of the maximum concentration of the test item in the pre-experiment without metabolic activation.
Solvent control: 7.41
2600 µg/mL: 7.39
- Osmolarity: osmolarity was determined in culture medium of the solvent control and of the maximum concentration of the test item in the pre-experiment without metabolic activation
Solvent control: 358 mOsm
2600 µg/mL: 380 mOsm

ACCEPTABILITY OF THE ASSAY:
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 10^6 cells found in the solvent controls fall within the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.
The data of this study comply with the above mentioned criteria and historical data.
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment and the results are outside the distribution of the historical negative control data.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency and the results are outside the distribution of the historical negative control data. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

In a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
In a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
please refer to the field "Additonal information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation of the test item was observed in the first experiment at 2000 μg/mL and above without metabolic activation and at 1000 μg/mL (culture I) and at 2000.0 μg/mL (culture II) with metabolic activation. In the second experiment precipitation was noted at 1000 μg/mL and above with metabolic activation.

MAIN EXPERIMENT (Experiment 1 & Experiment 2):
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration.
The threshold of three times the corresponding solvent control was exceeded in both cultures of experiment 1 without metabolic activation at 1000.0 μg/mL. The range of the historical solvent control data was also exceeded. However, no dose dependent increase was noted in culture I as indicated by the lacking statistical significance. In the second experiment without metabolic activation the threshold but not the historical range was exceeded at 500 μg/mL and at 1500 μg/mL. This effect however is based on a relatively low solvent control of just 6.4 colonies per 10^6 cells and therefore, has no biological relevance at all.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture of the first experiment without metabolic activation. This isolated trend was judged as irrelevant as it was neither reproduced in the parallel culture nor in the second experiment without metabolic activation. In the second experiment a significant trend was noted in the first culture without metabolic activation. However, this trend was of no biological relevance at all as it was reciprocal, going down versus increasing concentrations.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 6.4 up to 34.6 mutant colonies per 10^6 cells; the range of the groups treated with the test item was from 5.2 up to 64.9 mutant colonies per 10^6 cells.
Ethylmethane sulfonate (150 μg/mL) and 7,12-dimethylbenz(a)anthracene (2.2 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. The positive control in the first experiment with metabolic activation did not quite reach the historical range in culture I (68.8 colonies per 10^6 cells compared to a range of 77.7 – 2042.6 colonies per 10^6 cells). This deviation was judged as irrelevant as it was minor and the threshold of three times the corresponding solvent control was exceeded (induction factor of 5.5).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both cultures occurred in the second experiment at 1500 μg/mL in the absence of metabolic activation. The recommended cytotoxic range of approximately 10 – 20% relative cloning efficieny I or relative cell density was covered without metabolic activation. With metabolic activation precipitation was dose limiting as no cytotoxicity occurred at lower precipitating concentrations up to 2000 μg/mL.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, strontium neodecanoate is considered to be non-mutagenic in this HPRT assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A bacterial reverse mutation assay was performed to investigate the potential of strontium neodecanoate to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver S9. Each concentration, including the controls, was tested in triplicate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 μg/plate with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Strontium Neodecanoate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, strontium neodecanoate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

Strontium neodecanoate, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The highest applied concentration in this study (2654.0 μg/mL of the test item) was chosen with regard to the solubility properties of the test item. In Experiment I in the absence and presence of S9, no cytotoxicity was observed up to the highest evaluated concentration. In Experiment II in the absence of S9 clear cytotoxicity (67.9 %) and in the presence of S9 moderate cytotoxicity (44.0 %) was observed at the highest evaluated concentrations. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, strontium neodecanoate is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations.

 

Strontium neodecanoate was assessed in an in vitro HPRT mutation assay in V79 cells. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation and for 4 hours with and 24 hours without metabolic activation in a second experiment. The maximum concentration in the first main experiment was based on the solubility properties of the test item in culture medium (4000 μg/mL). No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, strontium neodecanoate is considered to be non-mutagenic in this HPRT assay.

Justification for classification or non-classification

Based on the guideline-conform studies conducted under GLP, the test substance should be considered void of genotoxicity. Thus, according to Regulation (EC) No 1272/2008 as amended, the test substance is not considered to be genotoxic, and hence no classification or labelling is required.