Strontium neodecanoate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Strontium neodecanoate

Cas Number:

106705-37-7

Molecular formula:

C10H19O2.xSr

IUPAC Name:

Strontium neodecanoate

Test material form:

other: granular solid

Details on test material:

- Name of test material: strontium neodecanoate
- Molecular weight: 429.6 g/mol
- Physical state: creme/pale yellow granular solid
- Storage condition of test material: cool, dry place (at room temperature)

Method

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

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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.

Controls

Controls 1

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

Controls 2

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 %)

Controls 3

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

Controls 4

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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'.

Applicant's summary and conclusion

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.