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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
; signed by the Depertment of Health of the Government of the UK (21.02.1996)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Strontium chloride-6-hydrate extra pure
- EC number: 233-971-6
- Molecular formula (if other than submission substance): SrCl6*6H2O
- Physical state: solid, white crystalline
- Storage condition of test material: at ambient temperature (<25°C), stored under artificial light

Method

Target gene:
not applicable
Species / strainopen allclose all
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/plate
Main study: 0, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile water
Controlsopen allclose all
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.

Results and discussion

Test resultsopen allclose all
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 FACTORS
No 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 data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No further details are reported.
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

In 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.