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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
17-feb-2010 to 20-apr-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
- The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Silicic acid(H2Si2O5), barium salt(1:1), lead-doped
- Substance type: white powder
- Physical state: powder
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Method

Target gene:
- Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
-RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24 hours treatment: 3, 10, 33, 100 and 333 µg/mL
Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 0.3, 1, 3, 10, 33, 100, 333 and 666 µg/mL
With S9-mix, 3 hours treatment: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A workable suspension could be obtained in DMSO and DMSO has been accepted and approved by authorities and international guidelines
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Positive control substance:
cyclophosphamide
Remarks:
with S9 Migrated to IUCLID6: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplo cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

RANGE-FINDING/SCREENING STUDIES:
-The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests
Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
after the prolonged treatment period
Vehicle controls validity:
valid
Positive controls validity:
valid
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 333 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 333 µg/mL in the absence of S9, 24 hours treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
- The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- After the 3 hours treatment period, no toxicity was observed at the highest precipitating tested dose level in the absence and presence of S9-mix.

- After the 24 hours treatment period, the relative total growth of the highest test substance concentration was reduced by 75% compared to the total growth of the solvent control .

Remarks on result:
other: strain/cell type: L5178Y/TK+/-3.7.2C
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

In the absence of S9-mix, Silicic acid(H2Si2O5), barium salt(1:1), lead-doped did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Silicic acid(H2Si2O5), barium salt(1:1), lead-doped did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

In conclusion, Silicic acid(H2Si2O5), barium salt(1:1), lead-doped is not mutagenic in the TK mutation test system under the experimental conditions described in the report.