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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:
experimental study
Adequacy of study:
key study
Study period:
08 Sep. 2010 - 24 Sep. 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Performed under GLP

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 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): crystalline calcium silicate hydrate (xonotlite - tobermorite)
- Physical state: White solid
- Analytical purity: Confidential
- Lot/batch No.: Confidential
- Stability under test conditions: H2O: 96h; EtOH; acetone; CH3CN; DMSO: unknown
- Storage condition of test material: The test item was stored in a closed vessel at room temperature (20 ± 5°C)

Method

Target gene:
His-gene: Amino acid histidine
Species / strain
Species / strain / cell type:
other: S. typhimurium TA1535, TA97a, TA98, TA100 and TA102
Details on mammalian cell type (if applicable):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
Male Sprague-Dawley rat liver S9 induced by Aroclor
Test concentrations with justification for top dose:
Experiment I (plate incorporation test): 50, 148, 497, 1508, and 5007 ug/plate
Experiment II (pre-incubation test): 309, 652, 1262, 2540, and 5008 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The test item was not sufficiently soluble in the accepted solvents for the Ames-test. Therefore, suspensions were prepared. The test item was autoclaved and suspended in deionised water. Water was chosen, because the test item could be suspended completely, and this solvent doesn't have any effects on the viability of the bacteria or the number of spontaneous revertants. The suspensions were stirred during the test.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: strains TA100 and TA1535: Sodium azide; Strains TA97a, TA98, and TA102: 4-Nitro-1,2-phenylene-diamine. With metabolic activation: strains TA97a, TA100, TA102, and TA1535: 2-Aminoanthracene; strain TA98: Benzo-a-pyrene.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes
- Selection time (if incubation with a selection agent): 48 hours

SELECTION AGENT (mutation assays): agar containing Histidine

NUMBER OF REPLICATIONS: Per strain and dose, 4 plates with and 4 plates without S9 mix were used.

NUMBER OF CELLS EVALUATED: 10*9

DETERMINATION OF CYTOTOXICITY
- Method: A reduction of the bacterial background lawn or a reduction of the revertant colonies.
Evaluation criteria:
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
Statistics:
Mean and Standard Deviation

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA1535, TA97a, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was autoclaved and suspended in deionised water.

COMPARISON WITH HISTORICAL CONTROL DATA: The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory).

ADDITIONAL INFORMATION ON CYTOTOXICITY: In both experiments no signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.
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 with metabolic activation
negative without metabolic activation

The test item did not show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only). Cytotoxicity of the test item was not detected. The background lawn was visible and the number of revertants was not significantly decreased. Therefore it can be stated, that under the test conditions, the test item crystalline calcium silicate hydrates (xonotlite - tobermorite) is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535.
Executive summary:

The mutagenic potential of crystalline calcium silicate hydrates (xonotlite - tobermorite) was determined in the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14. Two valid experiments were performed. In the first experiment 5 concentrations of the test item, suspended in deionised water were used (50, 148, 497, 1508, and 5007 ug/plate). Five genetically manipulated strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item didn't show any mutagenic effects in the first experiment. No signs of toxicity towards the bacteria could be observed. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

To verify the results of the first experiment a second experiment was performed, using 5 concentrations of the test item (309, 652, 1262, 2540, and 5008 ug/plate) and a modification in study performance (pre-incubation method). The test item didn't show mutagenic effects in the second experiment, either. No signs of toxicity towards the bacteria could be observed. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Under the conditions of the test, the test item didn't show mutagenic effects towards Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined. The test item crystalline calcium silicate hydrates (xonotlite - tobermorite) is considered as not mutagenic under the conditions of the test.