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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
no data available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented publication.

Data source

Reference
Reference Type:
publication
Title:
Ability of root canal antiseptics used in dental pratice to induce chromosome aberrations in human dental pulp cells
Author:
Nishimura, H.; et al.
Year:
2008
Bibliographic source:
Mut. Res. 649, 45-53

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Examination of the ability to induce chromosome aberrations in human dental pulp cells.
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
Calcium dihydroxide
EC Number:
215-137-3
EC Name:
Calcium dihydroxide
Cas Number:
1305-62-0
IUPAC Name:
calcium dihydroxide
Details on test material:
- Name of test material (as cited in study report): Calcium hydroxide
- Physical state: solid
- Purity: 99,9 % (Wako Pure Chemical)
No further details are given.

Method

Species / strain
Species / strain / cell type:
mammalian cell line, other: human dental pulp cells, designated as D824 cells
Details on mammalian cell type (if applicable):
The enzymazically released cells were suspended in alpha-minimum essential medium (alpha-MEM) supplemented with 20 % fetal bovine serum, 100 µM L-ascorbic acid phosphate magnesium salt n-hydrate, 2mM L-glutamine, 0.22 % NaHCO3, 100 units/mL penicilline and 100 µg/mL streptomycin and passed through a 70 µm nylon cell strainer to remove cell aggregates. After counting the number of cells, 1.5x10^5 cells were plated into 75cm² flasks. When confluent, cells were harvested with a solution containing 0.25 % trypsin and 0.1 % EDTA and replated into new flasks.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
30; 100 and 300 µM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Test substance was dissolved at 30 mM in glycerol at 65 °C.
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Cells were treated for 2 hours in the presence of 5% PMS

Migrated to IUCLID6: (50 µM)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: formalin (0.001 and 0.0003 µM)
Remarks:
CA in D824 cells induced by treatment for 30 hours.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): After treatment, cells were washed twice with fresh medium and subsequently incubated for a further 27-hour period, or cells were treated continuously for 30 hours.
- Fixation time (start of exposure up to fixation or harvest of cells): 3 hours before the end of the incubation, colcemid was administered at 0.02 µg/mL, and metaphase chromosomes were prepared for analysis.

NUMBER OF METAPHASES EVALUATED: 100 to 200

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was measured to select concentrations for analysis of chromosome aberrations. Cytotoxicity was determined as the number of cells treated with calcium hydroxide, relative to the number of cells in the control cultures x 100.
D824 cells after 6 to 8 passages were plated in triplicate onto 60-mm dishes at 8x10³ cells/cm², equivalent to 1.6x10^5 cells/dish, and incubated overnight. The cells were treated with calcium hydroxide (in absence or presence of metabolic activation) at varying concentrations for 3 hours. After two washings with 2 mL of fresh medium, cells were incubated for a further 24 hours and the number of cells was counted after harvesting cells with 0.25% trypsin.

EXAMINATIONS:
The aberrations scored were gaps, breaks, exchanges, dicentric chromosomes, ring chromosomes and fragmentations.
Evaluation criteria:
No data given
Statistics:
Statistical analysis was performed by qui-square test to assess the significance of the difference in the incidences of chromosome aberrations between control cultures and cultures treated with calcium hydroxide. The level of significance in the statistical analysis was determined at p<0.05.

Results and discussion

Test results
Species / strain:
mammalian cell line, other: D824 cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No increases in the levels of chromosome aberrations were observed in cells treated with calcium hydroxide.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Calcium hydroxide did not induce chromosome aberrations after 3 h. Since it is known that some substances induce chromosome aberrations only after longer exposure, a protocol with a continuous treatment for 30 hours was employed additionally. Results show that calcium hydroxide did not enhance the level of chromosome aberrations in D824 cells even after prolonged exposure.
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

Calcium hydroxide failed to induce chromosome aberrations in the absence or presence of exogenous metabolic activation.