Calcium dihydroxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable publication. Purity of the test material unknown.

Data source

Reference

Reference Type:

publication

Title:

Genotoxicity of antimicrobial endodontic compounds by single cell gel (comet) assay in Chinese hamster ovary (CHO) cells

Author:

Ribeiro DA, Scolastici C, de Lima PL, Marques ME, Salvadori DM

Year:

2005

Bibliographic source:

Oral Surg Oral Med Oral Pathol Oral Radiol Endod 99: 637-40

Materials and methods

Principles of method if other than guideline:

The purpose of this study was to investigate whether antimicrobial endodontic agents can induce DNA breakage in Chinese hamster ovary (CHO) cells evaluated by the single cell gel (comet) assay in vitro. CHO cells were chosen because the mechanism of DNA damage induced in these cells has been well documented.

GLP compliance:

not specified

Type of assay:

comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Calcium dihydroxide

EC Number:

215-137-3

EC Name:

Calcium dihydroxide

Cas Number:

1305-62-0

Molecular formula:

CaH2O2

IUPAC Name:

calcium dihydroxide

Details on test material:

- Name of test material (as cited in study report): Calcium hydroxide
- Physical state: powder
- Analytical purity: no data given

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Test concentrations with justification for top dose:

10 µl of a 100 µg/ml test substance solution to obtain final concentration ranging from 0.01 % to 1% after the mixture with the cells.

Details on test system and experimental conditions:

Treatment of cells
CHO K-1 cells were grown to confluence in 75-cm2 culture flasks (Corning, New York, NY) using Ham’s F-10 medium (Invitrogen Corporation; Grand
Island, NY) supplemented with 10% fetal calf serum and 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen Corporation) incubated in a 95% air, 5% CO2 atmosphere at 37°C. Cells were cultured for 5 days prior to treatment with test substances. Confluent cells were detached with 0.15% trypsin (Invitrogen Corporation) for 5 minutes after that, 2 mL complete medium was added and cells were centrifuged at 180g for 5 minutes. Cell suspension was counted using a Neubauer chamber and seeded in 96-well microtitre plates (Corning) at a density of 1x10exp+4 cells per well (at a concentration of 1 x 10exp+6/mL). Ten microliters of the test substance solution (alcium hydroxide) adjusted to 100 mg/mL concentration) was then added to CHOcells individually.Achlorhexidine (chlorhexidinedigluconate) 20% stock solution was used to prepare fresh successive dilutions in phosphate buffer solution (PBS, pH 7.4) to obtain the final concentrations ranging from 0.01% to 1% after the mixture with the cells. The concentrations of calcium hydroxide were selected through preliminary experiments performed with the drugs in order to verify only the genotoxicity of these compounds. The same volume was added to control cultures of either negative vehicle control (PBS) or of a reference alklylating agent MMS (methylmethasulfonate, Sigma Aldrich, St. Louis, Mo) at 1 mg/mL concentration (positive control). After incubating for 3 hours at 37°C, the cells were centrifuged at 180g for 5 minutes and washed twice with fresh medium and resuspended with fresh medium. Each treatment was repeated 3 times consecutively to ensure reproducibility.

Single cell gel (comet) assay
The protocol used for single cell gel (comet) assay followed the guideline purposed by Tice et al. Briefly, a volume of 10 mL of cells (ca.1 x 10exp+4 cells) was added to 120 mL of 0.5% low-melting point agarose at 37°C, layered onto a precoated slide with 1.5% regular agarose, and covered with a coverslip. After brief agarose solidification in the refrigerator, the coverslip was removed and slides immersed in lysis solution (2.5M NaCl, 100 mM EDTA, 10 mM Tris-HCl buffer, pH 10, 1% sodium sarcosinate with 1% Triton X-100 and 10% DMSO) for about 1 hour. The slides were left in alkaline buffer (pH 13) for 20 minutes and then electrophoresed for 20 minutes, at 25V (0.86V/cm) and 300 mA. After electrophoresis, the slides were neutralized
in 0.4 M Tris-HCl (pH 7.5), fixed in absolute ethanol, and stored at room temperature until analysis in a fluorescence microscope at 3400 magnification. To minimize extraneous DNA damage from ambient ultraviolet radiation, all steps were performed with reduced illumination.

DNA damage
An automatic analysis system (Comet Assay II, Perceptive Instruments; Haverhill, UK) was used to determine DNA damage. Two parameters were estimated: tail moment (product of tail DNA/total DNA by the center of gravity) and tail intensity (percentage of DNA in the tail) from 50 cells per treatment.

Cell viability test
Cell viability test for CHO cells was performed using trypan blue staining before the treatment.18 In brief, a freshly prepared solution of 10 mL trypan blue (0.05%) in distilled water was mixed with 10 mL of each cellular suspension for 5 minutes, spread onto a microscope slide, and covered with a coverslip. Nonviable cells appear blue-stained. At least 200 cells were counted.

Statistics:

Statistical analysis of treatments and controls were performed as recommended by Wiklund and Agurell.
The migration data were compared by 1-way analysis of variance (ANOVA), followed by a post hoc analysis using the software SPSS (version 1.0) for Windows. P value less than .05 was considered for statistical significance.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

DNA strand breaks were represented by the mean tail moment and tail intensity for 50 comets/sample.
Calcium hydroxide did not induce strand breaks in DNA. Samples were also incubated in the presence of chlorhexidine at concentrations that ranged from 0.01% to 1%. The mean cell viability for CHO cells was approximately 95%.

Remarks on result:

other: strain/cell type: CHO

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mean +/- SD of DNA damage (tail moment and tail intensity) in CHO cells:

Agent	DNA damage (n=3)
	Tail moment	Tail intensity
Negative control	0.27 ± 0.16	2.99 ± 1.06
Calcium hydroxide	0.22 ± 0.14	2.13 ± 0.63
Positive control	3.50 ± 1.23	22.59 ± 6.48