Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study similar to OECD TG 482

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
GLP compliance:
yes
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Test material

Constituent 1
Chemical structure
Reference substance name:
Triclosan
EC Number:
222-182-2
EC Name:
Triclosan
Cas Number:
3380-34-5
Molecular formula:
C12H7Cl3O2
IUPAC Name:
5-chloro-2-(2,4-dichlorophenoxy)phenol
Details on test material:
- Name of test material (as cited in study report): 39317
- Physical state: white powder
- Analytical purity: not specified in the study report, however, according to " Triclosan supplement I to EU dossier submitted 18 August 2009", the test substance named 39317 was described as being triclosan with a purity of 101%
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: Stability in vehicle was verified by analysis of dosing solutions.
- Storage condition of test material: at room temperature

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
hepatocytes: obtained from an adult male F344 rat
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Metabolic activation system:
intrinsic
Test concentrations with justification for top dose:
0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250 µg/mL
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Remarks:
untreated medium control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO control
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Details on test system and experimental conditions:
Potential to induce unscheduled DNA synthesis of Triclosan was evaluated in primary culture of rat hepatocytes.
Hepatocytes were isolated from the liver of a Fischer 344 rat by the two-step in situ perfusion. Aliquots of 1×10E+5 viable hepatocytes were inoculated into 12-well cluster dishes containing 15 mm diameter plastic coverslips and 2 mL Williams' medium E (WME) supplemented with 10% calf serum. The hepatocytes were allowed to attach for approximately 2 hours in an incubator humidified to 95-100% at 37°C in an atmosphere of 5% CO2 in air.
Triclosan dissolved in dimethylsulphoxide (DMSO), the solvent (DMSO) or the positive control substance were added to each well containing 2 mL of serum-free and 10 µCi ³H-thymidine (specific activity 50-80 Ci/mM). Each Triclosan treatment was representative of triplicate cultures; the concurrent controls were also evaluated in triplicates.
After18 to 20 hours of exposure, the cultures were washed and the cells on coverslips were fixed in 100% ethanol: glacial acetic acid (3:1), air-dried and mounted cell surface up on glass slides with Permaslip. Slides were dipped in NTB-2 photographic emulsion in the dark, allowed to dry overnight and stored at 4°C in light-proof slide boxes for one week. After seven days of exposure time, autoradiographs were developed. Unscheduled DNA synthesis, evidenced by a net increase in black silver grains over the nucleus, was quantified by determining nuclear and cytoplasmic grain counts using an Artek 880 automated colony counter with microscopic/video camera attachment interfaced to an Apple II computer for data acquisition. A total of 150 hepatocytes were scored for each dose level. The cytoplasmic grain count was subtracted from the corrected nuclear grain count to determine the net nuclear grains count (NNG).
Since microscopic examination of the coverslips indicated that Triclosan was cytotoxic at concentrations ≥ 5 µg/mL as evidenced by the low grain incorporation, 2.5 µg/mL was the highest concentration considered for evaluation of DNA repair test. Lower concentrations scored for unscheduled DNA synthesis included 0.25. 0.5 and 1 µg/mL.
Evaluation criteria:
Generally, solvent and/or untreated controls have a NNG count ≤ 0 with a percentage of cells in repair ranging from 0 to 10%. A positive response consists of a mean NNG count greater than 5 with 70-100% of the hepatocytes in repair.

Results and discussion

Test results
Species / strain:
hepatocytes: from an adult male F344 rat
Metabolic activation:
not applicable
Genotoxicity:
negative
Remarks:
the hepatocytes treated with Triclosan showed mean NNG counts below 0 with the percent of cells in repair ranging from 0 to 6%.
Cytotoxicity / choice of top concentrations:
other: >/= 5 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Autoradiographic analyses of the hepatocytes treated with Triclosan revealed mean NNG counts below 0 with the percent of cells in repair ranging from 0 to 6%. Similarly, the untreated media and DMSO controls had the mean NNG counts and the percent of cells in repair comparable with Triclosan-treated groups. The positive control (2AAF) yielded a mean NNG count of 21.2 ± 16.6 and 88.7% of the hepatocytes in repair.
Remarks on result:
other: strain/cell type: see above
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

UDS assay in primary rat hepatocytes

Treatment

Concentration [µg/mL]

NNGa
[mean ± SD]

Average % of nuclei with ≥ 5 grains

Untreated

0

-19.0 ± 12.5

0.0

DMSO

0

-21.6 ± 13.4

0.0

2AAF

0.022

+21.2 ± 16.6

88.7

Triclosan

0.25

-23.4 ± 13.3

0.7

0.5

-25.8 ± 15.2

0.0

1.0

-21.3 ± 14.2

2.7

2.5

-17.1 ± 14.3

6.0

a NNG = net nuclear grains

Applicant's summary and conclusion