Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Version / remarks:
(dated 1984)
GLP compliance:
yes
Type of assay:
chromosome aberration assay

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): FAT 80'023/Q
- Physical state: white solid
- Analytical purity: not specified in the study report; however, according to " Triclosan supplement I to EU dossier submitted 18 August 2009", the purity of CIBA-produced Triclosan exceeded 99%, and for FAT 80’023/Q, Batch No. EN 91390.76 a purity of 99.6% was reported.
- Lot/batch No.: EN 91390.76
- Expiration date of the lot/batch: November 1992
- Stability under test conditions: stable for at least 24 months
- Stability in solvent: stable for at least 48 h in ethanol, DMSO, DMF and hexane.
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SAVO med. Versuchstierzuchten GmbH, Kißlegg, Germany
- Age at study initiation: at least 7 weeks old
- Weight at study initiation: 140-160 g
- Assigned to test groups randomly: yes
- Housing: single, in Makrolon Type I cages, with wire mesh top.
- Fasting: Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum
- Diet (e.g. ad libitum): pelleted standard diet (ALTROMIN 1324)
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
1% aqueous carboxymethylcellulose
Details on exposure:
The test article was suspended in 1% carboxymethylcellulose. The volume administered orally was 10 mL/kg bw.
Duration of treatment / exposure:
6 h, 24 h and 48 h after a single administration of the test article the animals were sacrificed for bone marrow cells removal.
Frequency of treatment:
Single administration
Post exposure period:
see duration of treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
4000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Sex animals/sex/group
Control animals:
yes, concurrent no treatment
Positive control(s):
Cyclophosphamide, 20 mg/kg bw (in deionised water) was used as positive control substance.

Examinations

Tissues and cell types examined:
Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of cytogenetic damage. Per animal, 50 well spread metaphases were scored for gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION
The dose for the cytogenetic assay was determined in a pre-experiment for toxicity. 4000 mg/kg bw was the maximum tolerated dose (MTD).

TREATMENT AND SAMPLING TIMES
After 6 h, 24 h and 48 h following the single administration of the test article, the animals were sacrificed and the the bone marrow cells were collected for chromosome aberration analysis.

DETAILS OF SLIDE PREPARATION
The femora were removed, the epiphyses were cut off and the marrow was flushed out for collection of a cell suspension which was then incubated (20 min, 37°C). Following centrifugation and removal of the supernatant, the cell pellet was fixed (3:1 absolute methanol in glacial acetic acid fixative), gently resuspended and stored overnight at 4°C. Prior to making slides the fixative was changed and enough fixative was added to make a relatively thin cell suspension. The fixative-cell suspension was spread by flame-drying and stained with Giemsa solution. Cover slips were mounted with Eukitt. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS
Evaluation of the slides was performed microscopically. Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. At least 50 weIl-spread metaphases per animaI were scored for cytogenetic damage on coded slides. Only metaphases with the characteristic chromosome number of 42 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis; 1000 cells were scored) was determined. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal died in its test group spontaneously or due to gavage error.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of structural chromosomal aberrations or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of structural chromosomal aberration nor a statistically significant and reproducible positive response at anyone of the test points is considered non-mutagenic in this system.
However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
At no preparation interval the chromosome aberration frequency was significantly enhanced as compared to the negative control.
Toxicity:
no effects
Remarks:
The mitotic index in the preparations was not affected by the test substance.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRETEST
In the preliminary toxicity experiment, 4 animals per dose received 5000 or 4000 mg triclosan /kg bw peroral. At 5000 mg/kg bw, one male rat died after 48 h post dose. Apathy and reduction of spontaneous activity were observed. No mortality was observed in rats treated with 4000 mg/kg bw. Reduction of spontaneous activity was observed in some animals until 6 h post treatment. Thus, 4000 mg/kg bw was defined as the maximum tolerated dose (MTD) and was selected as test dose for the genotoxicity assay.

MAIN ASSAY
At no preparation interval the chromosome aberration frequency was significantly enhanced as compared to the negative control.
An appropriate reference mutagen was used as positive control and showed a distinct increase of induced aberration frequency.

Applicant's summary and conclusion