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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
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:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Lucirin TPO-L
- Physical state: Yellow liquid
- Analytical purity: > 98.7%
- Purity test date: 2004-02-09
- Lot/batch No.: 020012P050
- Expiration date of the lot/batch: Nov. 2004
- Storage condition of test material: Room temperature (protected from light)
- Stability under test conditions: verified in DMSO for 4h and in water for 96h

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM medium with glutamine supplemented with 10% (v/v) fetal calf serum (FCS), 1 % (v/v) penicillin/streptomycin (10 000 IU / 10 000 Ng/ml), 1 % (v/v) amphotericine B (250 Ng/ml) - No FCS was used during 4h treatments
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
aroclor induced rat liver S-9 fraction
Test concentrations with justification for top dose:
Mixed Population Method (MP):
24 hours exposure, 24 hours harvest time, without S-9 mix: 0 ; 6.25; 12.5; 25; 50; 75 ug/ml
4 hours exposure, 24 hours harvest time, with S-9 mix: 0 ; 6.25; 12.5; 25; 50; 100 ug/ml

Mitotic Shake Off Method (MSO) (24 hours mitotic shake off):
24 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, without S-9 mix: 0 ; 6 .25; 12.5; 25; 50; 75 ug/ml
4 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, with S-9 mix: 0; 6 .25; 12.5; 25; 50; 100 ug/ml
4 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, with S-9 mix: 0; 80; 100; 120 ; 140 ; 160 ug/ml (not scoreable due to cytotoxicity)
4 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, with S-9 mix: 0; 60; 70; 80; 90 ; 100 ug/ml
Vehicle / solvent:
Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the V79 in vitro micronucleus assay and for which historical control data is available.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24h (without S9), 4h (with S9)
- Expression time (cells in growth medium): 20h (with S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 24h (mixed population), 27h (mitotic shake off)

STAIN (for cytogenetic assays): Wrights solution (modified May-Gründwald solution)

NUMBER OF REPLICATIONS: 2 per experiment, 4 independent experiments

NUMBER OF CELLS EVALUATED: 1000 per culture, i.e., 2000 per test group

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, proliferation index
Evaluation criteria:
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed, at least to a large extent, the identification and evaluation of a sufficient number of analyzable cells.
- The proportion of cells with micronuclei in negative control (vehicle control) cultures was within the normal range of the historical control data.
- The positive control chemicals (with and without S-9 mix) induced a significant increase in the number of cells with micronuclei.

The test chemical is considered positive, if:
- A dose-related and reproducible significant increase in the number of cells containing micronuclei is observed
- The proportion of micronucleus-containing cells exceeds both the concurrent negative control range and the negative historical control range.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not affected
- Effects of osmolality: not affected
- Precipitation: from app. 90-100µg/ml onward

RANGE-FINDING/SCREENING STUDIES:
75µg/ml without S9 and 100µg/ml with S9 were selected as top doses based on cell count, cell attachment, assessment of slides and the proliferation index in a range finding assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak and dose dependent suppression of mitotic activity and the proliferation index was reported at and above 25µg/ml without S9. A dose dependent reduction in cell count was observed at and above 50µg/ml withough S9 and at and above 100µg/ml with S9. At the same concentrations, cell attachment was slightly reduced.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this assay,the substance is considered not to be a chromosome-damaging (clastogenic) agent nor does it to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells.