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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:
experimental study
Adequacy of study:
key study
Study period:
2006-05-26 to 2006-11-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD test guideline 473

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
19 May 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Phenobarbital/ß-Naphthoflavone induced)
Test concentrations with justification for top dose:
Exp. 1 without and with S9 (18 + 4 hours): 22.2, 44.4, 88.8, 177.5, 355.0, 710.0, 1420.0, 2480.0 µg/mL
Exp. 2 without S9 (18 + 18 and 28 + 28 hours): 44.4, 88.8, 177.5, 355.0, 710.0, 1420.0, 2480.0 µg/mL
Exp. 2 with S9 (28 + 4 hours): 44.4, 88.8, 177.5, 355.0, 710.0, 1420.0, 2480.0 µg/mL
The highest concentration was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or precipitation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility properties and its relative non-toxicity to the cell cultures
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphase plates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistical significance was confirmed by means of the Fisher s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 5.6 % polyploid cells).
Statistics:
Fisher’s exact test

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:
In this study, in the absence and presence of S9 mix, no cytotoxicity indicated by reduced cell numbers and/or mitotic indices of below 50 % of control was observed up to the highest applied concentration being far in the range of test item precipitation.
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 4.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.0 - 2.0 % aberrant cells, exclusive gaps) and within the range of the historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.
Two significant increases in the number of cells carrying structural chromosome aberrations were observed in Experiment II, at preparation interval 18 hrs, after treatment with 88.8 µg/mL and at preparation interval 28 hrs, after treatment with 355 µg/mL. Although these increases of 2.0 % and 4.0 % aberrant cells, respectively were statistically significant compared to the low responses in the solvent control data (0.0 and 0.5 % aberrant cells, respectively), the values were within the historical control data range (0.0 - 4.0 % aberrant cells). In addition, dose-related increases in the number of aberrant cells were observed at 88.8 to 355 µg/mL both, in Experiment I after 4 hrs treatment in the absence of S9 mix (0.0 %, 1.5 %, and 3.5 % aberrant cells) and in Experiment II, at preparation interval 28 hrs in the presence of S9 mix (1.0 %, 1.5 %, and 4.0 % aberrant cells). Since all values were within the range of the historical control data, the increases have to be regarded as biologically irrelevant.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion