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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:
March 14, 2006 to Janury 19, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4A: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test", dated May 19, 2000.
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
"Kanpoan No. 287 - Environmental Agency" "Eisei No. 127 - Ministry of Health & Welfare" "Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry".
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Test material form:
other: solid
Details on test material:
Colour: Black
Storage: At room temperature, in the desiccator

Method

Target gene:
chromosome
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
IA: Exposure period 4 hrs without S9 mix 156.3, 312.5, 625.0, 1250.0, 2500.0, 5000.0 µg/mL
II: Exposure period 18 hrs without S9 mix 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0 µg/mL
II: Exposure period 28 hrs without S9 mix: 312.5, 625.0, 1250.0, 2500.0 µg/mL
IA: Exposure period 4 hrs and Preparation interval 18 hrs with S9 mix: 4.9, 9.8, 19.5, 39.1, 78.1, 156.3 µg/mL
IB: Exposure period 4 hrs and Preparation interval 18 hrs with S9 mix 25.0, 50.0, 75.0, 100.0, 125.0, 150.0, 175.0, 200.0 µg/mL
II: Exposure period 4 hrs and Preparation interval 28 hrs with S9 mix: 9.8, 19.5, 39.1, 78.1, 156.3, 312.5 µg/mL
II: Exposure period 4 hrs and Preparation interval 28 hrs with S9 mix: 31.3, 62.5, 125.0, 250.0 , 500.0, 1000.0 µg/mL
Vehicle / solvent:
deionised water
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Large stocks of the V79 cell line were stored in liquid nitrogen. Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of standardized characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks. About 5 x 10E5 cells per flask were seeded into 15 mL of MEM supplemented with 10% fetal calf serum. The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
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 (9) (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:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the Fisher's exact test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
in the absence of S9 mix
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
in the presence of S9 mix
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Recipitation of the test item in culture medium was observed in the presence of S9 mix, in Experiment IA, at preparation interval 18 hrs with 78.1 µg/mL and above, in Experiment IB, at preparation interval 18 hrs with 50 µg/mL and above, and in Experiment II at preparation interval 28 hrs with 62.5 µg/mL and above.
Remarks on result:
other: strain/cell type: Chinese hamster cell line
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation
negative with metabolic activation

Under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in the absence of S9 mix.
Executive summary:

The test item, suspended (pre-experiment and) or dissolved (experiments IB and II) in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in three independent experiments.

In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (5000 µg/mL) was chosen with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

In the absence of S9 mix and in Experiment IB and II, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II, in the absence of S9 mix, at preparation interval 18 hrs, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In contrast, in the presence of S9 mix and in Experiment II, in the absence of S9 mix, at preparation interval 28 hrs, cytotoxicity was observed at the highest scorable concentrations.

In the absence of S9 mix, and in Experiment IB and II in the presence of S9 mix, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

In contrast, in the presence of S9 mix, a statistically significant and biologically relevant increase (6.0 % aberrant cells, excluding gaps) was observed at the highest scored concentration. The number of aberrant cells showed a dose-related increase, with the two highest concentrations exceeding our historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps). This observation was not verified in the confirmatory Experiment IB.

In Experiment II, in the absence of S9 mix, at preparation interval 18 hrs, two statistically significant increases in the number of aberrant cells (both 5.5 %) exceeding our historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps) were observed. In addition, at preparation interval 28 hrs, two statistically significant and biologically relevant increases in the number of aberrant cells, excluding gaps (9.0 and 10.5 %, respectively) were observed at the scored concentrations.

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the absence of S9 mix.