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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 03, 2006 - Jun 22, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells:Hoffmann-La Roche, Pharma, Basel, at May 27, 1997
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index: 16 to 17.5 hours
V79 cells have been successfully used in mutagenicity testing for many years. This cell line has a high proliferation rate and cloning efficiency. The cells have a relatively stable karyotype with a modal chromosome number of 22 ± 1, and an aberration rate of about 0-5 % of the metaphases. Since the cell line is not able to metabolize indirect mutagens to reactive forms, the test is performed both in the presence and absence of an external metabolizing system (liver S9 mix of rats pre-treated with Aroclor 1254).



Metabolic activation:
with and without
Metabolic activation system:
S9-mix with S9 from Aroclor 1254 induced rats
Test concentrations with justification for top dose:
In the first experimental series, test material concentrations ranging from 0.50 to 500 µg/mL were tested. The test material precipitated in the culture medium at concentrations ≥158 µg/mL in the absence and presence of S9 mix. No cytotoxic effects were seen. Quality control of the cell preparations did not show a relevant influence of the test material on the structur,e or spreading of the chromosomes in the concentration range investigated. A change in the pH or the osmotic value of the cell culture medium did not occur in the dose range tested.
For these reasons, cultures treated with the following test material concentrations were evaluated in absence and presence of S9 mix for the occurrence of chromosomal aberrations:
1st series: 15.8, 50.0, and 158 µg/mL
2nd series: 50.0, 88.9, 158, 281 and 500 µg/mL
Vehicle / solvent:
Acetone, 0.1%
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
other: Griseofulvin
Details on test system and experimental conditions:
No. of slides per concentration:
Solvent control: 4
Others: 2
No. of metaphases evaluated per slide: 100 (for structural aberrations); 1000 (for polyploidy)

Preparation times: 25 and 35hours

Exposure times:
- S9 mix: 5, 25 and 35 hours
+ S9 mix: 5 hours

Solvent for the test material: Acetone

Concentrations evaluated:
Test item first series: 15.8, 50.0 and 158 µg/mL
second series: 50.0, 88.9, 158, 281 and 500 µg/mL

Positive controls: - S9 mix: 250 and 500 µg Ethylmethansulfonat (EMS)/mL
31.6 and 88.9 µg Griseofulvin (GRIS)/mL

+S9 mix: 2.00 µg Cyclophosphamide (CPA)/mL
Rationale for test conditions:
Guideline settings are applied
Evaluation criteria:
Step 1: Evaluation of Slide Quality
Step 2: Evaluation of Chomosomal Aberrations

The decisive parameter for the evaluation of both, the treated and untreated cultures, is the number of aberrant metaphases (excluding gaps) per 100 cells. A basic prerequisite for the acceptance of a test series is (a) the correspondence of the actual negative controls with the historic negative controls of the laboratory and (b) a statistically significant and biologically relevant increase in the number of aberrant metaphases for the respective positive controls in relation to the actual negative controls. The discussion of biological relevance includes, among other things, considerations concerning the type and time dependent appearance of the observed chromosomal aberrations.
A test material is defined as being unambiguously negative or non-clastogenic in this test system if no statistically significant increase in the number of aberrant metaphases per 100 cells, as compared to the actual negative control, occurs at any test material concentration.
A test material is positive or clastogenic in this test system if
• a statistically significant, dose-related increase in the number of aberrant metaphases per 100 cells occurs or
• a statistically significant increase in the number of aberrant metaphases per
100 cells is reproduced at the same test material concentration in independent experiments.
In both cases, however, the number of aberrant metaphases should be above the range defined as the historical negative controls of the laboratory and the biological relevance of the results has to be discussed.
In all other cases, further decisions for testing strategies should be made following the scientific evaluation of all existing data including those of non­ toxicological investigations.
Statistics:
Fisher’s Exact Test

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment of V79 cell cultures with the test item, did not increase the proportion of cells with aberrant chromosomes. The test item was not clastogenic in this in vitro test system.