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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:
3.4.2012 - 20.9.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
See Overell remarks
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Centralit
- Physical state: white solid crystalline
- Analytical purity: 99.9 % (w/w)
- Lot/batch No.: 0500906
- Expiration date of the lot/batch: 16.9.2016
- Stability under test conditions: stable
- Storage condition of test material: in dry room at the temperature < 40°C

Method

Species / strain
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media:
RPMI 1640 with L-Glutamine
PHA-M (Phytohaemagglutinin M)
Foetal Bovine Serum

- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
500, 250, and 100 μg/mL (3h)
600, 500, 250, 100 and 50 μg/mL (24h) - concentrations of 500, 250, 100 μg/mL were used for analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was not soluble in culture medium.
Controls
Untreated negative controls:
yes
Remarks:
untreated culture
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (without metabolic activation), DMSO+S9 (with metabolic activation)
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: thiotepa
Remarks:
thiotepa (without metabolic activation), CPA+S9 (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: diluted in DMSO

DURATION
- Exposure duration: 3h, 24h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs

SPINDLE INHIBITOR (cytogenetic assays): colcemid solution
STAIN (for cytogenetic assays): Giemsa-Romanowski solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 metaphases per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
Cytotoxic effect of the test substance is was indicated by relative mitotic index. If the mitotic index is reduced more than 50 %, it will refer to cytotoxicity.
Mutagenic potential is indicated by increasing of number of chromosome aberrations in comparison with the negative solvent control (two-fold increase rule) and/or by dependence of increasing number of aberrations on dose (dose-response relationship).

Results and discussion

Test results
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentration 5000, 2500, 1000, 800 and 700 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

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

Under the above-described experimental design, the test substance Centralit was non mutagenic for the human peripheral blood lymphocytes in both experiments without and with metabolic activation.
Executive summary:

The In Vitro Mammalian Chromosome Aberration Test assayed the test substance Centralit for the mutagenicity. The performed test was based on EU Method B.10, Mutagenicity – In Vitro Mammalian Chromosome Aberration Test, which is analogous to the OECD Test Guideline No. 473.

The human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was diluted in dimethylsulfoxide and assayed in doses of 50 - 5000µg/mL, which were applied to cultures in volume of 20µL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance Centralit was non mutagenic for the human peripheral blood lymphocytes in both experiments without and with metabolic activation.