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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:
7 November 2003 to 25 February 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Details on test material:
Name: HSY-2701
Lot No.: 1004-1
Purity: 99.8 %
Impurity: Unknown substance 0.2 %
Storage condition: Stored at room temperature (measured temperature: 19.1 - 23.0 °C) protected from light.
Appearance at room temperature: Red powder
Stability: Stable at room temperature

Method

Target gene:
Not applicable for chromosome aberration studies.
Species / strain
Species / strain / cell type:
other: Chinese Hamster Lung CHL/IU
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimum essential medium (MEM) and MEM culture medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared using homogenate of the livers of Sprague Dawley rats treated with phenobarbital 4 times and 5,6-benzoflavone once.
Test concentrations with justification for top dose:
- S9 mix assay: 313, 625, 1250, 2500, 5000 µg/mL
+ S9 mix assay: 313, 625, 1250, 2500, 5000 µg/mL
24 hour assay: 313, 625, 1250, 2500, 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In the preliminary test for the selection of the solvent it was found that the tet substance did not dissolve and did not suspend uniformly in saline or in 1 % w/v sodium carboxymethylcellulose solution at 50 mg/mL. It suspended almost uniformly at 250 mg/mL in DMSo and in acetone. In the preparations using acetone, however precipitation was recognised sooner that when using DMSO. No heat, discolouration or foaming was observed when using DMSO.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: Without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: With S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 (continuous) or 6 hours (short term)
- Expression time (cells in growth medium): 18 hours in the short term assay
- Fixation time : Two hours prior to the end of culture.

SPINDLE INHIBITOR: Colcemid
STAIN: Giemsa's solution

NUMBER OF REPLICATIONS: Performed in duplicate

NUMBER OF CELLS EVALUATED: 100 cells per plate

DETERMINATION OF CYTOTOXICITY
- Method: Cell growth, 50 % cell growth inhibition, mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Polyploid cells with 35 or more centromeres (including endoreduplicated cells)
Evaluation criteria:
Negative: Both structural and numerical aberrant cells were obseved at lower than 5 % in the test substance treatment group.
Inconclusive: The structural or numerical aberrant cells were observed at 5 % or higher but lower than 10 % in the test substance treatment group.
Positive: The structural or numberical aberrant cells were observed at 10 % or higher in the test substance treatment group and increased dose-dependently.
Statistics:
Cell growth inhibition was not inhibited by more than 50% under any treatment condition. Therefore 50% cell growth inhibition concentration (IC50) was not calculated.
Measurement of growth index: A portions of the cells was used for cell counting with a haemocytometer. Cell growth index was calculated based on the negative control value (average as 100 %). Measurement of cell growth was not conducted for the positive controls.
Measurement of mitotic index and relative mitotic index: The number of metaphase cells was counted by examining 500 cells per one plate (i.e. 1000 cells per concentration). Mitotic index % was calculated for each dose according to the following equation:
Mitotic index = (number of metaphase cells)/(number of cells examined) x 100
From the mitotic index, relative mitotic index % at the dose was calculated according to the following equation.
Relative mitotic index = (Mitotic index in cells treated with the test substance)/(Mitotic index in cells treated with the negative control) x 100

No statistical analysis was performed.

Results and discussion

Test results
Species / strain:
other: Chinese Hamster Lung CHL/IU
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the cell growth inhibition test and the chromosomal aberration test, some of the test substance floated or precipitated in the treatment medium at the begining and the end of the treatment in each test substance treatment group in each treatment condition.

RANGE-FINDING/SCREENING STUDIES:
In the cell growth inhibition test, the test substance did not inhibit cell growth more than 50 % under any treatment condition.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In the attached document, the tabulated results are presented. Graphical representations of the results are also included.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative With and without metabolic activation.

HSY-2701 was considered not to have the ability to induce chromosomal aberration under the conditions employed in the study.
Executive summary:

In a GLP compliant study conducted to guideline OECD 473, the test substance HSY-2701 was considered not to have the ability to induce chromosomal aberrations in Chinese hamster lung cells (CHL/IU).