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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:
November 15, 2007 - January 15, 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study has been performed similar to OECD 473 Guideline and according to GLP principles. Deviations: - No information in the report to determine if an independent repeat was performed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Guideline for Genotoxicity Studies (Notificationn 1604 of Iyakushin, November 1, 1999, Ministry of Health, Labour and Welfare)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No information in the report to determine if an independent repeat was performed.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): CH-CA
- Description: Transparent liquid
- Stability under test conditions: Unknown
- Storage condition of test material: Stored at room temperature in a sealed container

Method

Species / strain
Species / strain / cell type:
mammalian cell line, other: Chinese Hamster Lung (CHL/IU)
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's MEM supplemented with 10% heat-inactivated fetal bovine serum was mixed with the penicillin-streptomycin mixture containing 10000 units/mL penicillin and 10000 µg/mL streptomycin at the ratio of 100:1.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, with a modal chromosome number of 25 and approximately 15 hours of cell cycle.
- The cells were subcultured until 16 passages and used for the growth inhibition test and cytogenetic test.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and 5,6-benzoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Growth inhibition test, with and without S9-mix: 0, 5, 10, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL (6h and 24h exposure, 24h fixation)
Cytogenetic test, without S9-mix: 350, 700 and 1400 µg/mL (6h exposure, 24h fixation)
Cytogenetic test, with S9-mix: 875, 1750 and 3500 µg/mL (6h exposure, 24h fixation)
Cytogenetic test, without S9-mix: 450, 900 and 1800 µg/mL (24h exposure, 24h fixation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Based on the information provided by the Sponsor, the solubility and dispersion characteristics of the test substance were assessed for the highest dose in water for injection, saline and DMSO. The test substance was insoluble in water for injection and saline, but was soluble in DMSO. Therefore, DMSO was determined as the vehicle for this study.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix

Migrated to IUCLID6: dose 0.05 µg/mL (6h and 24h exposure)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9-mix

Migrated to IUCLID6: dose 20 µg/mL (6h exposure)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: 6h (with and without S9 mix), 24h (without S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24h

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa solution diluted with phosphate buffer

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 200 metaphases/dose

DETERMINATION OF CYTOTOXICITY
- Method used in the growth inhibition test: Spectrophotometrical absorbance of solutions was measured using the ELISA reader (VERSA matTM, Molecular Devices, USA). Since cytotoxicity was observed, 50% inhibition concentration (IC50) was calculated to determine the highest dose for the cytogenetic test.
- Method used in the cytogenetic test: The number of cells of all well dishes were counted using hemocytometer and cell proliferation rate was calculated.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no

Evaluation criteria:
Mean frequencies of chromosome aberration cells: <5%: negative; >5%-<10%: equivocal; >10%: positive.
Statistics:
For the aberration cells data, Fisher's exact test was used for the negative control versus treated groups comparison, and the negative control versus positive control. Group comparisonwere performed at the 5.0% one-tailed probability level. The entire statistical analysis was performed using the statistical program (PC SAS BUNDLE, SAS (version 9.1.3, SAS Institute Inc., Cary, NC, USA)).

Results and discussion

Test results
Species / strain:
mammalian cell line, other: Chinese Hamster Lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
proliferation rate decreased at the highest tested dose in the short and continuous treatment time.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The deposition of the test substance was not observed in the short time treatment with and without S9-mix and in the continuous treatment of all doses of the test substance.
RANGE-FINDING/SCREENING STUDIES: As a result of the growth inhibition test, cytotoxicity was observed in the short time treatment with and without S9-mix and in the continuous treatment. Based on the growth inhibition test, 50% inhibition concentration (IC50) was calculated as 3563 and 1325 µg/mL for the short time treatment with and without S9-mix, respectively, and as 1721 µg/mL for the continuous treatment.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In a chromosome aberration test performed similar to OECD 473 and GLP, the test substance, CH-CA, did not show chromosome aberrations with and without S9-mix using chinese hamster lung cell (CHL/IU). The number of aberration cells in the positive controls were significantly increased when compared with the negative control.