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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:
July - October 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted under GLP conditions.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
See details in section : Principles of method if other than guideline.
Qualifier:
according to
Guideline:
other: European Economic Community (EEC). Directive 2000/32/EC, Part B.
Deviations:
yes
Remarks:
See details in section: Principles of method if other than guideline.
Qualifier:
according to
Guideline:
other: Japanese Ministry of Health, Labour and Welfare, Ministry of Economy. Trade and Industry and Ministry of the Environment.
Deviations:
yes
Remarks:
See details in section: Principles of method if other than guideline.
Principles of method if other than guideline:
REMARK From deviations :
The highest dose level tested (100 micrograms/ml) for the 24 and 48 h exposure time is determined by the solubility in the culture medium. Since the test article was tested at the highest dose level possible in this test system, restricted by the solubility in the solvent, this deviation of testing concentrations beyond the limit of solubility has no effect on the results of this study.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 7145
- Substance type: clear colorless liquid
- Physical state: liquid
- Analytical purity: ~94.5%
- Impurities (identity and concentrations): NA
- Lot/batch no.: Batch 142072:43
- Expiration date of the lot/batch: 30 December 2007
- Stability under test conditions: Stable at higher temperatures to 200 degrees C, maximum duration 24 hours. Stable in ethanol for atleast 96 hours.
- Storage condition of test material: At room temperature in the dark
- Other: pH: 7.2.
- Specific Gravity: 1.8

Method

Species / strain
Species / strain / cell type:
mammalian cell line, other: Peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: cultured medium consdidted of RPMI 1640 medium, supplemented with 20 (v/v) heat-inactivated (56 degrees C); 30 min) foetal calf serum, L-glutamine(2 nM), penicillin/streptomycin (50 U/ml and 50 micrograms/mL respectively) and 30 U/mL heparin.
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 (9000 x G supernatant)
Test concentrations with justification for top dose:
Dose-Range finding study: 1,3, 10, 33 and 100 micrograms/ml. Without S9 mix: 10, 33 and 100 micrograms/ml culture medium. With S9-mix: 10,33,66 and 100 micrograms/ml culture medium.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: excellent solubility characteristics
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Solvent for positive controls: Hank's Balanced Salt Soultion (HBSS) without calcium and magnesium.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
DURATION
- Exposure duration: 3 hours, 24 hours, 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells):24 hours/48 hours
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
Considered a positive response if a) It iduced a dose-related statistically significant increase in the frequencies of the number of cells with in chromosome abberations. b) A statistically significant and biologically relevant increase in the frequency of the number of cells with chromosome abberations was observed in the absence of a clear dose-response relathionship.
Statistics:
Chi-squared test, one-sided, p <0.5

Results and discussion

Test results
Species / strain:
mammalian cell line, other: human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
negative with and without metabolic activation up to 100 micrograms/mL
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
but tested up to limit concentrations (100 micrograms/mL)
Vehicle controls validity:
valid
Positive controls validity:
other: yes, mitomycin-C (without metabolic activation).
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test article is not clastogenic in human lymphocytes under the experimental conditions described in the report.
Executive summary:

The purpose of this study was to evaluate the ability of the test article to induce chromosome aberrations in cultured peripheral human lymphocytes. The most recent OECD Guidelines (OECD 473, 1997) were employed. In the first cytogenetic assay, the test article was tested up to 100 ug/mL for a 3-hour exposure with a 24-hour fixation time in the absence and presence of S-9 mix (1.8% v/v).The test article precipitated in the culture medium at this dose level. In the second cytogenetic assay, the test substance was tested up to 100 ug/mL for 24-hours and 48-hours of continuous exposure time with 24-hour and 48-hour fixation times in the absence of S9-mix. In the presence of S9-mix, the test material was tested up to 100 ug/mL for a 3-hour exposure time with a 48-hour fixation time. Positive control chemicals mitomycin C and cyclophosphamide both produced a statistically significant increase in the incidence of cells with chromosome aberrations indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test article did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix in two independently repeated experiments. No effects of the test article on the number of polyploid cells and cells with endoreduplicated chromosomes were observed in either the absence or presence of S9-mix. Therefore, it can be concluded that the test article does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under these experimental conditions. It is concluded that the test article is not clastogenic in human lymphocytes under the conditions described in this report.