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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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
No target gene
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: DME
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
mammalian microsomal fraction (S9 mix)
Test concentrations with justification for top dose:
Experiment A:
3/20 hours treatment/sampling time
without S9 mix: 61.73, 20.58, 6.86, 2.29, 0.76 μg/mL
with S9 mix: 185.18, 61.73, 20.58, 6.86, 2.29 μg/mL

Experiment B:
20/28 hours treatment/sampling time
without S9 mix: 20.58, 6.86, 0.76, 0.25 μg/mL

Experiment B:
3/28 hours treatment/sampling time
with S9 mix: 185.18, 61.73, 20.58, 6.86, 2.29 μg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: no data; however, DMSO is a standard solvent used in studies of this type and a concurrent solvent control was included.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Dulbecco's Modified Eagle's (DME) medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (1 % in DME medium)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Untreated negative controls:
yes
Remarks:
DME medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (1 % in DME medium)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 3 or 20 hours
- Expression time (cells in growth medium): 20 or 28 hours
- Fixation time (start of exposure up to fixation or harvest of cells): after 20 or 28 hours (after expression time)

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa stain (5 %)

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: at least 200 metaphase cells containing 2 N ± 2 centromeres

DETERMINATION OF CYTOTOXICITY
- Method: the viable cells were distinguished from non-viable cells on the basis of their appearance and shape

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
– the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data.
– the positive control items produce biologically relevant increases in the number of cells with structural chromosome aberrations.
Statistics:
A Chi square test was performed.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects
- Effects of osmolality: no effects
- Precipitation: no precipitation was noted for examined test concentrations

RANGE-FINDING/SCREENING STUDIES:
Based on the results of a pre-test (cytotoxicity) the concentration indicated in section "Test concentrations" were selected

COMPARISON WITH HISTORICAL CONTROL DATA:
In the presence of metabolic activation, although the chromosomal aberration numbers were not statistically significantly higher than solvent control, the mean values at 61.73 μg/mL (3 hours treatment and 20 hours sampling time) were slightly above the historical control range. The frequency of the cells with structural chromosome aberrations without gaps was within the historical control range in a second experiment with 3 hours treatment and 28 hours sampling time.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The cytotoxicity observed at a test item concentration of 185.18 µg/mL was too pronounced for a reliable analysis due to a limited number of available metaphases.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The validity of the test was shown using ethyl methanesulphonate (0.4 and1.0 μL/mL) and cyclophosphamide (6.0 μg/mL) as positive controls. The test item tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce significant structural chromosome aberrations in this test in Chinese Hamster lung cells.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item was non-clastogenic in this system.
Executive summary:

In a mammalian cell cytogenetics assay for chromosome aberration (Al-Sabti, 2010), V79 cell cultures were exposed to 1-Hexanol, 2-ethyl-, manuf. of, by-products from, distn. Residues in 1 % DMSO at (i) 0.76, 2.29, 6.86, 20.58, and 61.73 µg/mL (without S9 mix), 2.29, 6.86, 20.58, 61.73 and 185.18 µg/mL (with S9 mix) with 3 hours treatment and 20 hours harvest and at (ii) 0.25, 0.76, 6.86 and 20.58 µg/mL (without S9 mix) with 20 hours treatment and 28 hours harvest as well as at (iii) 2.29, 6.86, 20.58, 61.73 and 185.18 µg/mL (with S9 mix) with 3 hours treatment and 28 hours harvest. The results of a pretest on cytotoxicity were used as basis for dose level selection. Montanol 800 was tested up to cytotoxic concentrations with and without metabolic activation. Positive controls induced the appropriate responses. There was no evidence of chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement for test guideline OECD 473 for in vitro cytogenetic mutagenicity data.