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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test using a well-recognised assay method and a close chemical analogue. Sufficient experimental detail for assessment

Data source

Reference
Reference Type:
publication
Title:
Study on three kind of gasoline oxygenates - induced DNA damage in mice fibroblasts
Author:
Song et al
Year:
2002
Bibliographic source:
China J Hyg Occup Dis, October 2002, Vol 20. No5

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Comet assay based on recognised method.
Mouse fibroblast cells exposed to test substance for 1 hour, then harvested for single cell electrophoresis:
- cells layered on agarose-coated slides
- cells lysed to release DNA
- alkaline treatment to unwind DNA strands
- electrophoresis
- slides then washed and stained.
Cells were then microscopically observed, and percentage of comet cells recorded.
GLP compliance:
not specified
Type of assay:
comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
mammalian cell line, other: L-929 mouse fibroblasts
Metabolic activation:
without
Test concentrations with justification for top dose:
150mg/ml
Vehicle / solvent:
DMSO
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
Potassium permanganate
Details on test system and experimental conditions:
This procedure consists of several different stages:

Cell treatment:
during propagation of fibroblasts, 20 microlitres of DMC solution was added to the vial. Cell were treated with 0.25% pancreatin after 1 hour, collected by centrifugation and suspended in 2 mL PBS.

Slide preparation:
110 microlitres of 0.8(wt%) normal melting point agarose was deposited on to the slides and coverslips applied. Slides were refrigerated (4 degree C) for 10 minutes which allowed the agarose to solidfy. The second layer was prepared by adding 10 microlitres of PBS (containing 1000 treated cells) to 75 microlitres of low melting point agarose (LMPA) and mixing at 37 degree C. Coverslips were removed from the slides and the second layer was applied on to the first layer. The slides were re-covered with new coverslip and refrigerated (4 degree C) for 10 minutes to allow the second layer to solidify.

Lysis:
at ambient temperature, the coverslips on the slides were removed with care and the slides were carefully placed in a lysis solution for 1 hour at low temperature and protected from light. (Lysis solution consisted of 2.5 mol/L Sodium chloride, 100 mmol/L Na2-EDTA, 10mmol/L Tris-HCl, 1% (wt%) sarcosine Na with 1% Trition X-100. 10% (vol%) DMSO added just before use)

DNA strand unwinding:
the slides were removed from the lysis solution and washed with distlled water and placed in the electrophoresis bath on the anode side. Freshly prepared alkaline buffer (sodium EDTA 1 mmol/L, NaOH 300 mmol/L) was added the electrophoresis bath to a level 2mm higher than the slides. Conditions: low temperature, protected from for 20 minutes.

Electrophoresis:
Conditions: constant 22V and 180mA
Temperature: Low
Duration: 30 minutes
Special consideration: protect from light

Observation:
DNA image was visualised as orange under UV light using a Olympus fluorescent microscope. Slides were randomly observed and 100 cells selected for scoring (% comet cells). Up to 25 comet cells were selected for comet tail measurement using an integrated micrometer eyepiece microscope
Evaluation criteria:
Comparison of % observed comet cells and comet tail length in treated sample with vehicle (negative) and positive controls
Higher % of comet cells and longer comet tail, increased DNA damage.
Statistics:
- chi-squared test for incidence on comet cells
- variance analysis for comparison of tail length

Results and discussion

Test results
Species / strain:
mammalian cell line, other: L-929 mouse fibroblasts
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: not reported
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: L-929 mouse fibroblasts
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of Comet Assay

 Group  % of comet cells Mean length of Comet tail (um)
 Negative/vehicle control  5  6.1 (+/-0.8)
 DMC  6  6.2 (+/-1.1)
 Positive control  100  114.9 (+/-9.2)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative No DNA damage

The test substance did not induce DNA damage during the study. Therefore it is considered that the test substance is not mutagenic.
Executive summary:

Direct exposure of mammalian cells to dimethyl carbonate at a relatively high concentration (150 mg/mL) caused no significant DNA damage, visualised as comet cells. It is reasonable to predict that ethyl methyl carbonate would give a similarly negative result in this assay: as its predicted hepatic metabolites (ethanol, methanol, carbon dioxide) are generally recognised as non-mutagenic, omission of cell exposure in the presence of of a liver enzyme metabolising system is not considered to invalidate a conclusion of non-genotoxicity.