2-Propenoic acid, 2-[2-(ethenyloxy)ethoxy]ethyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between Feb. 23rd and May 25, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

see priniciples of method

Principles of method if other than guideline:

The wells were not washed after 4 hours in culture in the growth inhibition test. In this study, it was found that the test substance was dissolved into the vehicle and not precipitated. Moreover, the results of the absorbance of negative control were within the range of the standard deviation of the historical control dtaa in our laboratory. Therefore protocol deviation noted above was not considered to give an effect on the integrity and interpretation of the data or outcome of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: not stated Date of Signature: 25th May 2006

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-690-9

EC Name:

-

Cas Number:

86273-46-3

Molecular formula:

C9H14O4

IUPAC Name:

2-[2-(ethenyloxy)ethoxy]ethyl prop-2-enoate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 2-(2-Vinyloxyethoxy) ethyl acrylate
- Molecular formula (if other than submission substance): C9H1404
- Molecular weight (if other than submission substance): 186.20
- Substance type: Transparent liquid
- Physical state: liquid
- Analytical purity: 99,3%
- Lot/batch No.: 5B25
- Expiration date of the lot/batch: Dec. 31 , 2007
- Storage condition of test material: Light tight, Refrigerate
- Handling & notice: Mask and gloves should be worn

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Model chromosome number of Chinese hamster lung (CHL/IU) cells is 25, and cell-cycle is 15-17 hours. Cells were cultured with EMEM in the C02 incubator.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat induced liver PB + BF)

Test concentrations with justification for top dose:

A growth inhibition test was conducted in 9 dose levels, in which the highest dose of 5000 µg/mL was sequentially diluted to 8 additional lower doses (2.5, 5, 10, 105, 210, 420µg/mL) with the negative control groups.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO, Lot No. : K33960231 504, Merck, USA)
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was used as a vehicle since the test substance was dissolved through the pre-preparation of the test substance.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 Migrated to IUCLID6: (CP)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium


DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Short term exposure = 6 hours Continuous treatment = 24 hours

- Expression time (cells in growth medium):
18 hrs for 6 hrs exposure.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
24 hrs.


SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid


STAIN (for cytogenetic assays):
Stained with 5% Giemsa solution


NUMBER OF REPLICATIONS:
Duplicate cultures


NUMBER OF CELLS EVALUATED:
200/metaphases/dose


DETERMINATION OF CYTOTOXICITY
- Method:


-Scoring of Chromosome Damage:
In each slide, 100 metaphases (200 metaphases/dose) were examined using the biological microscope of differential interference type (BX51, Olympus, Japan) of 1 ODD-fold magnification. Structural aberration was classified as follows; chromatid break (ctb), chromatid exchange (cte), chromosome break (csb), chromosome exchange (cse) and other (0). Numerical aberration was classified as follows polyploidy (pol). Two types aberration such as chromatid and chromosome gap (g) were recorded separately. In the meta phase, if there are several gap or cuttings, it was recorded as a fragment (frg). For a numerical aberration, any cell with 1 or more aberration was counted as 1 aberrant cell. Evaluation of results did not include gaps while the gaps were recorded in raw data of structural aberration. For numerical aberration, any cell with 1 or more polyploidy (pol) aberration was counted as 1 aberrant cell.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells

Evaluation criteria:

The final decision of chromosome aberration in relation to the test substance was carried out in accordance with Toshio Sofuni and et al. [4]. If an an appearance rate was below 5% between 5 and 10% and over 10%, it was judged as a negative, equivocal, and positive, respectively

Statistics:

Statistical analysis was not performed. The mean value and standard deviation were calculated using the values measured from the test.

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

Remarks:

Refer to information on results and attached tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Range finding studies:
Growth inhibition test (Table 1)
A growth inhibition test was conducted in 9 dose levels, in which the highest dose of 5000 µg/mL is sequentially diluted to 8 additional lower doses (5, 10, 50, 100,250,500, 1000,2500 µg/mL) with the negative control groups. As the result of the growth inhibition test, the ICso was calculated as 9.2 µg/mL and 422.1 µg/mL for the short time treatment without and with metabolic activation system and 8.9 µg/mL for the continuous treatment.

2. In vitro chromosome aberration test (Table 2, 3)
On the basis of the growth inhibition test, the highest dose in chromosome aberration test was chosen as 10 µg/mL for the short time treatment without metabolic activation system and the continuous treatment, and 420 µg/mL for the short time treatment with metabolic activation system. Subsequently, the highest dose formulations were diluted by common ratio of 2 to produce 2 additional lower doses levels accompanied by a negative and positive control. Each dosing formulations were treated for 6 and 24 hours for the short time treatment regardless of metabolic activation and continuous treatment without metabolic activation, respectively; and specimens were prepared to examine chromosome aberration.

As a result, the structural and numerical chromosome aberrations were not observed in both the short time treatment with and without metabolic activation system and the continuous treatment as the negative control.

The cell proliferation rate in 0, 2.5, 5 and 10 µg/mL were calculated as 100.0, 102.7, 98.6 and 98.6 % for short time treatment without metabolic activation system, and in 0, 105, 210, 420 µg/mL were calculated as 100.0, 115.0, 115.0 and 82.5 % for short time treatment with metabolic activation system. In addition, the cell proliferation rate in 0, 2.5,5,10 µg/mL were calculated as 100.0,101.3,117.7, 72.2 % for continuous treatment system.

According to the chromosomal aberration test, the result was negative for the short time treatment with metabolic activation. In order to verify the result, the confirmation test was carry out in 6 hours after the cultivation, recovery time executed for 42 hours.

Result of confirmation test, the number of structural and numerical chromosome aberration was not increased as compared with that of a negative control.

Any other information on results incl. tables

This study was designed to examine the clastogenic potential of 2-(2-Vinyloxyethoxy) ethyl acrylate in the chromosome aberration test system using Chinese hamster lung cell.

The result of growth inhibition test showed that the IC50 was calculated as 9.2 µg/mL and 422.1 µg/mL for the short time treatment without and with metabolic activation system and 8.9 µg/mL for the continuous treatment. On the basis of the growth inhibition test, the highest dose in chromosome aberration test was chosen as 10 µg/mL for the short time treatment without metabolic activation system and the continuous treatment and 420 µg/mL for the short time treatment with metabolic activation system. Subsequently, the highest dose formulations were diluted by common ratio of 2 to produce 2 additional lower doses levels accompanied by a negative and positive control.

The structural and numerical chromosome aberrations were not observed in both the short time treatment with and without metabolic activation system and the continuous treatment as the negative control.

Form that the result showed negative for the short time treatment with metabolic activation, the confirmation test was performed to verify the negative result by 6 hours treatment and 42 hours recovery.

Through the confirmation test, it was also found that the number of structural and numerical chromosome aberration for the short time treatment with metabolic activation system was not increased as compared with that of a negative control.

It is confirmed that averages of chromosome aberration cells in negative and positive control group were within the range of the historical control data in our laboratory (Table 4).

In conclusion, 2-(2-Vinyloxyethoxy) ethyl acrylate did not show the chromosome aberrations regardless of application of metabolic activation system in the chromosome aberration test system using Chinese hamster lung cell (CHL/IU), under the conditions of this study. -

Applicant's summary and conclusion

Conclusions:

In conclusion, 2-(2-Vinyloxyethoxy) ethyl acrylate did not show the chromosome aberrations regardless of application of metabolic activation system in the chromosome aberration test system using Chinese hamster lung cell (CHL/IU), under the conditions of this study.

Executive summary:

This study was designed to examine a clastogenic potential of 2-(2-Vinyloxyethoxy)ethyl acrylate in the chromosome aberration test system using Chinese hamster lung cell (CHL/IU).

The result of growth inhibition test showed that the IC50 (Inhibition concentration 50%) was calculated as 9.2 µg/mL and 422.1 µg/mL for the short time treatment without and with metabolic activation system and 8.9 µg/mL for the continuous treatment. On the basis of the growth inhibition test, the highest dose in chromosome aberration test was chosen as 1 0 µg/mL for the short time treatment without metabolic activation system and the continuous treatment, and 420 µg/mL for the short time treatment with metabolic activation system. Subsequently, the highest dose formulations were diluted by common ratio of 2 to produce 2 additional lower doses levels accompanied by a negative and positive control.

Each dosing formulations were treated for 6 and 24 hours for the short time treatment regardless of metabolic activation and continuous treatm~nt without metabolic activation, respectively; and specimens were prepared to examine chromosome aberration.

As the result of chromosome aberration test, the number of structural and numerical chromosome aberration cells of treatment groups was not increased as compared with that of a negative control group regardless of application of metabolic activation system in the short time treatment and continuous treatment.

From that the result showed negative for the short time treatment with metabolic activation, the confirmation test was performed to verify the negative result by 6 hours treatment and 42 hours recovery.

Through the confirmation test, it was also found that the number of structural and numerical chromosome aberration for the short time treatment with metabolic activation system was not increased as compared with that of a negative control. It is confirmed that averages of chromosome aberration cells in negative and positive control group were within the range of the historical control data in the laboratory.

In conclusion, 2-(2-Vinyloxyethoxy)ethyl acrylate did not show the chromosome aberrations regardless of application of metabolic activation system in the chromosome aberration test system using Chinese hamster lung cell (CHL/IU), under the conditions of this study.