Vinyl 2-ethylhexanoate

Genetic toxicity: in vitro

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the O.E.C.D. test guideline 473 with GLP compliance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Vinyl 2-ethylhexanoate

EC Number:

202-297-4

EC Name:

Vinyl 2-ethylhexanoate

Cas Number:

94-04-2

Molecular formula:

C10H18O2

IUPAC Name:

ethenyl 2-ethylhexanoate

Constituent 2

Reference substance name:

Vinyl-2-ethylhexanoate

IUPAC Name:

Vinyl-2-ethylhexanoate

Constituent 3

Reference substance name:

Hexanoic acid, 2-ethyl-ethenyl ester

IUPAC Name:

Hexanoic acid, 2-ethyl-ethenyl ester

Test material form:

other: Clear liquid at room temperature.

Details on test material:

As per IUCLID5 Sections 1.1. 1.2. and 4.1.

Method

Target gene:

Whole chromosome

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 fraction with cofactors.

Test concentrations with justification for top dose:

Scored for chromosome aberrations: 4-hour treatment without S9, 25, 50 and 65 ug/mL; 4-hour treatment with S9 metabolic activation, 50, 100 and 200 ug/mL; 20-hour treatment without S9, 10, 25 and 50 ug/mL.

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide

Remarks:

Cyclophosphamide was the positive control with S9 metabolic activation.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Mitomycin C was the positive control with S9 metabolic activation.

Details on test system and experimental conditions:

Chinese hamster ovary (CHO K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20.

Exponentially growing CHO K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 1.5 mM L glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin and 2.5 µg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1C) in a humidified atmosphere of 5 ± 1% CO2 in air) for 16 24 hours.

Aroclor 1254-induced rat liver S9fraction was used as the metabolic activation system. The S9 (Lot No. 3244) was obtained from Molecular Toxicology Inc. (Boone, NC). Immediately prior to use, the S9fraction was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 µL S9 fraction per milliliter medium (McCoy's 5A serum-free medium supplemented with 100 units penicillin/mL, 100 µg streptomycin/mL, 1.5 mM L-glutamine and 2.5 µg/mL amphotericin B).

In a preliminary cytotoxicity screen CHO cells were exposed to vehicle alone and to nine concentrations of testsubstance with half-log dose spacing using single cultures. Precipitation of test article dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment.

Seven to ten dose levelsof the test substance were tested for chromosome aberration induction using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. Precipitation of test article dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose level evaluated for chromosome aberrations was selected to giv eapproximately 50% cytotoxicity (approximately 50% cell growth inhibition relative to the vehicle control).

Treatment was carried out in duplicate by re-feeding the cultures with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL of S9 cofactor pool) for the S9 fraction activated exposure, to which was added 50 µL of test substance dosing solution or vehicle alone. After the treatment period in the non-activated and the S9-activated studies, the treatment medium was aspirated, the cells washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium and returned to the incubator under standard conditions. Two hours prior to cell harvest, Colcemid® was added to all cultures at a final concentration of 0.1 µg/mL. Cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analyzed in the first division metaphase. The cells were trypsinized and counted and the cell viability was assessed using trypan blue dye exclusion.

To evaluate chromosome aberration s cells were collected by centrifugation, treated with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 3:1 v/v), capped and stored overnight or longer at 2 to 8C. To prepare slides, the cells were collected by centrifugation and the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with Giemsa and permanently mounted. To ensure that a sufficient number of metaphase cells are present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each treatment group selected for scoring chromosome aberrations. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads from each dose level (100 per duplicate culture) were examined and scored for chromatid-type and chromosome-type aberrations.

Evaluation criteria:

The frequency of cells with structural chromosome aberrations in the vehicle control must be within the historical control range. The percentage of cells with aberrations must be statistically increased (p < 0.05, Fisher's exact test) in the positive control relative to the vehicle control. The test substance was considered to have induced a positive response if it induced a statistically significant and dose dependent increase the frequency of aberrant metaphases (p< 0.05). If only Fisher's exact test was statistically significant without dose dependent increase, the result may be considered equivocal. If neither criterion was met, the results were considered to be negative.

Statistics:

Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the cytotoxicity screen substantial toxicity (> 50% cell growth inhibition, relative to the vehicle control) was observed at dose levels > 170 µg/mL in the non-activated 4 and 20-hour exposure groups, and at dose levels > 510 µg/mL in the S9-activated 4-hour exposure group.

Toxicity of Vinyl 2-ethylhexanoate (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the absence of S9 fraction activation was 54% at 65 µg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 65 µg/mL, was 9% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 25, 50, and 65 µg/mL. The percentage of cells with structural or numerical aberrations in the VEHA treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

Toxicity of Vinyl 2-ethylhexanoate (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the presence of S9 fraction metabolic activation was 54% at 200 µg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 200 µg/mL, was not reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 50, 100, and 200 µg/mL. The percentage of cells with structural or numerical aberrations in the VEHA treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

Toxicity of Vinyl 2-ethylhexanoate (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 20 hours in the absence of S9 fraction activation was 59% at 50 µg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 50 µg/mL, was only 1% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 10, 25, and 50 µg/mL. The percentage of cells with structural or numerical aberrations in the VEHA treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

Remarks on result:

other: strain/cell type: Chinese Hamster Ovary cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Tables presenting cytotoxicity, mitotic index and frequency of chromosme aberrations for all treatment conditions are attached below.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
other: Negative with and without S9 fraction metabolic activation up to cytotoxic doses.

The test substance, Vinyl-2-ethylhexanoate (VEHA) was negtive for the induction of chromosome aberrations in CHO cells in culture with and without a rat liver derived S9 fraction metabolic activation system. Therefore, VEHA is not genotoxic under the conditions of the study.

Executive summary:

The test substance, Vinyl-2-ethylhexanoate was evaluated for the potential to induce chromosome aberrations in an O.E.C.D. test guideline 473 study conducted in Chinese hamster ovary cells with GLP compliance. The test substance, Vinyl-2-ethylhexanoate (VEHA) was negtive for the induction of chromosome aberrations in CHO cells in culture with and without a rat liver derived S9 fraction metabolic activation system when tested up to cytotoxic concentrations. Therefore, VEHA is not genotoxic under the conditions of the study.