3,3'-[methylenebis(oxymethylene)]bisheptane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-[methylenebis(oxymethylene)]bisheptane

EC Number:

244-815-1

EC Name:

3,3'-[methylenebis(oxymethylene)]bisheptane

Cas Number:

22174-70-5

Molecular formula:

C17H36O2

IUPAC Name:

3,3'-[methylenebis(oxymethylene)]diheptane

Test material form:

other: liquid

Details on test material:

Test substance name: 2-ETHYLHEXYLAL
CETA sample number: 100/12
IUPAC name: 3,3'-[methylenebis(oxymethylene)]bisheptane
Molecular formula: C17H36O2
Molecular weight: 272.40
CAS No.: 22174-70-5
EEC number: 244-815-1
Batch No.: 1204051500
Purity: 99.766 % (w/w)
Impurities: 2-Ethylhexanol (0.0826 %), Formaldehyde (not found), Water (0.0370 %)
Storage: Store in supplied bottle in dark at the temperature under 25°C. Provide local exhaust or general room ventilation. Keep container closed when not in use. Keep away from heat.
Safety precautions: Do not breathe gas, fumes, vapour and spray. Use personal protective equipment (goggles, gloves).
Stability/Expiration: 3 years / 05.04.2015
The form of the test substance: colourless liquid with a characteristic odor

Method

Target gene:

Thymidine kinase (TK) gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were routinely maintained in R10 medium (RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 200 μg/ml pyruvate sodium and 10% horse serum; Gibco) in culture dishes (Nunc, #156340) in CO2 incubator (temp. 37°C, 5% CO2).
The cells were exposed in RPMI 1640 medium with 5% (R5) or 10% (R10) horse serum (for 3 or 24 hrs, respectively), while medium with 20% serum (R20) was used in the assay. Selective medium, i.e. R20 medium supplemented with 5-trifluorothymidine (TFT; 4 μg/ml, Sigma-Aldrich) was used for the mutant Tk-/- detection.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial S9 fraction (Cappel/MP Biomedicals, LOT #7663K) supplemented with cofactors was used as metabolic activation system (S9-mix).

Test concentrations with justification for top dose:

2-ETHYLHEXYLAL dissolved in culture medium at the concentration of 5 mM was used for the preparation of concentration range used in the main tests.
The test concentrations prepared from the stock solution of 5 mM were as follows:
1; 0.5; 0.25; 0.125 mM for both, 3 hr-exposure (without and with metabolic activation system) and 24 hr-exposure (without metabolic activation system).
The cell exposure (~1x107 cells/sample) was performed in RPMI 1640 medium supplemented with 5% (R5) or 10% (R10) horse serum for 3 and 24 hrs, respectively.
No significant changes in pH and osmolarity were observed at stock solution of 2-EH (5 mM).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none
- Justification for choice of solvent/vehicle:
Solubility of 2-EH in different vehicles (DMSO, ethanol) and then in RPMI was determined.
Very low solubility of the substance was observed (with/without using a vortex or a sonication bath) i.e.:
- the substance was insoluble or very slightly soluble in DMSO at 100 mM
- the substance was soluble in ethanol at 100 mM, however it “precipitated” when the solution was diluted further in the culture medium.
- the substance was insoluble in RPMI (with different FBS concentrations: 0-10%) after vortexing or sonication.

In each case (also after adding the 2-EH solution in ethanol to culture medium) big drops of 2-EH were observed in the culture medium. It seemed that only the concentrations below 1 mM did not form big visible drops, but rather a lot of small droplets seen under the microscope.
According to OECD 476, § 17, “relatively insoluble substances should be tested up to or beyond their limit of solubility under culture conditions. Evidence of insolubility should be determined in the final treatment medium to which cells are exposed.”

Based on the above results and the OCED recommendation, the concentration of 1 mM was applied as the highest one for the studies of 2-EH. The concentration of 1 mM was included to show that the potential mutagenicity was tested also at the concentrations showing obvious droplets of insoluble compound.
The culture medium was used as the solvent since neither ethanol nor DMSO was used to prepare 2-EH stock solution. Since prolonged sonication for 15 min did not improve the solubility of 2-EH, the vortexing for only 1 min was applied in order to exclude any potential physical damage to the test compound or culture medium.

Controls

Untreated negative controls:

yes

Remarks:

Culture medium was used as the negative control for test samples as well as for MMS.

Negative solvent / vehicle controls:

yes

Remarks:

Solution of DMSO at the concentration 0.1% was used as the solvent control for B[a]P (the exposure for 3 hrs with metabolic activation).

True negative controls:

no

Positive controls:

yes

Remarks:

Methyl methanesulfonate (MMS; 10 μg/ml for the 3-hr treatment and 5 μg/ml for the 24-hr treatment; Sigma Aldrich, #M4016) and benzo[a]pyrene (B[a]P; 3 μg/ml; Sigma-Aldrich #48564) were used in the test without and with metabolic activation, respectively.

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

The L5178Y TK+/- cells growing in exponential phase were exposed to test compound for 3 hours without and with metabolic activation system (S9 fraction) or for 24 hours without metabolic activation system. The additional 24-hour exposure time was performed according to the ICH recommending that negative results obtained in the test after the short treatment should be confirmed in the test using longer (24-hour) treatment without metabolic activation system (S9 fraction) (Moore et al., 2007).
After the exposure, derivative cultures were taken in order to determine cytotoxicity and to allow for expression of the mutant phenotype.
Cytotoxicity was determined on the basis of the measurement of the relative cloning efficiency (survival) and relative total growth of cultures after the treatment period. Cells treated with test substance were maintained in culture medium for 48 hours to provide them with optimal condition for phenotypic expression.
Mutant frequency was determined by seeding a known amount of cells in medium containing the selective agent (TFT) to detect mutant cells, and in medium without selective agent to determine the cloning efficiency (viability).
After 11-13 days of incubation colonies were counted. The mutant frequency was calculated based on the number of mutant colonies in selective medium and the number of colonies in non-selective medium.

Toxicity Assay - selection of test concentrations for the main study
Before the main study, effect of the test compound on proliferation and viability of L5178Y TK+/- cells was assessed using the Trypan blue exclusion test.

The aim of the study was to exclude concentrations evoking extreme cytotoxicity (concentrations evoking >80% decrease of cell number in the exposed population in comparison to the control population) as well as to determine non-cytotoxic concentrations.
The exposure was performed keeping conditions of the test similar to the conditions in the main study, i.e. for 3 hr-exposure the cell density in R5 medium was 5x105 cells/ml and for 24 hr-exposure the cell density in R10 was 2x105 cells/ml.
L5178Y TK+/- cells were incubated with the test compound at the wide range of concentrations for 3 hrs (without and with metabolic activation) as well as for 24 hrs (without metabolic activation). Thereafter, the cell suspension was mixed by pipetting several times and 100 μl of the cell suspension was mixed with 100 μl of Trypan blue solution. After thorough mixing, the suspension was put onto Bürker’s chamber and the cells were counted. Cell density (amount of cells in 1 ml) was counted according to the following formula:

Cell density = Average amount of cells (average from 3 squares) * 2 (dilution of cell suspension in Trypan blue) * 10000 (adjusting to 1 ml)

Cell viability was calculated based on % of viable cells, i.e. cells not stained with Trypan blue per total amount of counted cells (viable + dead cells, i.e. stained with Trypan blue).

Gene Mutation Assay - exposure of L5178Y TK+/- cells to 2-ETHYLHEXYLAL
L5178Y TK+/- cells, cleansed of cells deficient in tk gene (TK-deficient cells), were exposed to the test compound in the selected range of concentrations for 3 hours (without and with metabolic activation) as well as for 24 hours (without metabolic activation).

After 3- or 24-hr exposure the cells were washed twice using medium R10, then the cell density was counted and cell survival was measured (a). After 2-day period of expression (b) the measurement of viability (c) and mutant frequency (d) were preformed.

a. Survival measurement
After the exposure, 10 ml of cell suspension with density of 2x105 cells/ml in R10 medium was prepared from every exposed and control culture. Following dilution to 8 cell/ml in medium supplemented with 20% horse serum (R20), every cell suspension was plated onto 2 96-well plates (Nunc# 167008) in the amount of 200 μl/well (1.6 cell/well). Cell growth was determined after 11-13 days of incubation (37oC, 5% CO2).

b. Expression
After the exposure, the part of the cells (derivative cultures; 10 ml of cell suspension with density of 2x105 cells/ml) were taken and incubated for 48 hrs (37oC, 5% CO2) in order to allow for expression of the mutant phenotype before selection.
To ensure optimal growth condition for the cells, following 24-hr incubation density of every culture was counted and adjusted to 2x105 cells/ml by diluting with R10 medium. At 48th hour of expression, cell density of every culture was counted again and the cells were designed for viability and mutant frequency measurements.

c. Viability measurement
Measurement of viability was performed similarly to survival measurement, but the cells were used after expression period, i.e. 48 hours after exposure.
Following 48 hours of expression, 50 ml of cell suspension of the density of 2x104 cells/ml was prepared in R20 from every culture. Part of cell suspension was diluted to density of 8 cells/ml in R20 and plated onto 2 96-well plates in the amount of 200 μl/well (1.6 cell/well).
Cell growth was determined after 11-13 days of incubation (37°C, 5% CO2). The remaining part of cell suspension (2x104 cells/ml) was designed for mutant frequency measurement.

d. Mutant frequency measurement
Following 48 hours of expression, cell suspension of the density of 2x104 cells/ml was prepared in R20, and then diluted to density of 1x104 cells/ml in R20 supplemented with selective agent - trifluorothymidine (TFT; final concentration: 4 μg/ml). Suspension was plated onto 4 96-well plates in the amount of 200 μl/well (2x103 cells/well) under light protected conditions. Cell growth was determined after 11-13 days of incubation with distinction between large colonies (colonies covering >1/2 of well area) and small colonies (colonies covering less than 1/4 of well area).

Evaluation criteria:

Please refer to "Any other information on M&M" below

Statistics:

A statistical analysis was performed according to the method developed by Robinson et al. (1989) for the United Kingdom Environmental Mutagen Society (please refer to

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

STATISTICAL ANALYSIS

A statistical analysis was performed according to the method developed by Robinson et al. (1989) for the United Kingdom Environmental Mutagen Society.

 

1. Based on the results obtained, calculated values of viability (Hv) and mutation (Hm) heterogeneity factors for all 2-EHconcentrations used were not 10.8-fold higher than the current values of the heterogeneity factors.

Therefore all concentrations were included in further analysis as meeting acceptance criteria.

 

2. No statistically significant differences in mutant frequency between the control and the 2 -EHtreatment concentrations were found (one-sided Dunnett's test at p<0.05).

 

3. Test for the dose-effect relationship (the linear trend for mutant frequency with dose) showed no evidence of an increase in mutant frequency with increase in dose (p<0.05).

 

4. Strong mutagenic effects induced by positive control substances used in the study confirmed reliability of the results obtained.

 

To sum up, no significant mutagenic effect of 2-EHwas observed in L5178Y Tk+/-cells after 3 hr exposure without and with metabolic activation system, as well as after 24 hr exposure without metabolic activation system.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Performed MLA test did not reveal any significant mutagenic effect of 2-ethylhexylal after 3 hr exposure without and with metabolic activation system, as well as after 24 hr exposure without metabolic activation system.
Obtained results showed that 2-ethylhexylal did not induce mutations of the kinase thymidine gene in L5178Y TK+/- mouse lymphoma cells under the experimental conditions used.

Executive summary:

2-ETHYLHEXYLAL (2-EH) was evaluated in the in vitro mouse lymphoma (L5178Y TK+/-) gene mutation assay (MLA). The genotoxic potential of the test substance was assessed in two independent assays, i.e. in the absence and presence of an externally supplied metabolic activation (S-9).

Influence of 2-EH on density and viability of L5178Y TK+/- cells was assessed with Trypan blue exclusion in the preliminary study. The cells were exposed to eight 2-EH concentrations ranging from 1 to 0.0078 mM, for 3 and 24 hrs. Following 3 hr-exposure (with or without metabolic activation system) no significant differences in density and viability were observed between control and test cultures. However, after exposure for 24 hours (without metabolic activation system) a weak decrease (~20%) in the number of exposed L5178Y TK+/- cells in comparison to control, with no effect on viability, was observed at the highest concentration of 2-EH tested (1 mM). Similar decrease was observed in cultures exposed for 3 hrs to the highest concentration of 2-EH with subsequent post-exposure incubation for additional 21 hr-incubation.

In the main study, L5178Y TK+/- cells were exposed to 2-EH at the concentration range of 1 – 0.125 mM for 3 hrs, with or without metabolic activation system, as well as for 24 hrs without metabolic activation system. As the negative control RPMI 1640 culture medium was used. As the positive controls methylmethanesulfonate (MMS; 10 μg/ml for the 3-hr treatment and 5 μg/ml for the 24-hr treatment) and benzo[a]pyrene (B[a]P; 3 μg/ml) were used in the tests without or with metabolic activation, respectively.

The measurements performed did not reveal any significant mutagenic effect of 2-EH after exposure of the cells neither for 3 hrs (with or without metabolic activation system), nor for 24 hrs without metabolic activation system.