Bis(2-ethylhexyl) carbonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

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: Comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Evaluation of di(2-ethylhexyl) phthalate and its major metabolites in the Ames test and L5178Y mouse lymphoma mutagenicity assay.

Author:

Kirby PE et al

Year:

1983

Bibliographic source:

Environ. Mutagen. 5, 657-663

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexan-1-ol

EC Number:

203-234-3

EC Name:

2-ethylhexan-1-ol

Cas Number:

104-76-7

Molecular formula:

C8H18O

IUPAC Name:

2-ethylhexan-1-ol

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 2-Ethylhexanol

Method

Target gene:

Thymidine Kinase (TK) locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fisher's medium for leukemic cells
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 activation mixture (FoP, nicotinamide adenine dinucleotide diphosphate, isotric acid, and Aroclor 1242/ 1254-induced liver microsomes prepared from Spaague-Dawley rats)

Test concentrations with justification for top dose:

with and without metabolic activation 10 concentrations from 0.013 to µL/mL to 0.24 µl/ml were tested, concentration selection was based on initial toxicity tests in which the substance demonstrated complete toxicity at concentrations >/= 1.0 µL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethansulfonate, Dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: incubated for 4 hours at 37 °C
- Expression time (cells in growth medium): 48 hours at 37°C
- Selection time (if incubation with a selection agent): 10-12 days in presence of Trifluorothymidine at 37°C

SELECTION AGENT (mutation assays): TFT (Trifluorothymidine)

NUMBER OF CELLS EVALUATED: 600 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Mutation frequency is expressed as the number of TK-/- mutant per 10exp4 surviving cells.
Criteria for postive result: significant increase of mutation frequency, at least a doubling of the background mutation frequency

Statistics:

no details reported

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

complete toxicity at concentrations>/= 1.0 µL/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

None of the cultures treated with 2-Ethylhexanol, at any dose level, exhibited mutation frequencies that were significantly greater (twofold greater than background) than that of the corresponding ethanol solvent control. Total growth at highest concentration without and with metabolic activation was approximately 10% and 40 %, respectively.
The positive control chemicals, on the other hand, demonstrated significant increases in mutation frequencies for both S9 activated and nonactivated cultures. These results show that under the experimental conditions, 2-Ethylhexanol is not mutagenic in the L5178Y TK+/- mammalian mutagenicity assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

None of the cultures treated with 2 -Ethylhexanol at any dose-level, exhibited mutation frequencies that were significantly greater (two-fold greater than background) than that of the corresponding ethanol solvent control. Thus, 2-Ethylhexanol was not mutagenic in the L5178Y TK+/- mammalian mutagenicity assay.

Executive summary:

2-Ethylhexanol was tested in an in vitro gene mutation assay using mammalian cells cultures both in the absence and presence of metabolic activation (S9 mix), according to a protocol similar to the OECD Guideline 476.

Mouse lymphoma L5178Y cells (clone 3.7.2) cultured in vitro were exposed to 2-Ethylhexanol (99.97 %) at 10 concentrations from 0.013 to µL/mL to 0.24 µL/mL in ethanol. Total growth at highest concentration without and with metabolic activation was approximately 10% and 40 %, respectively. Concentration selection was based on an initial toxicity test in which the substance demonstrated complete toxicity at concentrations >/= 1.0 µL/mL. Appropriate positive controls were used. After a 48 rest period, cells were then incubated mutagenicity evaluation with trifluorothymidine during 10-12 days.

None of the cultures treated with 2 -Ethylhexanol at any dose-level, exhibited mutation frequencies that were significantly greater (two-fold greater than background) than that of the corresponding ethanol solvent control.

The positive control chemicals, on the other hand, demonstrated significant increases in mutation frequencies for both S9 activated and non-activated cultures.

Under these experimental conditions, 2 -Ethylhexanol was not mutagenic in the L5178Y TK+/- mammalian mutagenicity assay. This study is considered as acceptabe. This study although performed prior to implementation of the corresponding guideline satisfies to a great extent the requirements for an in vitro gene mutation study in mammalian cells and is considered as of high reliability and adequate to evaluate the mutagenicity of the test substance in this mamalian cell test system.