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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Mutagenic activity of 2-ethylhexanoic acid (the source substance of the read across approach) was investigated in one bacterial reverse mutation assay (Ames test; test strains used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A), in an in vitro gene mutation study in mammalian cells (Chinese Hamster Ovary cells; CHO) and in one in vitro chromosome aberration study in rat lymphocytes. Negative results were obtained in all tests with and without metabolic activation. For read across justification please refer to the attached document, IUCLID Chapter 13.
Link to relevant study records
Reference
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
Study period:
2007-08-30 till 2007-09-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no rescrictions, fully adequate for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO (Chinese hamster ovary) cell line (substrain K3) (1, 2) is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.
Stocks of the CHO cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254
Test concentrations with justification for top dose:
1 - 10 - 50 - 100 - 250 - 500 - 750 - 1000 - 1500 μg/mL
Vehicle / solvent:
Due to the limited solubility of the test substance in water, dimethylsulfoxide (DMSO) was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT test and for which historical data are available.
The final concentration of the vehicle DMSO in the culture medium was 1% (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
without metabolic activation: positive control substance; ethylmethanesulphonate. With metabolic activation: positive control substance: methylcholanthrene.
Details on test system and experimental conditions:
Day 1: Seeding of the cells pretreated with "HAT" medium: in 175 cm² flasks (1x106 cells in 20 mL) and in 25 cm² flasks (200 cells in 5 mL)
Day 2: Test substance incubation (20 – 24 hours after seeding); exposure period (4-hour and 24-hour); washing of the cultures (4-hour exposure);
1st cytotoxicity determination (cloning efficiency 1: survival)
Day 3: Washing of the cultures (24-hour exposure)
Day 5: 1st passage of the treated cells
Day 9: 2nd passage of the treated cells with seeding in the selection medium ("TG" medium); 2nd cytotoxicity determination (cloning efficiency 2: viability)
From day 16: Drying, fixation, staining and counting of the selected colonies
In this study all incubations were performed at 37°C with a relative humidity of > 90% in a 5% (v/v) CO2 atmosphere.
Evaluation criteria:
• The absolute cloning efficiencies of the negative controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative controls should fall within our historical negative control data range of 0 – 15 mutants per 106 clonable cells.
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies.
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.
Statistics:
no
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix; 10 μg/mL) induced clearly increased mutant frequencies as expected. The values of the corrected mutant frequencies (MFcorr.: 91.24 – 553.18 per 106 cells) were clearly within our historical positive control data range (without S9 mix 4 hours treatment: MFcorr.: 48.83 –587.77 per 106 cells; without S9 mix 24 hours treatment: MFcorr.: 272.94 – 331.63 per 106 cells; with S9 mix: MFcorr.: 29.06 – 413.54 per 106 cells.

The osmolarity was not influenced by test substance treatment. The pH of the stock solutions were adjusted to physiological values using small amounts of 2 N NaOH.
In this study, in the absence and the presence of S9 mix no precipitation of the test substance in culture medium was observed up to the highest applied test substance concentration.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions described, 2-ethylhexanoic acid does not induce forward mutations in vitro in the CHO/HPRT forward mutation assay in the absence and the presence of metabolic activation.
Based on a read-across approach, sodium 2-ethylhexanoate is not considered to be a mutagenic in the HPRT test
Executive summary:

A read across was performed from the source substance 2 -ethylhexanoic acid to the target substance sodium 2 -ethylhexanoate (for read across justification please refer to attached document, IUCLID Chapter 13).

The substance 2-Ethylhexanoic Acid was tested for its ability to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of Aroclor-induced rat liver S9 mix (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following doses were tested and evaluated in the main experiments:

1st Experiment (4-hour exposure period)

- without S9 mix

0; 125; 250; 500; 1 000; 1 500 μg/mL

- with S9 mix

0; 125; 250; 500; 1 000; 1 500 μg/mL

2nd Experiment

- without S9 mix (24-hour exposure period)

0; 125; 250; 500; 1 000; 1 500 μg/mL

- with S9 mix (4-hour exposure period)

0; 500; 750; 1 000; 1 250; 1 500 μg/mL

After an attachment period of 20 – 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 7 – 9 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both of the positive control substances, i.e. EMS and MCA, led to the expected increase in the frequencies of forward mutations. Due to a pH shift observed in the pre-experimental phase the stock solutions were adjusted with small amounts of NaOH to physiological values. No precipitation of the test substance in culture medium occured. In none of the experiments in the absence and the presence of metabolic activation cytotoxicity indicated by reduced relative cloning efficiency of below 20% control was observed. On the basis from the results of the present study, the test substance did not cause any

biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions described, 2-Ethylhexanoic Acid does not induce forward mutations in vitro in the CHO/HPRT forward mutation assay in the absence and the presence of metabolic activation.

Based on a read-across approach, sodium 2 -ethylhexanoate is not considered to be mutagenic in the HPRT test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A read across was performed from the source substance 2 -ethylhexanoic acid to the target substance sodium 2 -ethylhexanoate (for read across justification please refer to attached document, IUCLID Chapter 13).

Valid in vitro studies concerning mutagenic properties are available for 2-ethylhexanoic acid.

2 -Ethylhexanoic acid showed negative results in the study for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The study was performed with the test strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A. Test concentrations up to the limit concentration of 5000 µg/plate were tested in the experiment. The test compound proved to be not mutagenic to the bacterial strains.

2 -Ethylhexanoic acid also yielded negative results in an in vitro gene mutation study in mammalian cells in concentrations up to 1500 µg/ml with and without metabolic activation.

 

2 -Ethylhexanoic acid was assessed for its potential to induce chromosome aberrations in rat lymphocytes in vitro. The test item did not induce chromosome aberrations. Therefore, 2- ethylhexanoic acid is considered to be non-mutagenic in this in vitro chromosome aberration test when tested up to 5000 µg/mL.

Justification for selection of genetic toxicity endpoint
This study is selected as key study representing the toxicological endpoint "Genetic toxicity" since it was performed using mammalian cells and examines the most sensitive genotoxic mechanism. The study was performed according to the current OECD Guideline 476 and und GLP.

Justification for classification or non-classification

In conclusion, 2 -ethylhexanoic acid is not mutagenic in the bacterial reverse mutation assay, the in vitro gene mutation study in Chinese hamster CHO cells and the in vitro chromosome aberration in the presence and absence of metabolic activation up to the tested concentrations. 

2 -Ethylhexanoic acid does not have to be not classified for mutagenicity since this substance did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation in concentrations up to 5000 µg/plate, in the in vitro gene mutation assay (up to 1500 µg/mL) or in the in vitro chromosome aberration study in concentrations up to 5000 µg/mL.

Based on a read-across approach, sodium 2-ethylhexanoate is not considered to be a eye irritant and is therefore not subjected for labelling and classification requirements according to regulatory requirements.