Dodecanoic acid, 2,2-dimethyl-3-oxopropyl 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

February 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

19 May 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

468-880-2

EC Name:

-

Cas Number:

102985-93-3

Molecular formula:

C17H32O3

IUPAC Name:

2,2-dimethyl-3-oxopropyl dodecanoate

Test material form:

liquid

Method

Target gene:

No target gene.

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully for many years in in vitro experiments. Especially the high proliferation rate (doubling time of clone V79/D3 in stock cultures: 12 hrs, determined on January 13, 2003) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

without S9-mix:
4 hrs exposure period: 6.3, 12.5, 25.0, 50.0, 100.0 and 200.0 µg/mL
18 hrs exposure period: 1.6, 3.1, 6.3, 12.5, 25.0 and 50.0 µg/mL
28 hrs exposure period: 6.3, 12.5, 25.0 and 50.0 µg/mL

with S9-mix:
4 hrs (I) exposure period: 12.5, 25.0, 50.0, 100.0, 200.0 and 400.0 µg/mL
4 hrs (II) exposure period: 6.3, 12.5, 25.0, 50.0, 100.0 and 200.0 µg/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:

ethylmethanesulphonate

Remarks:

Without 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:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; with and without metabolic activation (rat liver S9-mix)

DURATION
- Exposure duration: 4, 18, 28 hrs
- Expression time (cells in growth medium): 5 x 10E05 cells per flask

SELECTION AGENT (mutation assays): no

NUMBER OF REPLICATIONS: two per dose

NUMBER OF CELLS EVALUATED: 100 metaphase plates

DETERMINATION OF CYTOTOXICITY
- Method: precipitation:

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps) and/or
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps). and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher´s exact test (9) (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications.The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

NA

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 23.4 and 3000 µg/mL were applied. In the absence of S9 mix, clear toxic effects were observed after 4 hrs treatment with 46.9 µg/mL (44 % of control) and above and after 24 hrs continuous treatment with 23.4 µg/mL (26 % of control) and above. In contrary, in the presence of S9 mix no clear toxic effects were observed after 4 hrs treatment up to the highest applied test item concentration .

In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 187.5 µg/mL and above in the absence and in the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 350 mOsm, pH 7.4 versus 357 mOsm and pH 7.2 at 3000 µg/mL).

In experiment I, in the presence of S9 mix, precipitation of the test item in culture medium was observed after 4 hrs treatment with 400 µg/mL. In experiment II at preparation interval 28 hrs precipitation of the test item in culture medium was observed after 28 hrs continuous treatment with 25 µg/mL and above in the absence of S9 mix and after 4 hrs treatment with 200 µg/mL in the presence of S9 mix.

In this study, in the absence of S9 mix toxic effects indicated by clearly reduced cell numbers or mitotic indices were observed in all experimental parts. In detail, strongly reduced cell numbers were observed in experiment II after 18 hrs continuous treatment with 25 µg/mL (48 % of control) . In all additional experimental parts, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage .      In contrary, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied test item concentration being in the range of test item precipitation . In both cytogenetic experiments, in the absence and the presence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed . The aberration rates of the cells after treatment with the test item (0.0 - 4.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.5 - 2.5 % aberrant cells, exclusive gaps) and within the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).                                  

In both experiments,EMS (200 and 400 µg/mL, respectively) and CPA (1.0 and 1.4 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations, being in the range of the historical control data.

In conclusion, it can be stated that under the experimental conditions reported, the test item 2,2-Dimethyl-3-lauroyloxy-propanal did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to cytotoxic concentrations (without metabolic activation) and to clearly precipitating concentrations (with metabolic activation).

Applicant's summary and conclusion

Conclusions:

The test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, 2,2-Dimethyl-3-lauroyloxy-propanal is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations (without metabolic activation) or to precipitating concentrations (with metabolic activation).

Executive summary:

The test item 2,2-Dimethyl-3-lauroyloxy-propanal, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamsterin vitroin the absence and the presence of metabolic activation by S9 mix.

Two independent experiments were performed. In experiment I, the exposure period was 4 hrs with and without metabolic activation. In experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (exp. I and II) and 28 hrs (exp. II) after start of treatment with the test item.

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the positive control in experiment I, without metabolic activation, where only 50 metaphase plates were scored and for the test item concentrations 50 and 100 µg/mL in experiment II, with metabolic activation, where 200 metaphase plates were scored.

In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 23.4 and 3000 µg/mL were applied. In the absence of S9 mix, clear toxic effects were observed after 4 hrs treatment with 46.9 µg/mL (44 % of control) and above and after 24 hrs continuous treatment with 23.4 µg/mL (26 % of control) and above. In contrary, in the presence of S9 mix no clear toxic effects were observed after 4 hrs treatment up to the highest applied test item concentration.

In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 187.5 µg/mL and above in the absence and in the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 350 mOsm, pH 7.4 versus 357 mOsm and pH 7.2 at 3000 µg/mL).

In experiment I, in the presence of S9 mix, precipitation of the test item in culture medium was observed after 4 hrs treatment with 400 µg/mL. In experiment II at preparation interval 28 hrs precipitation of the test item in culture medium was observed after 28 hrs continuous treatment with 25 µg/mL and above in the absence of S9 mix and after 4 hrs treatment with 200 µg/mL in the presence of S9 mix.

In this study, in the absence of S9 mix toxic effects indicated by clearly reduced cell numbers or mitotic indices were observed in all experimental parts. In detail, strongly reduced cell numbers were observed in experiment II after 18 hrs continuous treatment with 25 µg/mL (48 % of control). In all additional experimental parts, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In contrary, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied test item concentration being in the range of test item precipitation.

In both cytogenetic experiments, in the absence and the presence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 4.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.5 - 2.5 % aberrant cells, exclusive gaps) and within the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).                                  

In both experiments, EMS (200 and 400 µg/mL, respectively) and CPA (1.0 and 1.4 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations, being in the range of the historical control data.

In conclusion, it can be stated that under the experimental conditions reported, the test item 2,2-Dimethyl-3-lauroyloxy-propanal did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to cytotoxic concentrations (without metabolic activation) and to clearly precipitating concentrations (with metabolic activation).