N-(2-ethoxyphenyl)-N'-(4-isododecylphenyl)oxamide

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:

weight of evidence

Study period:

21 July - 22 Aug 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform GLP study without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

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:

N-(2-ethoxyphenyl)-N'-(4-isododecylphenyl)oxamide

EC Number:

279-979-3

EC Name:

N-(2-ethoxyphenyl)-N'-(4-isododecylphenyl)oxamide

Cas Number:

82493-14-9

Molecular formula:

C28H40N2O3

IUPAC Name:

N-(2-ethoxyphenyl)-N'-[4-(10-methylundecyl)phenyl]ethanediamide

Method

Species / strain

Species / strain / cell type:

other: Human lymphocytes

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from a female donor (22 years old).
Blood samples were drawn by vernous puncture and collected in heparinized tubes.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 5.5, 9.6, 16.8, 29.4, 51.4, 89.9, 157.4, 275.4, 481.9, 843.4, 1475.9, 2582.9, 4520.0 µg/mL
Experiment II: 5.5, 9.6, 16.8, 29.4, 51.4, 89.9, 157.4, 275.4, 481.9, 843.4, 1475.9, 2582.9, 4520.0 µg/mL

Without metabolic activation:
Experiment I: 5.5, 9.6, 16.8, 29.4, 51.4, 89.9, 157.4, 275.4, 481.9, 843.4, 1475.9, 2582.9, 4520.0 µg/mL
Experiment II: 5.5, 9.6, 16.8, 29.4, 51.4, 89.9, 157.4, 275.4, 481.9, 843.4, 1475.9, 2582.9, 4520.0 µg/mL

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Ethylmethane sulfonate (EMS), Cyclophosphamide (CPA)

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in DMSO

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Results and discussion

Test results

Key result

Species / strain:

other: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item Hostavin 3206 (solvent free), dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments.

In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations.
The highest applied concentration in this study (4520.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the the molecular weight of the test item and with respect to the current OECD Guideline 473.
Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473.
In the absence and presence of S9 mix, no clear cytotoxicity was observed up to the highest applied concentration.
In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

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 with and without metabolic activation

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, test substance is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.

Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after the start of treatment with the test item.

In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.

The highest treatment concentration in this study, 4520.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.

In Experiment I, visible precipitation of the test item in the culture medium was observed at 9.6 µg/mL and above in the absence and presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II at 51.4 µg/mL and above in the absence of S9 mix and at 16.8 µg/mL and above in the presence of S9 mix. No relevant influence pH value was observed; the osmolarity was was slightly decreased at the highest applied concentration (Exp. I: solvent control: 466 mOsm, pH 7.4 versus 397 mOsm and pH 7.4 at 4520.0 µg/mL; Exp. II: solvent control: 454 mOsm, pH 7.5 versus 357 mOsm and pH 7.4 at 4520.0 µg/mL).

In this study, at both preparation intervals, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices could be observed. However, in Experiment II after continuous treatment in the absence of S9 mix, the mitotic index was decreased to 67.2 % of control at the highest evaluated concentration.

In both experiments, in the absence and presence of S9 mix, no 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 - 2.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

In both experiments, either(660 or 825 µg/mL) or CPA (7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.