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Toxicological information

Genetic toxicity: in vitro

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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:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Fatty acids, tall-oil, reaction products with diethylenetriamine, maleic anhydride, tetraethylenepentamine and triethylenetetramine
CAS No.: 68990-47-6
Batch no.: TEE3316/22
Physical state at RT: solid
Colour: dark
Purity: Date of analysis 11 February 2010
97% (w/w)
Storage Conditions: at room temperature, protected from light
Solubility in Water: very low
Safety Precautions: Routine hygienic procedures will be sufficient to assure personnel health and safety.

Method

Target gene:
V79 cells
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 (ATCC, CCL-93) cells are stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking for mycoplasma infections was carried out before freezing.
For the experiment, thawed cultures were set up in 75 cm2 cell culture plastic flasks at 37 oC in a 5% carbon dioxide atmosphere (95% air). 5 x 105 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FCS (foetal calf serum) and subcultures were made every 3 - 4 days.
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Experiment I:
with and without metabolic activation: 1000, 2500 and 5000 μg/mL
Experiment II:
with metabolic activation: 3000, 4000 and 5000 μg/mL
without metabolic activation: 250, 500 and 1000 μg/mL
Controls
Untreated negative controls:
yes
Remarks:
cell culture medium alone
Negative solvent / vehicle controls:
yes
Remarks:
cell culture medium alone
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate without S9 and Cyclophosphamide with S9

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1 mg/ml in experiment II without S9, a relevant decrease of the relative mitotic index (<70%)was noted
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The selection of the concentrations used in experiment I and II was based on data from the solubility test and the pre-experiment.

In experiment I with and without metabolic activation 5000 μg/mL was selected as highest dose groups for the microscopic analysis of chromosomal aberrations. In experiment II without metabolic activation 1000 μg/mL and with metabolic activation

5000 μg/mL were selected as highest dose groups for the microscopic analysis of chromosomal aberrations.

The following concentrations were evaluated for microscopic analysis:

Experiment I:

with and without metabolic activation:

1000, 2500 and 5000 μg/mL

Experiment II:

with metabolic activation: 3000, 4000 and 5000 μg/mL

without metabolic activation: 250, 500 and 1000 μg/mL

Precipitation:

The test item was suspended in cell culture medium. Precipitate of the test item was noted in all dose groups evaluated in experiment I and II.

Toxicity:

In experiment I with and without metabolic activation, no biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at any concentration. The cell density was not decreased.

In experiment II without metabolic activation, a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at 1000 μg/mL (47% at 1000 μg/mL. The cell density was not decreased. With metabolic activation, no biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at the concentrations evaluated. No decrease of the cell density was noted up to the highest dose evaluated.

Clastogenicity:

In experiment I without metabolic activation the aberration rate of the negative control (1.5%) and all the dose group tested with the test item (1.5% (1000 μg/mL), 0.5% (2500 μg/mL) and 2.5% (5000 μg/mL)) were within the historical control data of the

testing facility (0.0% – 4.0%. With metabolic activation, the aberration rates of the negative control (2.0%) and all dose groups treated with the test item 3.0% (1000 μg/mL), 2.0% (2500 μg/mL) and 2.0% (5000 μg/mL) were within the historical

control data of the testing facility (0.0% – 4.0%, Table 10). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. In addition, no doseresponse

relationship was observed.

In experiment II without metabolic activation the aberration rate of the negative control (2.0 %) and all dose groups treated with the test item (0.5 % (250, 500 and 1000 μg/mL)) were within the historical control data of the testing facility (0.0% –4.0%. With metabolic activation the aberration rates of the negative control (2.0%) and all dose groups treated with the test item (1.5% (3000 μg/mL), 2.5% (4000 μg/mL) and 3.5 (5000 μg/mL)) were within the historical control data of the

testing facility (0.0% – 4.0%. The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. In addition, no dose-response relationship was

observed.

Polyploid cells:

No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

EMS (400 and 600 μg/mL) and CPA (0.83 μg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item Fatty acids, tall-oil, reaction products with diethylenetriamine, maleic anhydride, tetraethylenepentamine and triethylenetetramine is considered to be non-clastogenic.