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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: other: clastogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-14 to 2012-02-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
Phosphoric acid, dodecyl ester, potassium salt
EC Number:
254-414-3
EC Name:
Phosphoric acid, dodecyl ester, potassium salt
Cas Number:
39322-78-6
IUPAC Name:
potassium dodecyl hydrogen phosphate
Test material form:
semi-solid (amorphous): gel
Remarks:
migrated information: paste

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment:
with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL

Experiment I:
without metabolic activation: 15.6, 250, 500 and 1000 µg/mL
with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL

Experiment II:
without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL
with metabolic activation: 900, 1600 and 1800 µg/mL
Vehicle / solvent:
-Vehicle (s)/solvent(s) used: cell culture medium (MEM)
-Justification for choice of solvent/vehicle: The test item was prepared in cell culture medium followed by ultrasound for around 5 minutes prior to treatment. After that the test item was well suspended. The solvent was compatible with the survival of the cells and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation Migrated to IUCLID6: 400 and 900 µg/mL
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 0.83 µg/mL
Details on test system and experimental conditions:
TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10^4 - 5 x 10^4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture)
except for 15.6 µg/mL (experiment I with metabolic activation) and 31.3 µg/mL (experiment II without metabolic activation): 300 cells
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell density
Evaluation criteria:
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (with and without metabolic activation)).


Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

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
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Results of chromosome analysis
without metabolic activation
     Cytotoxicity Chromatid aberrations         Isochromatid aberrations       rel. Mitotic index (%) rel. Cell density (%)  Poly-ploidy mean % aberrant cells
Scored cells  gaps breaks  inter-changes  other  gaps breaks  inter-changes  other incl. Gaps excl. Gaps
Experiment I                              
negative control 200 - 5 4 0 0 0 0 1 1 100 100 2 4.5 2.5
15.6 µg/mL 200 no 5 0 0 0 0 0 0 0 103 96 3 2.5 0.0
250 µg/mL 200 no 6 2 0 1 0 0 0 0 81 78 3 3.5 1.5
500 µg/mL 200 yes 4 3 2 0 0 0 2 0 53 55 1 5.0 3.0
1000 µg/mL 200 yes 4 1 0 0 1 0 0 0 51 44 0 3.0 0.5
EMS (900 µg/mL) 200 - 7 10 8 2 0 1 2 0 85 94 3 12.5 9.5
Experiment II                                  
negative control 200 - 1 4 0 0 0 0 0 0 100 100 2 2.5 2.0
15.6 µg/mL 200 no 6 3 0 1 0 0 0 0 76 102 3 4.5 2.0
31.3 µg/mL 300 no 5 7 1 1 0 0 0 0 74 94 1 4.7 3.0
62.5 µg/mL 200 yes 2 2 0 0 0 0 1 0 51 89 1 2.5 1.5
125.0 µg/mL 200 yes 1 2 0 0 0 0 1 0 30 57 1 2.0 1.5
EMS (400 µg/mL) 200 - 7 17 3 3 3 0 0 0 72 103 0 13.0 10.5
Results of chromosome analysis
with metabolic activation
     Cytotoxicity Chromatid aberrations         Isochromatid aberrations       rel. Mitotic index (%) rel. Cell density (%)  Poly-ploidy mean % aberrant cells
Scored cells  gaps breaks  inter-changes  other  gaps breaks  inter-changes  other incl. Gaps excl. Gaps
Experiment I                              
negative control 200 - 6 2 0 0 0 0 0 0 100 100 0 4.0 1.0
15.6 µg/mL 300 no 4 3 3 1 0 0 2 0 96 102 1 4.0 3.0
500 µg/mL 200 no 2 0 2 0 0 0 0 0 90 86 0 2.0 1.0
1000 µg/mL 200 yes 3 0 0 0 0 0 0 0 62 85 1 1.5 0.0
1500 µg/mL 200 yes 2 1 0 0 1 0 0 0 54 63 1 2.0 0.5
CPA (0.83 µg/mL) 200 - 3 12 11 1 0 1 1 0 104 85 3 12.0 10.5
Experiment II                                  
negative control 200 - 4 5 0 1 1 0 1 0 100 100 2 5.5 3.5
900 µg/mL 200 no 1 3 0 0 0 0 0 0 71 88 0 2.0 1.5
1600 µg/mL 200 yes 3 3 0 0 0 0 0 0 54 75 0 2.5 1.5
1800 µg/mL 200 yes 0 2 0 0 0 0 0 1 45 56 0 1.5 1.5
CPA (0.83 µg/mL) 200 - 3 9 3 3 1 1 2 1 96 100 0 10.0 9.0

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item Afilan V5756 did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item Afilan V5756 is considered to be non-clastogenic in this chromosome aberration test.
Executive summary:

To investigate the potential of Afilan V5756 to induce structural chromosome aberrations in Chinese hamster V79 cells, anin vitrochromosome aberration assay was carried out.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation.

Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations, except for 15.6 µg/mL (experiment I with metabolic activation) and 31.3 µg/mL (experiment II without metabolic activation): 200 cells for the 1st culture and 100 cells for the 2nd culture.

The test item was prepared in cell culture medium followed by ultrasound for around 5 minutes prior to treatment. After that the test item was well suspended.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 15.6, 250, 500 and 1000 µg/mL

with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL

Experiment II:

without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL

with metabolic activation: 900, 1600 and 1800 µg/mL

Precipitation of the test item was observed with and without metabolic activation in both experiments.

Toxic effects of the test item were observed in experiment I without metabolic activation at concentrations of 500 µg/mL and higher, with metabolic activation at concentrations of 1000 µg/mL and higher. In experiment II without metabolic activation (long time exposure) toxic effects of the test item were observed at concentrations of 62.5 µg/mL and higher, with metabolic activation at concentrations of 1600 µg/mL and higher.

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

EMS (400 and 900 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.

The positive controls induced the appropriate responses.

There was no evidence of Afilan V5756 induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data. 

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