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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: other: clastogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-28 to 2012-06-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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
Chemical structure
Reference substance name:
Amines, C16-22-alkyl
EC Number:
268-215-4
EC Name:
Amines, C16-22-alkyl
Cas Number:
68037-92-3
Molecular formula:
Not applicable
IUPAC Name:
docosan-1-amine; hexadecan-1-amine; icosan-1-amine; octadecan-1-amine
Details on test material:
Name: C16-22-(even numbered)alkylamines
CAS No.: 68037-92-3
Chemical Name: C16-22-(even numbered)alkylamines
Batch No.: S001453
Physical State at RT: solid
Colour: white to yellow
pH: 11.5 at 5% in ethanol:water (1:1) DIN EN 1262
Molecular Weight: approx. 293 g/mol
Active Components: see CoA from 2011-07-12
Date of Analysis: 12 July 2011
Storage Conditions: put nitrogen gas into bottle before closing it to prevent test item from oxidizing
Expiry Date: at least to 20180421

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: 0.0125, 0.025 and 0.050 mM
with metabolic activation: 0.031, 0.063 and 0.125 mM

Experiment II:
without metabolic activation: 0.010, 0.025, 0.03 and 0.04 mM
with metabolic activation: 0.025, 0.05 and 0.10 mM

Vehicle / solvent:
-Vehicle (s)/solvent(s) used: cell culture medium (MEM)
-Justification for choice of solvent/vehicle: The test item could be suspended at a concentration of 5 mg/mL (17.06 mM) followed by ultrasound for 5 - 15 minutes. 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 at 0.04 mM (experiment II without metabolic activation): 53 cells for the 1st and 29 for the 2nd culture.
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
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.

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 5 0 1 1 0 0 0 100 100 1 5.5 3.0
0.0125 mM 200 no 2 4 0 2 0 1 0 0 98 123 0 3.0 2.0
0.025 mM 200 no 2 3 0 0 0 0 0 0 78 89 0 2.5 1.5
0.050 mM 200 yes 1 2 1 0 0 0 0 0 48 99 3 2.0 1.5
EMS 900 µg/mL 200 - 2 12 4 1 0 1 0 0 78 97 0 9.0 9.0
Experiment II                                  
negative control 200 - 0 2 1 0 0 1 1 0 100 100 3 2.0 2.0
0.010 mM 200 no 2 1 0 0 0 0 0 0 100 98 3 1.5 0.5
0.025 mM 200 yes 3 5 0 1 0 0 0 0 55 98 3 3.0 1.5
0.03 mM 200 yes 2 3 1 1 0 0 0 0 38 103 0 3.0 2.0
0.04 mM 82 yes 2 1 0 0 1 0 1 0 36 84 0 6.1 2.4
EMS 400 µg/mL 200 - 5 15 4 0 1 0 1 0 90 88 0 12.0 9.5
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 - 3 2 0 0 0 0 0 0 100 100 1 2.5 1.0
0.031 mM 200 no 2 1 0 0 0 0 0 0 108 97 0 1.5 0.5
0.063 mM 200 no 1 1 0 0 0 0 0 0 87 98 0 1.0 0.5
0.125 mM 200 yes 2 0 1 0 1 0 0 1 49 103 2 2.5 1.0
CPA 0.83 µg/mL 200 - 5 9 4 2 1 0 1 0 87 102 1 11.0 8.0
Experiment II                                  
negative control 200 - 3 1 0 0 0 0 0 0 100 100 2 2.0 0.5
0.025 mM 200 no 1 1 0 0 0 0 1 0 91 91 3 1.5 1.0
0.05 mM 200 no 1 1 0 0 0 0 1 0 79 95 1 1.5 1.0
0.10 mM 200 yes 2 0 0 0 0 0 0 0 37 51 2 1.0 0.0
CPA 0.83 µg/mL 200 - 5 17 7 1 1 0 0 0 98 91 2 13.5 11.5

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 C16-22-(even numbered)alkylamines did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item C16-22-(even numbered)alkylamines is considered to be non-clastogenic in this chromosome aberration test.
Executive summary:

To investigate the potential of C16-22-(even numbered)alkylamines 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 at 0.04 mM (experiment II without metabolic activation): 53 cells for the 1st and 29 for the 2nd culture.

The test item was suspended cell culture medium (MEM).

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

Experiment I:

without metabolic activation: 0.0125, 0.025 and 0.050 mM

with metabolic activation: 0.031, 0.063 and 0.125 mM

Experiment II:

without metabolic activation: 0.010, 0.025, 0.03 and 0.04 mM

with metabolic activation: 0.025, 0.05 and 0.10 mM

In both experiments without metabolic activation no precipitation of the test item was observed at the concentrations evaluated. With metabolic activation precipitation of the test item was noted at concentrations of 0.063 mM and higher in experiment I and at a concentration of 0.10 mM in experiment II. Precipitation was assessed after incubation with the test item by unaided eye.

In experiment I without metabolic activation, toxic effects of the test item were noted at concentrations of 0.050 mM, with metabolic activation at concentrations of 0.125 mM.

In experiment II without metabolic activation, toxic effects of the test item were observed at concentrations of 0.025 mM and higher. With metabolic activation, toxic effects of the test item were noted at concentrations of 0.10 mM.

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 test item 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.