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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

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:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Amides, C8-18 (even numbered) and C18-unsatd., N-(hydroxyethyl)
EC Number:
931-330-1
Cas Number:
69227-24-3
Molecular formula:
The alkyl chain length of the amide ranges between 8 and 18 carbon atoms
IUPAC Name:
Amides, C8-18 (even numbered) and C18-unsatd., N-(hydroxyethyl)
Constituent 2
Chemical structure
Reference substance name:
2,2'-iminodiethanol
EC Number:
203-868-0
EC Name:
2,2'-iminodiethanol
Cas Number:
111-42-2
Molecular formula:
C4H11NO2
IUPAC Name:
2,2’-iminodiethanol
Test material form:
solid: flakes

Method

Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/mL respectively) and 30 U/mL heparin.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared after inducing metabolizing enzymes by injection of rats with phenobarbitone and β-naphthoflavone.
Test concentrations with justification for top dose:
Dose range finding study:
- At 3 h exposure time: 3, 10, 33, 100 and 333 µg/mL culture medium with and without S9-mix.
- At 24 and 48 h continuous exposure time: 3, 10, 33, 100, 333 and 1000 µg/mL culture medium without S9-mix

Experiment 1 (First cytogenetic assay):
Without and with S9-mix: 33, 100 and 200 µg/mL culture medium (3 h exposure time, 24 h fixation time)

Experiment 2 (Second cytogenetic assay):
- Without S9-mix: 10, 50, 100, 150, 175, 200, 225, 250, 275 and 300 µg/mL culture medium (24 h exposure time, 24 h fixation time)
10, 50, 75, 100, 125, 150, 175 and 200 µg/mL culture medium (48 h exposure time, 48 h fixation time)
- With S9-mix: 50, 100 and 200 µg/mL culture medium (3 h exposure time, 48 h fixation time)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material was soluble in DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation: 10 µg/mL 3 h exposure period (24 h fixation time)
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation: 0.1 µg/mL (48 h exposure), 0.2 (24 h exposure) and 0.5 µg/mL (3 h exposure)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 3h (Experiment 2 with S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 48 h (Experiment 2 with S9 mix)

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.5 µg/mL medium)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: 1000 cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
Evaluation criteria:
Evaluation criteria
A test substance was considered clastogenic if:
a) A dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations
b) A significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations in the absence of a clear dose-response relationship

A test substance was considered non-clastogenic if:
a) None of the tested concentrations induced a statistically significant increase in the number of cells with chromosome aberrations.
Statistics:
Statistics
One sided, Chi-square test to calculate dose-related statistically significant increase in the number of cells with chromosome aberrations

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: Human
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:
RANGE-FINDING/SCREENING STUDIES:
At the 24 and 48 h exposure time, test material was tested beyond the limit of solubility to obtain adequate toxicity data. - Precipitate of the test material was seen at 333 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA:
Yes, test data were within the laboratory historical control data range

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Increased number of polyploid cells in the absence of S9-mix in a dose dependent manner in the first cytogenetic assay indicating potential to inhibit mitotic processes and to induce numerical chromosome aberrations.

Any other information on results incl. tables

The doses selected for scoring of chromosome aberrations:

Without S9-mix: 50, 100 and 150 µg/mL culture medium (24 h exposure time, 24 h fixation time).

50, 100 and 125 µg/mL culture medium (48 h exposure time, 48 h fixation time)

With S9-mix:50, 100 and 200 µg/mL culture medium (3 h exposure time, 48 h fixation time)

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro.
Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C8-18 and C18-unsatd. MEA, according to OECD Guideline 473 and EU Method B. 10, in compliance with GLP. Peripheral human lymphocytes were treated with the test substance (experiment 1: 33, 100 and 200 µg/mL without and with S9-mix; experiment 2: 10–300 µg/mL without S9-mix, 50, 100 and 200 µg/mL with S9-mix) for either 3, 24 or 48 h. The frequency of cells with aberrations in the vehicle control group was within the historical control data range. Both of the positive control substances induced significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any significant or biologically relevant increases in the frequency of cells with chromosome aberrations in the presence or absence of metabolic activation, in either independent repeat experiment. No effects on the number of polyploid cells were observed both in the absence and presence of S9-mix. The substance did not disturb the mitotic processes, cell cycle progression and did not induce numerical chromosome aberrations. Under the study conditions, the test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro (Verspeek-Rip, 2009).

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