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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
July 22, 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name of test substance: Laurylacrylate 1214
Batch identification: 110014P040
Purity/composition: 93.9 corr. area-% as a sum of two main compounds
Homogeneity: The homogeneity of the test substance was guaranteed by mixing before preparation of the test substance preparations.
Storage stability: The stability of the test substance under storage conditions throughout the study period was guaranteed
Date of production: 18 Aug 2011
Molecular weight: 250 g/mol
Physical state, appearance: Liquid, colorless, clear
Storage conditions: Room temperature

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Cell line and storage
The V79 cell line is a permanent cell line derived from the Chinese hamster and has a
− high proliferation rate (doubling time of about 12 - 14 hours),
− high plating efficiency (≥ 90%),
− stable karyotype (modal number of 22 chromosomes).

Stocks of the V79 cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for

− mycoplasma contamination,
− karyotype stability,
− plating efficiency (=colony forming ability) incl. vital staining.


Culture media
MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with
− 10% (v/v) fetal calf serum (FCS)
− 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
− 1% (v/v) amphotericine B (250 μg/mL)
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (Phenobarbital/beta-naphthoflavone iduced rat liver of Wistar rats)
Test concentrations with justification for top dose:
1st experiment:
with and without S9 mix (4 hours exposure)
1.25 μg/mL, 2.50 μg/mL, 5.00 μg/mL, 10.00 μg/mL, 20.00 μg/mL, 40.00 μg/mL, 80.00 μg/mL

2nd experiment:
without S9 mix (24 hours exposure)
0.39 μg/mL , 0.78 μg/mL , 1.56 μg/mL , 3.13 μg/mL , 6.25 μg/mL, 12.50 μg/mL
with S9 mix (4 hours exposure)
6.25 μg/mL,12.50 μg/mL, 25.00 μg/mL, 50.00 μg/mL, 100.00 μg/mL

3rd experiment:
without S9 mix (24 hours exposure)
3.13 μg/mL, 6.25 μg/mL, 12.50 μg/mL, 25.00 μg/mL, 50.00 μg/mL, 100.00 μg/mL
with S9 mix (4 hours exposure)
12.50 μg/mL, 25.00 μg/mL, 50.00 μg/mL, 100.00 μg/mL, 200.00 μg/mL, 400.00 μg/mL
Vehicle / solvent:
- Ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
Without metabolic activation: 500 and 600 μg/mL ethyl methanesulfonate, With metabolic activation: 2.5 μg/mL cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Three independent experiments were performed with different expsoure times and test concentrations.
In Experiment I the exposure period was 4 hours with and without metabolic activation.
In Experiment II the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation.
In Experiment III the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation

The cells were prepared 24 hours after start of treatment with the test item.

DURATION
- Exposure duration: 4 or 24 h

STAIN (for cytogenetic assays): Wrights solution (modified May-Grünwald solution)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 1 000 cells per culture, means at least 2 000 cells per dose group

DETERMINATION OF CYTOTOXICITY
For additional information about the cytotoxic potential of the test substance cells were seeded in flasks (2.5 x 10E5 cells per 25 cm2 flask) about 24 – 30 hours prior to exposure. The cells were treated similar to the slides using the same media and test substance concentrations. At the end of recovery period single cell suspensions were prepared from each test group (all test groups, except the positive controls) and the cells were counted using a cell counter. Based on these data the relative increase in cell count (RICC) was calculated.

OTHER EXAMINATIONS:
pH value
Changes in the pH were recorded by a change in the color of the indicator in the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two top doses and for the vehicle control with and without S9 mix.
Osmolarity
Osmolarity was measured, at least for the top dose and for the vehicle control with and without S9 mix.
Solubility
Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically/ microscopically).
Evaluation criteria:
Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells.
• The number of cells containing micronuclei in the vehicle control was within the range of the historical negative control data
• The positive control substances both with and without S9 mix induced a significant increase in the number of micronucleated cells

Assessment criteria
A test substance is considered "positive" if the following criteria are met:
• A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
• The number of micronucleated cells exceeds both the value of the concurrent vehicle control and the range of the historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
• The number of micronucleated cells in the dose groups is not significant increased above the concurrent vehicle control value and is within the range of the historical negative control data
Statistics:
The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced
- Effects of osmolality: not influenced
- Water solubility:
- Precipitation: not observed up to the highest applied test substance concentration

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC), decrease in proliferation index (PI) or non-scorable slides was observed at least at the highest applied test substance concentrations in the 1st and 3rd Experiment.

OTHER:
The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells.
Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions chosen here, the conclusion is drawn that the test substance has not the potential to induce micronuclei (clastogenic and / or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
Executive summary:

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Three independent experiments were carried out with and without the addition of liver S9 mix from induced rats (exogeneous metabolic activation).

Based on the observations and the toxicity data of a previously performed pretest for a HPRT study the following concentrations were tested. The test groups printed in bold type were evaluated.

1st Experiment

4 hours exposure; 24 hours harvest time; without S9 mix

0; 1.25; 2.50; 5.00; 10.00; 20.00; 40.00; 80.00 µg/mL

4 hours exposure; 24 hours harvest time; with S9 mix

0; 1.25; 2.50; 5.00; 10.00; 20.00; 40.00; 80.00 µg/mL

2nd Experiment

24 hours exposure; 24 hours harvest time; without S9 mix

0; 0.39; 0.78; 1.56; 3.13; 6.25; 12.50 µg/mL

4 hours exposure; 24 hours harvest time; with S9 mix

0; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL

3rd Experiment

24 hours exposure; 24 hours harvest time; without S9 mix

0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL

4 hours exposure; 24 hours harvest time; with S9 mix

0; 12.50; 25.00; 50.00; 100.00; 200.00; 400.00 µg/mL

A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group.

The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.

Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC), decrease in proliferation index (PI) or non-scorable slides was observed at least at the highest applied test substance concentrations in the 1st and 3rd Experiment.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.