Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline and GLP compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals No. 487, 22 Jul 2010, “In vitro Mammalian Cell Micronucleus Test”
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
liquid brownish

Method

Target gene:
not applicable
Species / strain
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
1st Experiment
4 hours exposure, 24 hours harvest time, without S9 mix
0, 31.3, 62.5, 125, 250, 500, 1000 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix
0, 31.3, 62.5, 125, 250, 500, 1000 μg/mL

2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0, 7.8, 15.6, 31.3, 62.5, 125, 250 μg/mL

4 hours exposure, 44 hours harvest time, with S9 mix
0, 15.6, 31.3, 62.5, 125, 250, 500 μg/mL

Vehicle:
culture medium
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and conditions:
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 24h
- Exposure duration: 4h or 24h
- Fixation time (start of exposure up to fixation or harvest of cells): See table below.
At the end of the exposure period, the medium was removed and the cultures were rinsed twice with 5 mL HBSS (Hanks Balanced Salt Solution). Subsequently, 5 mL MEM (incl. 10% [v/v] FCS) supplemented with Cytochalasin B (final concentration: 3 μg/mL; stock: 0.6 mg/mL in DMSO;
AppliChem, Cat.No. A7657) was added and incubated at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity for the respective recovery time.
In the case of 24-hour continuous exposure, CytB was added to the treatment medium at start of treatment, and cell preparation was started directly at the end of exposure. At 44 hours preparation interval in the presence of S9mix the supplementation of CytB was 24 hours before preparation of the cultures.

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): Before scoring, the slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. D9542) and propidium iodide (stock: 5 mg/mL; Sigma-Aldrich, Cat.No. P4170) in Fluoroshield™ (Sigma-Aldrich,
Cat.No. F6182) at a concentration of 0.25 μg/mL each. By the use of the combination of both fluorescence dyes it can be differentiated between DNA (DAPI; excitation: 350 nm, emission: 460 nm) and cytoplasm (PI; excitation: 488 nm, emission: 590 nm).

NUMBER OF CELLS EVALUATED: 2000

DETERMINATION OF CYTOTOXICITY
- Method: Proliferation index
The cytokinesis-block proliferation index (CBPI) is a direct measure of the proliferative activity of the cells and it was determined in at least 1000 cells per culture (at least 2000 cells per test group). This value indicates the average number of cell cycles per cell during the period of
exposure to the actin polymerisation inhibitor cytochalasin B.
The number of mononucleated, binucleated and multinucleated cells were recorded.

OTHER: MICRONUCLEUS ANALYSIS
The cytospin slides were scored by fluorescence microscopy (Axio Imager.Z2, Zeiss, 37081 Göttingen, Germany).
As a rule, at least 1000 cells per culture, means at least 2000 cells per test group, were evaluated and the number of micronuclei-containing binucleated cells was recorded.
The analysis of micronuclei was carried out following the criteria of Countryman and Heddle:
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only cells clearly surrounded by a nuclear membrane were scored.
Slides were coded before microscopic analysis by MUVIKE software, BASF SE. Cultures with few isolated cells were not analysed for micronuclei.
Since the absolute values shown were rounded but the calculations were made using the unedited values, there may be deviations in the given relative values.
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 evaluation of a sufficient number of analyzable cells
either in both control groups (vehicle/positive) or in at least three exposed test groups.
• Sufficient cell proliferation was demonstrated in the vehicle control.
• The number of cells containing micronuclei in the vehicle control was within the range of our
laboratory’s historical negative control data (95% control limit). Weak
outliers can be judged acceptable if there is no evidence that the test system is not “under
control”.
• The positive control substances both with and without S9 mix induced a distinct, statistically
significant increase in the number of micronucleated cells in the expected range.

Assessment Criteria
A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle
control and the range of our laboratory’s historical negative control data (95% control limit).
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the number of cells containing
micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent
vehicle control value and within the range of our laboratory’s historical negative control data
(95% control limit).
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.
If the results of this test were statistically significant compared with the respective vehicle
control, labels (* p ≤ 0.05, ** p ≤ 0.01) have been printed in the tables.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes

Any other information on results incl. tables

Experiment Exposure/Preparation interval Concentration S9 mix Precipitation Micronucleated cells* Proliferation index cytostasis (CBPI) cell count
          [%] [%] [%]
1 4/24 hrs negative control - n.d. 0.6 0.0 100.0
31.3 µg/mL - - 0.5 1.2 95.9
62.5 µg/mL - - 0.2 1.9 96.3
125 µg/mL - - 0.7 3.3 104.0
250 µg/mL - - n.s. n.s. 117.1
500 µg/mL - - n.p. n.p. -102.6
1000 µg/mL - - n.p. n.p. -159.5
    positive control - n.d. 2.1s 10.8 98.3
2 24/24 hrs negative control - n.d. 0.4 0.0 100.0
7.8 µg/mL - - n.d. n.d. 80.9
15.6 µg/mL - - n.d. n.d. 67.4
31.3 µg/mL - - n.d. n.d. 71.8
62.5 µg/mL - - 0.3 1.0 76.0
125 µg/mL - - 0.4 3.6 82.8
    250 µg/mL - n.d. 0.3 25.7 -0.7
positive control - n.d. 2.7s 19.4 92.3
1 4/24 hrs negative control + n.d. 0.5 0.0 100.0
31.3 µg/mL + - n.d. n.d. 98.6
62.5µg/mL + - 0.7 0.0 90.0
125 µg/mL + - 0.5 -1.1 84.6
250 µg/mL + - 0.6 4.2 106.5
500 µg/mL + - n.p. n.p. -143.9
1000 µg/mL + - n.p. n.p. -246.4
    positive control + n.d. 2.8s 21.6 89.4
2 4/44 hrs negative control + n.d. 0.4 0.0 100.0
15.6 µg/mL + - n.d. n.d. 122.1
31.3 µg/mL + - n.d. n.d. 128.7
62.5 µg/mL + - 0.4 -1.2 136.2
125 µg/mL + - 0.6 2.2 134.7
250 µg/mL + - 0.4 3.4 127.9
    500 µg/mL + - n.s. n.s. -258.0
     positive control  + n.d.  4.2s  -10.8  103.0
               
               
               
               

*Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

s Frequency statistically significant higher than corresponding control values

n.d. Not determined

n.s. Not scorable due to low cell quality / technical reasons

n.p. No cytospin slides prepared due to strong cytotoxicity

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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, Octadecanoic acid, sulfo-, potassium salt 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.