Fatty acids, C16-18 and C18-unsatd., Me esters, epoxidized, reaction products with ethylene glycol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals, July 22, 2010, Guideline No. 487 “In vitro Mammalian Cell Micronucleus Test".

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Soybean oil, epoxidized, reaction products with methanol

EC Number:

287-837-7

EC Name:

Soybean oil, epoxidized, reaction products with methanol

Cas Number:

85586-35-2

Molecular formula:

C H4 O . Unspecified

IUPAC Name:

Epoxidized Soybean Oil, reaction products with methanol

Details on test material:

- Test Item: Soybean oil, epoxidized, reaction products with methanol
- BASF Test Item No.: 12/0114-1
- Batch Number: CE61910022
- Purity: 100%
- Expiration Date: December 2013
- Physical state, appearance: Liquid, yellow
- Storage conditions: At room temperature
- tability in Solvent: Not indicated by the Sponsor

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Stocks of the V79 cells (obtained from LMP; Technical University Darmstadt, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank. Before freezing each batch is screened for mycoplasm contamination and checked for karyotype stability.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

See "any other information on materials and methods"

Vehicle / solvent:

The final concentration of DMSO in the culture medium was 0.5 % (v/v). The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Griseofulvin

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

DOSE-SELECTION:
With respect to the solubility of the test item, 4000.0 µg/mL of Soybean oil, epoxidized, reaction products with methanol was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 7.8 and 4000.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. No precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation in the presence of S9 mix, this preliminary test was designated Experiment IA. The experimental part without S9 mix was repeated with the same top concentration due to the low response of the positive control MMC and designated Experiment IB. Since no clear cytotoxicity and test item precipitation was observed in Experiment IA and IB, the same concentrations were applied in Experiment II.

EXPERIMENTAL PERFORMANCE:
- Culture Medium and Conditions: For seeding and treatment of the cell cultures the culture medium was MEM (minimal essential medium) containing Hank’s salts, glutamine, Hepes (25 mM) and 10 % (v/v) fetal bovine serum (FBS). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL). All cultures were incubated at 37°C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
- Seeding of the Cultures: Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37°C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes, which contained microscopic slides. The cells were seeded into Quadriperm dishes, which contained microscopic slides. Into each chamber 1.0 x 10^5 – 1.5 x 10^5 cells were seeded with regard to the preparation time.

TREATMENT:
- Exposure period 4 hours: The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL medium was added. Concurrent solvent and positive controls were performed. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. Then the cells were cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 20 hours.
- Exposure period 24 hours: The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item without S9 mix. The medium was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.

PREPARATION OF THE CULTURES:
For the micronucleus analysis, 24 hours after the start of the exposure, the cells were treated on the slides in the chambers of the quadriperm dishes with deionised water for 1 to 1.5 min at 37°C. Afterwards the cells were fixed twice with a solution containing 3 parts ethanol, 1 part acetic acid and 1.25 % (v/v) formaldehyde. After preparation the cells were stained with Giemsa and labelled with a computer-generated random code to prevent scorer bias.

ANALYSIS OF MICRONUCLEI AND CYTOTOXICITY:
Evaluation of the cultures was performed manually using NIKON microscopes with 40x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). Briefly the micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 1000 cells in two parallel cultures were scored for micronuclei, so that at least 2000 cells from clones with 2-8 cells were analysed per test group. The frequency of micronucleated cells was reported as % micronucleated cells. Cytotoxicity was assessed via counting the number of clones consisting of 1 cell (c1), 2 cells (c2), 3-4 cells (c4), and 5-8 cells (c8) among the cells that were scored for the presence of micronuclei. These clusters represented the cells that have divided 1, 2, or 3 times within the experiment. From these data, a proliferation index (PI) was calculated. Only those cultures were evaluated which showed a PI > 1.3, in order to guarantee for a sufficient cell proliferation during treatment and recovery.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY:
The micronucleus assay is considered acceptable if it meets the following criteria:
a) The number of micronucleated cells carrying one or more micronuclei found in the solvent controls falls within the range of the historical laboratory control data range.
b) The positive control substances produce a significant increase (at least two-fold the respective control value) in the number of micronucleated cells exceeding the historical laboratory solvent control data range.

EVALUATION OF RESULTS:
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical control data and
- either a statistically significant concentration-related increase in three test groups or a significant increase of micronucleated cells in at least one test group is observed.
A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated test groups is in the range of the historical control data and
- no statistically significant concentration-related increase in the number of micronucleated cells is observed.

Statistics:

Statistical significance was confirmed by means of the Chi square test

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

No precipitation of the test item in the culture medium was observed. Phase separation was observed in Experiment IA, IB and II in the presence of S9 mix at 125.0 µL/mL and above. In Experiment II in the absence of S9 mix phase separation was observed at 250.0 µg/mL and above. No relevant influence on pH value was observed. The osmolarity was slightly increased at the highest applied concentration in Experiment II. In the absence and presence of metabolic activation no clear cytotoxicity measured as reduced proliferation index was observed. No mutagenicity was observed at the concentrations evaluated. In Experiment IA and IB in the absence and presence of S9 mix two statistically significant increases were observed after treatment with 1000 µg/mL (1.20 and 1.30 %). The values are in the range of the laboratory historical control data (0.15-1.50 % and 0.05-1.70 % micronucleated cells, respectively) and therefore biologically irrelevant. In Experiment II in the presence of S9 mix two statistically significant increases were observed after treatment with 62.5 and 2000.0 µg/mL (1.55 and 1.45 %). The values are in the range of the laboratory historical control data (0.05 - 1.70 % micronucleated cells) and therefore biologically irrelevant. In the presence of S9 mix one single value (1.60 %) exceeded the range of the laboratory historical control data (0.05-1.50 % micronucleated cells) after treatment with 1000.0 µg/mL. The value is not statistically significant and therefore biologically irrelevant. Mitomycin C (0.1 µg/mL), Griseofulvin (8.0 µg/mL) or CPA (15.0 µg/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.