Fatty acids, vegetable-oil, Me esters, sulfurized

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 05 OCT 2017 to 03 SEP 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, vegetable-oil, Me esters, sulfurized

EC Number:

276-337-4

EC Name:

Fatty acids, vegetable-oil, Me esters, sulfurized

Cas Number:

72102-30-8

IUPAC Name:

Fatty Acids, vegetable-oil, Me-Esters, sulfurized

Test material form:

liquid

Details on test material:

- Density: 0.95 g/mL
- Batch no.: 71012767

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MKU Chemie GmbH batch 71012767
- Expiration date of the lot/batch: 31. Aug. 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature: (20 ± 5°C)
- Stability under test conditions: unkown
- Solubility and stability of the test substance in the solvent/vehicle: unkown
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: unkown

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
Trinova Biochem GmbH, Gießen
- method of preparation of S9 mix
The S9 mix preparation was performed according to Ames et al. (1975).
- concentration or volume of S9 mix and S9 in the final culture medium
The final concen-tration of the S9 fraction during treatment is 0.4

Test concentrations with justification for top dose:

According to OECD 476, the highest concentration should be 0.01 M or 2 mg/mL or 2 μL/mL (whichever is the lowest), unless limited by the solubility or toxicity of the test item. Relative survival values below 20 % are considered toxic. For cytotoxic test items the maximum con-centrations should result in 10 to 20 % relative suspension growth (RSG) or relative total growth (RTG) and the analysed concentrations should cover a range from maximum to little or no cytotoxicity. When solubility is a limiting factor, the maximum concentration analysed, if not limited by cytotoxicity, should produce visible turbidity or precipitates in the cultures at the end of the treatment.
According to the results of the pre-test, 6 concentrations were chosen for the main experi-ment and tested with and without metabolic activation.

Experiment I
Approach with metabolic activation
Nominal concentrations of
test solutions (μL/mL) 63 31 16 8 4 2 1
Resulting nominal concentra-
tions in experiment (μL/mL) 0.31 0.16 0.08 0.04 0.02 0.01 0.005
Approach without metabolic activation
Nominal concentrations of
test solutions (μL/mL) 63 31 16 8 4 2 1
Resulting nominal concentra-
tions in experiment (μL/mL) 0.31 0.16 0.08 0.04 0.02 0.01 0.005

Experiment II Approach without metabolic activation
Nominal concentrations of
test solutions (μL/mL) 8 4 2 1 0.5 0.25
Resulting nominal concentra-
tions in experiment (μL/mL) 0.04 0.02 0.01 0.005 0.0025 0.00125

Vehicle / solvent:

- Solvent(s) used: DMSO; ethanol; aqueous solvents (water or saline or culture medium)]

- Justification for choice of solvent:
A solubility test for the determination of a suitable solvent for the test item was performed in a non-GLP pre-test, the solubility of the test item was determined in cell culture medium (DMEM) and dimethyl sulfoxide (DMSO), ethanol absolute and acetone. The test item was sufficiently soluble in ethanol absolute and acetone at the required concentration. Since more historical data are available, ethanol absolute was used as solvent for the test item.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
The assay was performed in two independent experiments, using two parallel cultures each (duplicates).

METHOD OF TREATMENT/ EXPOSURE:
- Cells at seeding: 1*10^6

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
The cells were thawed 6 - 8 d prior treatment and cultivated in DMEM complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
- Exposure duration/duration of treatment:
In experiment I the test item was incubated for 4 hours. In experiment II the incubation time with the test item was 24 hours.

FOR GENE MUTATION:
- Expression time : 168 h
- Selection time: 7 d
- Selective agent used: 6-thioguanine, final concentration: 2 μg/mL, 7 days of cell exposure after a total expression time of at least 168 h.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: After a total expression time of at least 168 h, the cells were counted again and seeded into 10 cm culture dishes (5 * 10^5 ± 10^4 cells) for the evaluation of the mutagenicity in selection medium containing 6-TG (final concentration: 2 μg/mL) and into 6 cm culture dishes (500 ± 10 cells) for the evaluation of the viability in complete culture medium. Both plates were incubated for further 7 days. After this incubation time the cell colonies were stained with 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution and counted for the calculation of the cloning efficiency II and the mutation frequency.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method cloning efficiency: 500 cells were exposed to each concentration of the test item for 4 hours with and without metabolic activation (duplicate cultures per concentration and approach). Following treat-ment, the cells were washed twice with PBS Dulbecco (2.5 % HS). After an expression period of 7 d the cells were stained with methylene blue. Afterwards the colonies were counted and the absolute and the relative cloning efficiency values were calculated.

Rationale for test conditions:

Turbidity was observed at the test item concentrations 0.31 μL/mL, 0.16 μL/ml, 0.08 μL/mL, 0.04 μL/mL in experiment I (+S9 and -S9) and at the concentration 0.04 μL/mL in experiment II. For that reason, according to the criteria of the OECD 476, 0.04 μL/mL is the highest analysable test item concentration in experiment I and Exp II.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase com-pared with the concurrent negative control,
• the increase is concentration-related when evaluated with an appropriate trend test,
• any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly neg-ative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mamma-lian cells in this test system.
However, in a case by case evaluation this decision depends on the level of the correspond-ing solvent control data. If there is by chance a low spontaneous mutation rate within the laboratories historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study is also taken into consideration.
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, Fatty Acids, vegetable-oil, Me-Esters, sulfurized did not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation.
The recorded data in this study declare the test item Fatty Acids, vegetable-oil, Me-Esters, sulfurized as not mutagenic.