Oligo

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:

28 Jul - 16 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted in 2016

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerirsches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids C16-18 (even numbered), mono, di and triesters with sucrose, UVCB

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

Fatty acids C16-18 (even numbered), mono, di and triesters with sucrose, UVCB

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature protected from light
- Solubility and stability of the test substance in the solvent/vehicle: 5 mg/mL in 0.25% THF

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution of the test item was made with 0.25% THF. This stock solution was then diluted in RPMI and 5% HS.
- Final preparation of a solid: The test solution was then soniticated for 10 minutes at 37 degree C.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Solution

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins Munich cell bank
- Cell cycle length, doubling time or proliferation index: 10-12 hr doubling time
- Whether whole blood or separated lymphocytes were used if applicable: lymphoma cells
- Methods for maintenance in cell culture if applicable: Thawed stock cultures are maintained in RPMI 1640 complete medium.
- Modal number of chromosomes: 40

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

10, 20, 50, 100, 200, and 300 µg/mL.
Top dose based on results of toxicity pre-experiment which found precipitation at concentrations of 400 µg/mL and above.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: THF was chosen due to the solubility of the test item in that solvent as compared to other solvents.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension of 11 mL RPMI medium and 5% horse serum
- Cell density at seeding (if applicable): 1 x 10E+07

DURATION
- Preincubation period: 1-3 days
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days (8 days for cloning efficiency experiment)

SELECTION AGENT (mutation assays): RPMI with 20% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B, 5 µg/mL TFT

NUMBER OF REPLICATIONS: 4

NUMBER OF CELLS EVALUATED: all

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The test substance is considered mutagenic if the following criteria were met:

induced mutant frequency of >= 126 mutants per 10E06 cells
dose-dependent increase in mutant frequency
increased occurrence of small colonies >= 40% of all colonies

Statistics:

Mean mutant frequency
Mean induced mutant frequency
p-value

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, at highest dose level of 300 µg/mL

RANGE-FINDING/SCREENING STUDIES: A range-finding study was performed at concentrations of 10, 30, 100, 200, 400, and 1000 µg/mL with and without metabolic activation. Precipitation was seen at the 400 and 1000 µg/mL concentrations both with and without metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Ethylmethanesulfonate (300 µg/mL): range - 318.7-2919.0, mean - 726.5, standard deviation - 203.5
Methylmethanesulfonate (10 µg/mL): range - 376.4-2416.1, mean - 763.4, standard deviation - 421.6
Benzo[a]pyrene (2.5 µg/mL): range - 303.6-1267.2, mean - 635.9, standard deviation - 167.8
- Negative (solvent/vehicle) historical control data:
Negative without S9: range - 50.1-170.3, mean - 87.9, standard deviation - 25.5
Negative with S9: range - 50.1-165.9, mean 85.1, standard deviation - 24.3

Any other information on results incl. tables

Mouse Lymphoma Assay Results – Without Metabolic Activation

Concentration	Relative Cloning Efficiency (%)	Relative Total Growth (%)	Mutant Frequency (mutants/10E+06 cells)	Induced Mutant Frequency (mutants/10E+06 cells)
Control 1	102.9	106.4	69.5	--
Control 2	98.0	100.3	69.5	--
Solvent Control	100.0	100.0	64.9	--
10 µg/mL	106.5	109.8	71.8	6.9
25 µg/mL	85.1	86.7	64.6	-0.3
50 µg/mL	102.9	97.3	86.5	21.6
100 µg/mL	87.7	81.2	73.9	9.0
200 µg/mL	104.7	86.6	69.0	4.2
300 µg/mL (Precipitate observed)	93.4	84.1	61.6	-3.3
Ethylmethanesulfonate*	91.9	73.0	649.0	584.2
Methylmethanesulfonate*	62.8	46.6	631.5	566.7

* Global Evaluation Factor exceeded and statistically significant increase in mutant frequency seen

Mouse Lymphoma Assay Results – With Metabolic Activation

Concentration	Relative Cloning Efficiency (%)	Relative Total Growth (%)	Mutant Frequency (mutants/10E+06 cells)	Induced Mutant Frequency (mutants/10E+06 cells)
Control 1	94.4	89.4	63.7	--
Control 2	107.0	105.6	63.7	--
Solvent Control	100.0	100.0	80.2	--
10 µg/mL	89.0	96.6	91.8	11.6
25 µg/mL	112.3	119.5	71.2	-8.9
50 µg/mL	91.6	84.8	68.4	-11.8
100 µg/mL	100.4	95.5	49.6	-30.6
200 µg/mL	98.9	96.2	72.7	-7.4
300 µg/mL (Precipitate observed)	114.2	102.7	60.4	-19.8
Benzo[a]pyrene*	90.3	64.4	702.5	622.4

* Global Evaluation Factor exceeded and statistically significant increase in mutant frequency seen

Applicant's summary and conclusion

Conclusions:

The test substance is non-mutagenic.

Executive summary:

The mutagenicity of the test substance was determined in an OECD Guideline 490 study. The mutagenicity of the test substance was determined at concentrations of 10, 20, 50, 100, 200, and 300 µg/mL both with and without metabolic activation. Tetrahydrofuran was used as solvent. Negative, solvent, and positive controls were used. Precipitation was seen at the highest test concentration of 300 µg/mL. The test substance was not cytotoxic, nor was it mutagenic.