methyl 2-hydroxybut-3-enoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-05-19 to 2017-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted with the purified but non-stabilised form of the test substance. Storage conditions were not specified.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008-05-30

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

methyl 2-hydroxybut-3-enoate

EC Number:

831-109-9

Cas Number:

5837-73-0

Molecular formula:

C5H8O3

IUPAC Name:

methyl 2-hydroxybut-3-enoate

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Mouse Lymphoma L5178Y cells (clone TK+/- -3.7.2C)
- Suitability of cells: Yes, recommended according to OECD 490 because they are heterozygous at the thymidine kinase (TK) locus, because of their high proliferation rate, good cloning efficiency and stable karyotype
- Normal cell cycle time (negative control): 10-12 h doubling time

For cell lines:
- Absence of Mycoplasma contamination: Yes. Each cell batch is routinely checked for mycoplasma infection.
- Methods for maintenance in cell culture: Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.
- Doubling time: 10-12 h
- Modal number of chromosomes: 40 +/- 2 chromosomes
- Periodically ‘cleansed’ of spontaneous mutants: Yes


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
Complete culture medium (CCM): RPMI 1640 supplemented with 10% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES and 2.5 µg/mL amphotericin B
Treatment medium: CCM, but with 5% horse serum instead of 10%
Selective medium: CCM, but with 20% horse serum instead of 10% and 5 µg/mL TFT
Temperature: 37°C
Humidity: 95%
CO2 concentration: 5%

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) induced rats
- Method of preparation of S9 mix: The cofactor solution was prepared by mixing 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate and 5 mM NADP in ice-cold 100 mM sodium phosphate buffer pH 7.4. An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL
- Concentration or volume of S9 mix and S9 in the final culture medium: Volumes were chosen to achieve a final protein concentration of 0.75 mg/mL in the cultures (approx. 2% S9 in the final culture medium)
- Quality controls of S9: Biological activity was tested in the Salmonella typhimurium assay using 2-aminoanthracene, in the mouse lymphoma assay using benzo[a]pyrene and in the chromosome aberration assay using cyclophosphamide. Sterility was also tested (not specified).

Test concentrations with justification for top dose:

Pre-experiment: 0.2, 0.5, 2.5, 5.0, 7.5, 10 mM (with and without S9 mix)
Main experiment (with S9 mix): 0.10, 0.15, 0.30, 0.35, 0.40 and 0.45 mM
Main experiment (without S9 mix): 0.10, 0.2, 0.3, 0.4, 0.5 and 0.6 mM

Vehicle / solvent:

- Vehicle used: Treatment medium (test item, EMS), 0.9% NaCl (MMS), DMSO (Benzo[a]pyrene)

- Justification for choice of solvent/vehicle: The vehcile was chosen based on the results of the solubility test.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Same as untreated negative control as treatment medium was used as solvent (test item, EMS). For MMS, 0.9% NaCl was used as solvent control and for Benzo[a]pyrene, DMSO was used

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Remarks:

Without S9:
EMS: 300 µg/mL
MMS: 10 µg/mL
With S9:
BEP: 1.5 µg/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Single
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1 x 10^7 cells in 11 mL medium were exposed with the test item. During the expression period, the cell density was determined each day and adjusted to 3 x 10^5 cells/mL. After the expression period the cloning efficiency (CE) was determined by seeding 1.6 cells/well. For selection, 2000 cells/well were seeded in 200 μL selective medium.
- Test substance added in: Medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours (with and without metabolic activation)
- Harvest time after the end of treatment: After 4 hours of treatment, cells were incubated for a expression and growth period of 2 days. After the expression period, cells were incubated for at least 6 days for CE determination. Additionally, cells were seeded in selective medium and incubated for approx. 12 days


FOR GENE MUTATION:
- Expression time: 2 days
- Selection time: Approx. 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): Approx. 20 days
- Method used: Microwell method
- Selective agent used Trifluorothymidine (5 µg/mL, cells were treated for 12 days)
- Criteria for small (slow growing) and large (fast growing) colonies: Approx. ≤ ¼ of well diameter for small colonies and approx. > ¼ of well diameter for large colonies

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative total growth (RTG)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT.

Rationale for test conditions:

The test concentrations were selected based on a preliminary test for toxicity.

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10^6 cells and
- a dose-dependent increase in mutant frequency is detected

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations. According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result. A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH-value detected with the test item was within the physiological range.
- Data on osmolality: The osmolality was within the physiological range.
- Water solubility: Yes.
- Precipitation and time of the determination: No precipitation of the test item was noted in the pre-experiment and main experiment at the time of evaluation.

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 10 mM. Six concentrations [0.2, 0.5, 2.5, 5.0, 7.5, 10 mM] were tested without and with metabolic activation. After a 2-day growth period the relative suspension growth (RSG) of the treated cell cultures was calculated according to the method of Clive and Spector. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. 0.6 mM (without metabolic activation) and 0.45 mM (with metabolic activation) were selected as the highest concentrations.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: Yes.
- Statistical analysis; p-value: Statistical significant increase in mutant frequency compared to negative controls (Mann Whitney test , p<0.05) at all concentrations without metabolic activation and at 0.30 mM and above with metabolic activation.
- Any other criteria: The mutant frequencies obtained from all experiments were compared with the Global Evaluation Factor (GEF). For the microwell method the GEF was defined to be 126. The GEF was exceeded at 0.5 mM and above without metabolic activation and at 0.35 mM and above with metabolic activation.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: The relative total growth (RTG) was 13.1% (without metabolic activation) and 12.3% (with metabolic activation) for the highest concentration evaluated.
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: See "Attached background material"

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: See "Details on test system and experimental conditions"
o Number of cells plated in selective and non-selective medium: See "Details on test system and experimental conditions"
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: See "Attached background material"
o Colony sizing for the negative and positive controls and test chemical, related mutant frequency and GEF evaluation: Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. An extension of the GEF by the induced mutant frequency in combination with an increased occurrence of small colonies is an indication for potential clastogenic effects and/or chromosomal aberrations. The positive controls MMS and B[a]P induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥40%), thus proving the ability of the test system to indicate potential clastogenic effects. In the main experiment without and with metabolic activation the percentage of small colonies in the negative controls was found to be lower than 40%. Due to the increased number of small colonies and corresponding mutagenicity in the two highest dose groups (without metabolic activation) and in the highest dose group (with metabolic activation), these concentrations of the test items were considered as clastogenic.

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Applicant's summary and conclusion

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item is considered to be mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

The test item was assessed for its potential to induce mutations according to OECD guideline 490 and GLP at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The selection of the concentrations used in the main experiment was based on data from the pre- experiment. The test item was investigated at the following concentrations:

 

 

Without metabolic activation: 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mM

 

and with metabolic activation: 0.10, 0.15, 0.30, 0.35, 0.40 and 0.45 mM

 

No precipitation of the test item was noted in the experiment. Growth inhibition was observed in the main experiment without and with metabolic activation: The relative total growth (RTG) was 13.1% (without metabolic activation) and 12.3% (with metabolic activation) for the highest concentration evaluated.

Biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was exceeded by the induced mutant frequency at concentrations of 0.5 mM and higher (without metabolic activation) and at concentrations of 0.35 mM and higher (with metabolic activation). Moreover, a dose-response relationship was observed. Additionally, colony sizing showed clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation). EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

 

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item is considered to be mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.