2-butoxyethyl methacrylate

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:

19-05-2020 to 11-08-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

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:

2-butoxyethyl methacrylate

EC Number:

236-885-7

EC Name:

2-butoxyethyl methacrylate

Cas Number:

13532-94-0

Molecular formula:

C10H18O3

IUPAC Name:

2-butoxyethyl 2-methylprop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: F1-9K024C
- Expiration date of the lot/batch: 28 September 2020
- Purity test date: 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4oC in the dark

Method

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Suitability of cells: The Chinese hamster V79 cell line is recognized in the OECD 476 Test Guideline as being a suitable cell line for this test.

For cell lines:
- Absence of Mycoplasma contamination: Master stocks of cells were tested and found to be free of mycoplasma.
- Methods for maintenance in cell culture: grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS))
- Cell cycle length, doubling time or proliferation index : doubling time 12 - 16 h in stock cultures
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: yes, The cells were cleansed of mutants by culturing in HAT medium for four days


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: 37 °C with 5% CO2 in humidified air.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Moltox, Lot No. 4127, Expiry July 2021 and Lot No. 4222, Expiry 12 March 2022

- method of preparation of S9 mix : mixing S9 with a 0.1 M phosphate buffer containing NADP (5 mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% S9 concentration

- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9 when dosed at a 10% volume was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): tested and quality control certificate was provided

Test concentrations with justification for top dose:

4-hour without S9: 58.13, 116.25, 232.5, 465, 581.25, 697.5, 813.75 μg/mL
4-hour with S9 (2%): 58.13, 116.25, 232.5, 465, 813.75 μg/mL

The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by test item precipitate, as recommended by the OECD 476 guideline.

Preliminary Cytotoxicity Test
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. The preliminary cytotoxicity test was performed on cell cultures plated out at 1 x 107 cells/225 cm2 flask on the day before dosing. This was demonstrated to provide at least 20 x 106 cells available for dosing in each flask using a parallel flask, counted at the time of dosing. On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium (MEM). One flask per concentration was treated for 4-hours without metabolic activation and for 4-hours with metabolic activation (2% S9). The concentration range of test item used was 7.27 to 1860 μg/mL. Exposure was for 4 hours at approximately 37 °C with a humidified atmosphere of 5% CO2 in air, after which the cultures were washed twice with phosphate buffered saline (PBS) before being detached from the flasks using trypsin. Cells from each flask were suspended in MEM with 10% FBS, a sample was removed from each concentration group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks in 5 mL of MEM with 10% FBS and incubated for 6 days at approximately 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give relative survival (RS). A comparison of the test item to vehicle control relative survivals gave the relative toxicity of each test item dose level.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl Sulfoxide (DMSO)

Positive controls:

yes

Remarks:

Ethylmethanesulphonate (EMS) in Absence of S9-mix Dimethyl benzanthracene (DMBA) in Presence of S9-mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate
- Number of independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2 x 106 cells/225 cm2 flask 2 days before being exposed to the test or control items.
- Test substance added in serum free media

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 2 days
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment (sampling/recovery times): At the end of the exposure period the cultures were washed twice with PBS, detached from the flasks with trypsin and the cells suspended in MEM with 10% FBS. A sample of each concentration group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 106 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask to obtain the cloning efficiency, for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10% FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.


METHODS FOR MEASUREMENT OF CYTOTOXICITY and GENE MUTATION
Cytotoxicity flasks were incubated for 6 days then fixed with methanol and stained with Giemsa. Colonies were manually counted to give relative survival (RS). A comparison of the test item to vehicle control relative survivals gave the relative toxicity of each test item dose level. During the 7 Day expression period the cultures were sub-cultured on days 2 and 5 to maintain logarithmic growth. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 6 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 105 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 μg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 to 8 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.
The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes. The 4-hour exposure group in the absence of S9 was repeated three times due to test system failure as a result of the cells not growing satisfactorily. Only data from the final 4-hour exposure in the absence of S9 is reported.

Evaluation criteria:

The following criteria will be used to determine a valid assay:
i) The background (spontaneous) mutant frequency of the solvent controls is generally within the historical range. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays.
ii) The concurrent positive controls should induce responses that are comparable with those generated in the historical positive control range and produce a toxicologically significant increase compared with the concurrent negative control.
iii) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in a positive response.
iv) The criteria for selection of the maximum concentration have been met. The upper test item concentrations will be 10mM, 2 mg/mL or 2μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCBs) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL. Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point. In the absence of precipitate and if toxicity occurs, the highest concentration should lower the relative survival (RS) to approximately 10 to 20 % of survival.
v) Adequate numbers of cells and concentrations are analyzable. Mutant frequencies are normally derived from sets of ten petri dishes for the mutant colony counts and three flasks for cloning efficiency. To allow for contamination losses / technical errors it is acceptable to score a minimum of eight mutant selection dishes and two cloning efficiency flasks.
vi) A minimum of four analyzed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.

Statistics:

When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

At the end of the exposure period, precipitate of the test item was observed at 813.75 μg/mL and 930 μg/mL in both the absence and presence of metabolic activation. Therefore, the lowest precipitating dose level was plated for relative survival growth and expression of induced mutants and the subsequent dose level was discarded as it was considered surplus to requirements.
The Day 0 relative survival and Day 7 cloning efficiencies for the exposure groups in the absence and presence of metabolic activation are presented in Table 2 and Table 3. There were no marked concentration-related reductions in the relative survival values in the presence of metabolic activation. However, marked concentration-related reductions were observed in the absence of metabolic activation and near optimum levels of toxicity were achieved at the lowest precipitating dose level. There was no evidence of any reductions in the Day 7 cloning efficiencies in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred.
The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 2 and Table 3. The test item did not induce any statistically significant increases in the mutant frequency at any of the concentration levels in the main test using a dose range that included the lowest precipitating dose level, in either the absence or presence of metabolic activation.
The group vehicle control mutant frequency values were marginally lower than the current historical range for a vehicle but were all considered to be acceptable for inclusion within the historical control data base. The positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

Any other information on results incl. tables

See attached:

Table 1 Preliminary Cytotoxicity Test Results

Table 2 and 3 main experiment results

Appendix 1 Historical Background Data

Appendix 2 Quality Control & Production Certificates

Applicant's summary and conclusion

Conclusions:

2-Butoxyethil methacrylate did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test

Executive summary:

The potential mutagenicity of 2-Butoxyethil methacrylate, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line, was assessed during a GPL study compliant to the OECD TG 476.

Chinese hamster (V79) cells were treated with the test item at up to eight concentrations, in duplicate, together with vehicle (DMSO) and positive controls in both the absence and presence of metabolic activation (S9). The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by test item precipitate, as recommended by the OECD 476 guideline.

The vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.                2-Butoxyethil methacrylate did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was, therefore, considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test