2-butoxyethyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro bacterial reverse mutation assay (Ames Test) was performed with Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 and Escherichia coli strain WP2uvrA using both Ames plate incorporation and pre-incubation techniques. 2-Butoxyethyl methacrylate was considered to be non-mutagenic under the conditions of the test.

 

In accordance with Annex VIII of REACH, since the result of the in vitro gene mutation study was negative, the potential of 2-Butoxyethyl methacrylate to induce structural chromosomal aberration in vitro was determined in accordance with the OECD Guideline for Testing of Chemicals 487 under the principles of GLP. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three conditions were used for the study: a 4-hour exposure in the absence or presence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity although it coincided with the onset of toxicity. The 4- hour exposure group in the absence of S9 was repeated on a separate day due to excessive precipitate. The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the lowest precipitating dose level. Therefore, 2-butoxyethyl methacrylate was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

 

Moreover, 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

 

 

Link to relevant study records

Reference

Reference 1

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

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

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

Target gene:

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

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

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.

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.

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

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

 

 

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 August 2019 - 16 september 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: human donor
- Suitability of cells: peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Normal cell cycle time: Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours.



For lymphocytes:
- Sex, age and number of blood donors: Preliminary Toxicity Test: male, aged 33 years Main Experiment: male , aged 24 years Main Experiment repeat (4-hour without S9): female , aged 28 years
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: not addressed


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Purchased from Moltox and Lot no, 4061 expiry date 14 February 2021.
- method of preparation of S9 mix: The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).
- concentration or volume of S9 mix and S9 in the final culture medium :The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 was pre-tested for acceptability by the supplier prior to purchase and was supplied with a relevant Quality Control & Production Certificate

Test concentrations with justification for top dose:

4-hour exposure experiments (without S9-mix,): 0, 232.5, 465, 523.125, 631.25, 930, 1162.5 and 1860 μg/mL.μg/mL.
4-hour exposure experiments (without S9-mix,): 0, 58.13, 116.25, 232.5, 465, 631.25, 864 and 930 μg/mL.
24-hour exposure experiment: 0, 58.13, 116.25, 232.5, 465,631.25, 864 and 930 μg/mL.μg/mL. The maximum dose level was 1863 μg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level
The short exposure group without metabolic activation (S9) did not return enough non-precipitating dose levels in the previous experiment and, therefore, was repeated using the amended dose range 0, 29.07, 58.13, 116.25, 232.5, 465, 930 and 1860 μg/mL.Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture for the preliminary toxicity test, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the exposure groups of the main experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in aqueous media at 18.63 mg/mL but was miscible in DMSO at 186.3 mg/mL in solubility checks performed in-house.
- Justification for percentage of solvent in the final culture medium: There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate, vehicle (quadruplicate cultures)
- Number of independent experiments : Two independant experiments with one repeat of the main experiment

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in suspension

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
- Harvest time after the end of treatment (sampling/recovery times): At the end of the 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v).

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Cytochalasin B at a final concentration of 4.5 μg/mL, incubated for a further 24 hours following each exposure period.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry with gentle warming. Each slide was permanently labeled with the appropriate identification data.When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls.The micronucleus frequency in 1000 binucleated cells was analyzed per culture (2000 binucleated cells per concentration for the test item and positive control and 4000 binucleated cells for the vehicle controls) except for CP 5 μg/mL where an additional 1000 binucleate cells were assessed due to a poor response in the first 1000 cells.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: The selection of the maximum dose level for the Main Experiment was based mainly on precipitate but acknowledged the onset of toxicity and was 930 μg/mL for the 24-hour continuous exposure and 4-hour exposure group in the presence of S9 and was 1860 μg/mL for the 4-hour exposure group in the absence of S9.

Rationale for test conditions:

In accordance with the OECD 487 guideline.The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by precipitate even though it coincided with the onset of toxicity.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.The dose-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive controls). A linear regression model was then applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.

Key result

Species / strain:

lymphocytes: Peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: mainly on precipitate but acknowledged the onset of toxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No significant change in pH when the test item was dosed into media: For all the tested concentrations the pH was as follows 0 µg/ml = pH 7.46, 7.28µg/ml = pH7.41, 14.55µg/ml = pH7.41, 29.11µg/ml = pH 7.43, 58.22 µg/ml = pH 7.41,116.44 µg/ml = pH 7.43,232.88 µg/ml = pH 7.45,465.75 µg/ml = pH 7.45,931.5 µg/ml = pH 7.43,1863 µg/ml = pH 7.43.
- Data on osmolality: The osmolality did not increase by more than 50 mOsm. For all the tested concentrations the osmolality was as follows 0 µg/ml =476 Osm, 7.28µg/ml = 487Osm, 14.55µg/ml = Not performed, 29.11µg/ml = 490Osm, 58.22 µg/ml = Not performed,116.44 µg/ml = 498Osm,232.88 µg/ml = 482Osm,465.75 µg/ml = 481Osm,931.5 µg/ml = Not performed,1863 µg/ml =456Osm.
- Precipitation and time of the determination: A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 931.5 μg/mL in all three exposure groups. The selection of the maximum dose level for the Main Experiment was based mainly on precipitate. The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test but the precipitate appeared at dose levels lower than expected. A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 930 μg/mL in 4-hour exposure group in the absence of S9, at and above 631.25 μg/mL in the 4-hour exposure group in the presence of S9 and at and above 465 μg/mL in the 24-hour continuous exposure group.
- Definition of acceptable cells for analysis: Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.
- Other confounding effects: The short exposure group without metabolic activation (S9) did not return enough non-precipitating dose levels in the previous experiment and, therefore, was repeated using the amended dose range 0, 29.07, 58.13, 116.25, 232.5, 465, 930 and 1860 μg/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : See attached background documents

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method: CBPI or RI; distribution of mono-, bi- and multi-nucleated cells : see attached background material

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate : See attached background document

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: See attached background documents
- Negative (solvent/vehicle) historical control data: See attached background documents

Conclusions:

The test item, 2-butoxyethyl methacrylate (CAS 13532-94-0 / EC 236-885-7) was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

The potential of 2-Butoxyethyl methacrylate to induce structural chromosomal aberration in vitro was determined in accordance with the OECD Guideline for Testing of Chemicals 487 under the principles of GLP. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle (quadruplicate cultures) and positive controls (duplicate cultures). Three conditions were used for the study: a 4-hour exposure in the absence or presence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity although it coincided with the onset of toxicity. The 4- hour exposure group in the absence of S9 was repeated on a separate day due to excessive precipitate. The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the lowest precipitating dose level. Therefore, 2-butoxyethyl methacrylate was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 12, 2017 to January 12, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

Version / remarks:

24 November 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus in the genome of Salmonella typhimurium and tryptophan locus in the genome of Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Experiment 1 (Ames plate incorporation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (Pre-incubation method): 15, 50, 150, 500, 1500, 5000 µg/plate
- Salmonella typhimurium strain TA100 required a repeat second experiment analysis due to elevated untreated/ vehicle control counts and, as toxicity was noted, the dose range was amended:
TA100 (presence of S9-mix): 15, 50, 150, 500, 1500, 5000 µg/plate
TA100 (absence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Test Item Preparation and Analysis

The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer. No correction was required for purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

- Dose Selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

- Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

- With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

- Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method

As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

- Dose Selection
The dose range initially used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500, 5000 µg/plate. Six test item dose levels per bacterial strain were originally selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation. Salmonella strain TA100 required repeat second experiment analysis due to elevated untreated/ vehicle control counts and, as toxicity was noted in the second mutation test, the dose range for this particular tester strain was amended as follows:

TA100 (presence of S9-mix): 15, 50, 150, 500, 1500, 5000 µg/plate
TA100 (absence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate

- Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

- With Metabolic Activation
The procedure was the same as described previously (see 3.3.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

- Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A test item precipitate (greasy in appearance) was noted in both experiments at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in Experiment 1 (presence of S9-mix) at 50 µg/plate. However, this response was within the in-house historical vehicle/untreated control values for the bacterial strain and was, therefore, considered of no biological relevance. Subsequently, there were no toxicologically meaningful increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Remarks on result:

other: The toxic limit of the test item (1500 µg/plate) was employed in the absence of S9 for this strain only

Conclusions:

2-Butoxyethyl methacrylate was considered to be non-mutagenic under the conditions of this bacterial reverse mutation assay (Ames Test) in Salmonella typhimurium and Escherichia coli.

Executive summary:

An in vitro study was undertaken to determine the capacity of the 2-butoxyethyl methacrylate to induce reverse gene mutation at the histidine or tryptophan locus in five bacterial strains: Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2uvrA). The experiment was performed without deviation according to guidelines published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guideline 471 (Bacterial Reverse Mutation Test), Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

An initial experiment (Experiment 1) was performed using the Ames plate incorporation technique at pre-determined dose levels of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (n = 3) with and without metabolic activation, which consisted of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). A subsequent test (Experiment 2) was performed using the pre-incubation method based on the outcome of Experiment 1, with dose levels amended to 15, 50, 150, 500, 1500, 5000 µg/plate in order to achieve four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology. Strain TA100 required repeat analysis as part of Experiment 2 due to elevated vehicle control counts, and as toxicity was noted in the second mutation test, the dose range for this strain was amended to 15 - 5000 µg/plate (presence of S9-mix) and 1.5 to 1500 µg/plate (absence of S9-mix). The vehicle control was untreated and contained dimethyl sulphoxide. Five positive control substances were prepared in addition. 

According to the results of the vehicle, positive controls and negative controls, the sensitivity of the assay and the efficacy of the S9-mix were validated. No toxicologically meaningful increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 and 2. As 2-butoxyethyl methacrylate did not have the capacity to significantly induce gene mutation in bacteria under the conditions of this test, it can be regarded as non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)