methyl acrylate; methyl propenoate

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:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

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 acrylate

EC Number:

202-500-6

EC Name:

Methyl acrylate

Cas Number:

96-33-3

Molecular formula:

C4H6O2

IUPAC Name:

methyl prop-2-enoate

Specific details on test material used for the study:

Name of test substance: Methylacrylate
Test substance No.: 04/0652-7
Batch identification: 25.09.2018 17:11:11
CAS No.: 96-33-3
Purity/composition: 99.96 % (calculated)
(see analytical data, study code 1000339252)
Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance solutions.
Storage stability: The stability of the test substance under storage conditions was guaranteed until 24 Sep 2019 as indicated by the manufacturer, and the manufacturer holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.

Method

Target gene:

heterozygous autosomal thymidine kinase (TK) locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

line of 3-methylcholanthrene induced tumors (thymus) in DBA/2 mice and has a
- high proliferation rate (doubling time of about 9 - 10 hours)
- high plating efficiency (about 90%)
- stable karyotype with a near diploid number of 40 ± 1 chromosomes.
Stocks of the mouse lymphoma L5178Y TK+/- cell line (1-mL portions) will be maintained at
- 196°C in the gas phase above the liquified nitrogen using 7% (v/v) DMSO in FCS as a
cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma
contamination previously.

Metabolic activation:

with and without

Metabolic activation system:

1mM Glutathione

Test concentrations with justification for top dose:

The dose selection was based on a previously performed pretest (99M0652/04M026; data not
shown), where it could be shown that Methylacrylate induces significant cytotoxicity from 25
μg/mL (25 μg/mL: 27% RTG) and above and the published data.

Dose levels:
Without 1mM Glutathione (GSH) 4-hour exposure:
Vehicle control DMSO
5.0 μg/mL
10.0 μg/mL
15.0 μg/mL
20.0 μg/mL
25.0 μg/mL
30.0 μg/mL
35.0 μg/mL
MMS 15.0 μg/mL

Dose levels:
With 1mM Glutathione (GSH) 4-hour exposure:
Vehicle control DMSO
5.0 μg/mL
10.0 μg/mL
15.0 μg/mL
20.0 μg/mL
25.0 μg/mL
30.0 μg/mL
35.0 μg/mL

Vehicle / solvent:

Due to the limited solubility of the test substance in ultrapure water, dimethyl sulfoxide (DMSO)
was selected as vehicle, which has been demonstrated to be suitable in the mouse lymphoma
assay and for which historical control data are available.
The final concentration of the vehicle DMSO in culture medium was 1% (v/v).

Controls

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide (DMSO)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

TEST SUBSTANCE PREPARATION:
The test substance was handled under light protection conditions.
The substance was dissolved in DMSO.
The test substance was weighed and topped up with the chosen vehicle to achieve the required
concentration of the stock solution.
To achieve a solution of the test substance in the vehicle, the test substance preparation was
shaken thoroughly.
The further concentrations were diluted according to the planned doses.
All test substance formulations were prepared immediately before administration.

EXPERIMENTAL PROCEDURE:
Controls:
Vehicle control:
The vehicle controls were run in parallel and contained the vehicle without the test substance
at the same volume and concentration as used in the test cultures.

Positive controls:
The following concentration of the positive control was used to demonstrate the sensitivity of
the test method:
15 μg/mL methyl methanesulfonate (MMS; MERCK-SCHUCHARDT, Cat.No. 820 775)
MMS was dissolved in RPMI-0 medium (1500 μg/mL).
MMS (1) is a well-established mutagen whose stability is well-defined under the selected
culture conditions. The positive control stock solutions can be stored at -80°C and thawed
shortly before application. The preparation of a positive control is documented in a specific
record sheet.

Glutathione (GSH):
30.73 mg/mL Glutathione (Sigma; Cat.No. PHR1359) was dissolved in ultrapure water. 10 μL
of this solution were added per mL culture medium (200 μL per culture) resulting a
concentration of 1 mM.

Schedule:
Day 1: Seeding of the cells pretreated with “THMG /THG” medium; start of test substance
treatment;
after 4 hours: Washing of test cultures; 1st passage of the treated cells and seeding
for cloning efficiency 1 (CE1; survival).
Day 2: 2nd passage of the treated cells
Day 3: 3rd passage of the treated cells with seeding in selection medium (mutagenicity) and
seeding for cloning efficiency 2 (CE2; viability).
From
day 10: Evaluation of cloning efficiency 1 (CE1; survival).
From
day 12: Evaluation of the cultures in selection medium (mutagenicity) (only for the cultures
treated with the test substance in the presence of GSH); evaluation of cloning efficiency 2 (CE2;
viability).
In this study all incubations were performed at 37°C with a relative humidity of ≥ 90% in a
5% (v/v) CO2 atmosphere.

Preparation of test cultures:
Logarithmically growing cells in suspension culture (3 x 10^5 cells per 75 cm² flask in a total
volume of 30 mL in exponential growth per treatment group required) were incubated 4 - 5 days
prior to the start of the experiment.

Pretreatment of cells:
During the week prior to treatment, spontaneous TK deficient mutants (TK-/-) were eliminated
from the stock cultures by incubating 3 x 10^5 cells per 75 cm² flask in a total volume of 30 mL
for 1 day in “THMG" medium (pretreatment medium A), and for the following 3 days in
“THG" medium (pretreatment medium B).

Treatment of test cultures and expression period:
Following centrifugation and resuspension the cells were dispensed into 75 cm² flasks
(4-hour exposure: 1.5 x 10^7 cells per culture; 24-hour exposure: 1 x 10^7 cells per culture). Two
cultures were treated in parallel for each test group. Subsequently the treatment medium was
added. The cultures were incubated for the respective exposure period.

Treatment of the cultures:
At the end of the exposure period, the cells were transferred in tubes, centrifuged for 5 minutes
at 800 rpm (134 g) and were resuspended in RPMI-5 medium. The washing of the cells was
repeated at least once. Then the cells were centrifuged at 800 rpm (134 g, 5 min) and
resuspended in RPMI-10 medium. From each culture a sample of treated cells
(2 x 10^5 cells/mL or 6 x 10^6 cells/flask) was pipetted in 75 cm² flasks and incubated for a 2-day
expression period.
To maintain exponential growth during this phase, each culture was counted daily and the cell
numbers were adjusted at each day to 2 x 10^5 cells/mL in 30 mL RPMI-10 medium. The cell
numbers were determined using a cell counter (CASY, Roche Applied Science, Mannheim,
Germany).

Selection period:
For the selection of the mutants, the cells were centrifuged (134 g, 5 min) and 5 x 10^5 cells
from each culture were resuspended in 50 mL selection medium (“TFT" medium;
1 x 10^4 cells/mL). Per culture 200 μL were dispensed in each well of two 96-well plates
(2000 cells/well). After incubation for at least 9 days, both the number of negative wells and
the number of wells containing small or large colonies were scored for calculation of the mutant
frequency (MF).
No selection was performed for the test cultures without the addition of Glutathione, except for
the positive control MMS.

Cytotoxicity determination:
Cloning efficiency 1 (survival):
At the end of the exposure period, the cells were centrifuged (134 g, 5 min) and 400 cells from
each test group were resuspended in 50 mL RPMI-20 medium (8 cells/mL). Per culture 200 μL
were dispensed in each well of two 96-well plates (1.6 cells/well). After incubation for
9 - 11 days the plates were scored for empty wells.

Cloning efficiency 2 (viability):
After the expression period, 2 days after end of exposure, the cells were centrifuged (134 g,
5 min) and 400 cells from each culture were resuspended in 50 mL RPMI-20 medium
(8 cells/mL). Per culture 200 μL were dispensed in each well of two 96-well plates
(1.6 cells/well). After incubation for at least 9 days the plates were scored for empty wells.

Relative suspension growth and relative total growth:
For calculation of the relative suspension growth (RSG) and the relative total growth (RTG) the
cell counts determined within the expression period at 2nd and 3rd passage after 4-hour
exposure were used.

Rationale for test conditions:

The dose selection was based on a previously performed pretest (99M0652/04M026; data not
shown), where it could be shown that Methylacrylate induces significant cytotoxicity from 25
μg/mL (25 μg/mL: 27% RTG) and above and the published data.

Due to the limited solubility of the test substance in ultrapure water, dimethyl sulfoxide (DMSO)
was selected as vehicle, which has been demonstrated to be suitable in the mouse lymphoma
assay and for which historical control data are available.
The final concentration of the vehicle DMSO in culture medium was 1% (v/v).

Evaluation criteria:

Cytotoxicity:
The number of empty wells of the 96-well plates was scored and recorded.
Cloning efficiency 1 (survival)
The cytotoxicity of the test substance after the exposure period was determined for each test
group and is given as absolute and relative cloning efficiency (CE1 and RCE1, respectively).

Cloning efficiency 2 (viability)
The cytotoxicity of the test substance at the end of the expression period was determined for
each test group and is given as absolute and relative cloning efficiency (CE2 and RCE2,
respectively).

Relative total growth:
The RTG is the standard measure of cytotoxicity. This measure includes the relative growth in
suspension (RSG) during the expression period and the relative cloning efficiency (RCE2;
viability) at the time the mutants are selected.
For taking into account any loss of cells during the 4-hour treatment period a relative growth
during treatment factor (RGDT, %) was calculated by comparing the growth of each treated
culture relative to the control.

Mutant frequency:
The number of empty wells and the number of wells containing colonies were scored and
reported. The colonies are classified into large colonies (indication of gene mutation) and small
colonies (indication of chromosome breakage). Small colonies are defined as less than 1/4 of
the diameter of the well. Size is the key factor and morphology (the optical density of the small
colonies is considerably higher) should be secondary.

Uncorrected mutant frequency:
The uncorrected mutant frequency per 10^6 cells was calculated for each test group.

Corrected mutant frequency:
The corrected mutation frequency was calculated regarding the values of CE2

Determination of borderline mutant frequency based on GEF:
The GEF (global evaluation factor) evaluation method requires that the MF exceeds a value
based on the global distribution of the background MF of the test method. The borderline mutant frequency was calculated for each experiment separately.

Statistics:

An appropriate statistical method to test for linear trend (MS EXCEL function RGP; 10) was
performed to assess a possible linear dose-relation in mutant frequencies. The dependent
variable was the corrected mutant frequency and the independent variable was the
concentration. A trend was judged as statistically significant whenever the one-sided p-value
(probability value) was below 0.05 and the slope was greater than 0.
However, both, biological and statistical significance have been considered together.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

4-hour exposure period; without 1mM Glutathione

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

4-hour exposure period; with 1mM Glutathione

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

4-hour exposure period; with 1mM Glutathione

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TREATMENT CONDITIONS:
Osmolality and pH values were not relevantly influenced by test substance treatment.
In this study, in the absence and the presence of 1 mM Glutathione no precipitation in culture
medium was observed up to the highest applied test substance concentration.

CYTOTOXICITY:
After four hours treatment in the absence of 1 mM Glutathione a dose dependent reduction of
the relative total growth was observed from 5 μg/mL onward up to 25.0 μg/mL (RTG: 81.3% -
31.3%). At 30.0 μg/mL a reduced relative total growth of 14.1% of control was observed. For
35.0 μg/mL the parameters RTG and cloning efficiency could not be determined due to severe
toxicity.
After the addition of 1 mM Glutathione relevant cytotoxic effects indicated by reduced relative
total growth of below 20% (RTG: 83.0 – 107.7%) of control were not observed.

MUTANT FREQUENCY:
No selection was performed for the test cultures without the addition of Glutathione, except for
the positive control MMS.
After the addition of 1mM Glutathione, no dose-related increases in the corrected mutation
frequency were observed as determined by testing for linear trend. In the main
experiment, the values of all test groups (MFcorr.: 49.9 – 98.5 per 10^6 cells) were below the
respective calculated threshold for a mutagenic effect based on the GEF (196 mutant colonies
per 10^6 cells). The concurrent vehicle control was at MFcorr.: 70.4 per 106 cells. The statistical
analyses by testing for linear trend led to clearly negative findings.
The positive control substance MMS known to induce gene mutations (15 μg/mL; MFcorr.:
916.9) led to a clearly increased mutant frequency as expected. The value exceeded the
respective calculated thresholds for a mutagenic effect based on the GEF (126 plus the mutant
frequency of the respective negative control). In addition, the corrected mutant frequency was
comparable with the historical positive control data range. Besides, the
obtained value fulfilled the criteria for positive controls as mentioned in the current OECD
Guideline 490.

Any other information on results incl. tables

DISCUSSION:
Moore et al have described a mutagenic effect of Methylacrylate in the TK assay. The induction of mutant colonies was also closely associated with the induced cytotoxicity. It has been previously reported, that Glutathione has a high affinity for Methylacrylate. This could lead to a depletion of the intracellular levels of Glutathione. In this study the cultures were treated with the same levels of the test substance as previously described to be cytotoxic and mutagenic. Furthermore, a group of the cultures were replenished with Glutathione in order to assess the impact of Glutathione supplementation on mutagenicity and cytotoxicity. The results in the absence of supplementary Glutathione confirmed the data from Moore et al. However, the addition of 1 mM Glutathione abrogated this observed cytotoxicity. Furthermore, according to the results of the present in vitro study, the test substance Methylacrylate did not lead to a relevant increase in the number of mutant colonies after the addition of 1 mM Glutathione The mutant frequencies at any concentration were close to the range of the concurrent vehicle control value and within the range of our historical negative control data without S9 mix.
The mutant frequencies at any concentration were below the respective thresholds of 126 colonies per 10^6 cells (GEF) above the concurrent negative control values.
The mutation frequencies of the vehicle control groups were within our historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study.
The proficiency of the laboratory to perform the mouse lymphoma assay with L5178Y TK+/- cells was demonstrated by the laboratory’s historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data.
The increase in the frequencies of mutant colonies induced by the positive control substance MMS clearly demonstrated the sensitivity of the test method. The value was within the range of the historical positive control data. Thus, the acceptance criteria of the current OECD Guideline 490 were fulfilled.

Applicant's summary and conclusion

Conclusions:

CONCLUSION:
Thus, under the experimental conditions chosen here, the conclusion is drawn that
Methylacrylate does not exert a cytotoxic or mutagenic response at the tested levels under
in vitro conditions in the mouse lymphoma assay with L5178Y TK+/- cells after the addition of
1mM Glutathione.

Executive summary:

The ability of the test substance Methylacrylate to induce gene mutations at the thymidine kinase (TK) locus or structural chromosome aberrations at chromosome 11 in L5178Y TK+/- mouse lymphoma cells in vitro with the microwell method in the presence of 1 mM.
Glutathione was assessed. One experiment carried out without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats (exogenous metabolic activation), but with or without the addition of 1mM Glutathione.
The following concentrations were tested. The test groups printed in bold type were evaluated for gene mutations.

Main Experiment:
4-hour exposure period; without 1mM Glutathione
0; 5.0; 10.0; 15.0; 20.0; 25.0; 30.0; 35.0 μg/mL

4-hour exposure period; with 1mM Glutathione
0; 5.0; 10.0; 15.0; 20.0; 25.0; 30.0; 35.0 μg/mL

Cells were treated with the test substance for 4 hours in the absence of metabolic activation with or without the addition of 1mM Glutathione. Subsequently, cells were cultured for an expression period of about 48 hours and then only the cultures treated in the presence of Glutathione and the positive control MMS were cultured in selection medium for another approx. 10 days. Finally, the number of large and small colonies was determined for these cultures. The vehicle control and the dose groups without the addition of Glutathione were only assessed for the induction of cytotoxicity.
The vehicle control with the addition of Glutathione gave mutant frequencies within the range expected for the L5178Y TK+/- mouse lymphoma cell line. The positive control methyl methanesulfonate (MMS) led to the expected increase in the frequencies of forward mutations.
A dose-dependent cytotoxicity indicated by reduced relative total growth (RTG) of below 20% of control was observed in the absence of 1mM Glutathione at 30 μg/mL and above. At 35.0 μg/mL no RTG could be determined due to strong cytotoxicity. In the presence of
Glutathione, no relevant cytotoxicity was observed up to the highest tested concentration.
Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies without S9 mix after adding 1 mM Glutathione.
Thus, under the experimental conditions described, Methylacrylate did not induce forward mutations or structural chromosome aberrations in vitro in the mouse lymphoma assay with L5178Y TK+/- cells in the presence of 1mM Glutathione without the addition of a metabolic activation.