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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04/2020-06/2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 440/2008 B13/14
Version / remarks:
30-05-2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
ninth addendum to OECD 471; corrected 26-06-2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl methacrylate
EC Number:
202-597-5
EC Name:
Ethyl methacrylate
Cas Number:
97-63-2
Molecular formula:
C6H10O2
IUPAC Name:
ethyl methacrylate
Test material form:
liquid
Specific details on test material used for the study:
TEST MATERIAL:
- Name of Test material: Ethyl methacrylate
- physical state: liquid, colorless

Method

Target gene:
his locus
Species / strainopen allclose all
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:
Phenobarbital/ beta-naphthoflavone induced rat liver S9 Mix
Test concentrations with justification for top dose:
In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. The pre-experiment is reported as experiment I. Since only slight toxic effects were observed 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.

Experiment I (plate incorporation test): 3/ 10/ 33/ 100/ 333/ 1000/ 2500/ 5000 µg/plate
Experiment II (pre-incubation test): 33/ 100/ 333/ 1000/ 2500/ 5000 µg/plate
Vehicle / solvent:
On the day of experiment, the test item was dissolved in DMSO (purity >99%). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. All formulations were prepared freshly before treatment and used within two hours of preparation.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene: with metabolic activation: all strains; 4-nitro-o-phenylene-diamine: without metabolic activation: TA 1537 and TA 98
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation) and pre-incubation

EXPERIMENTAL PERFORMANCE:
For each strain and dose level, including the controls, three plates were used.
Experiment I (Plate incorporation): Test solution at each dose level (solvent or reference mutagen solution (positive control)), S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), bacteria suspension and overlay were mixed in a test tube and poured onto the selective agar plates.
Experiment II (Pre-incubation): The test solution at each dose level, the S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation) and the bacteria suspension were mixed in the test tube and incubated at 37 +/- 1.5°C for 60 minutes.
After the pre-incubation, 2.0 ml overlay agar was added to each tube. The mixture was poured on minimal agar plates. After solidification, the plates were incubated upside down for at least 48 hours at 37 +/- 1.5°C in the dark.
In parallel to each test, a sterile control of the test item was performed and documented in the raw data. Therefore, sterile stock solution, S9 mix/ S9 mix substitution buffer were mixed with 2.0 ml overlay agar and poured on minimal agar plates.
The colonies were counted using the validated computer system, which was connected with a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to the precipitation of the test item the colonies were partly counted manually.

DETERMINATION OF CYTOTOXICITY:
 Pre-Experiment for Toxicity: To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I since the acceptance criteria are met.

Evaluation criteria:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

- regular background growth in the negative and solvent control;
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data;
- the positive control substances should produce an increase above the threshold of twofold (strains TA 98, TA 100, and WP2 uvrA) or threefold (strains TA 1535 and TA 1357) the colony count of the corrresponding solvent control;
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
Statistics:
no appropriate statistical method available

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment II, cytotoxic effects were observed at 5000 µg/plate with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment I, cytotoxic effects were observed at 5000 µg/plate without S9 mix. In experiment II, cytotoxic effects were observed at 5000 /plate with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment II, cytotoxic effects were observed at 5000 µg/plate with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment II, cytotoxic effects were observed at 5000 µg/plate without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Ethyl methacrylate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of Ethyl methacrylate to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/ Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

No precipitation of the test item occured up to the highest investigated dose. 

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. 

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occured in experiment I in strain TA 1537 in the absence of S9 mix and in experiment II in strains TA 1535, TA 1537, and TA 98 in the presence and absence of S9 mix and in strain WP2 uvrA in the absence of S9 mix. 

No substantial increase in revertant colony number of any of the five tested strains was observed following treatment with Ethyl methacrylate at any dose, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below generally acknowledged border of biological relevance. 

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base par changes or frameshifts in the genome of the strains used.

Therefore, Ethyl methacrylate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

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