Methyl 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:

2020-10-06 to 2020-11-06

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

EPA 712-C-98-221, August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“
“Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“.

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:

Methyl methacrylate

EC Number:

201-297-1

EC Name:

Methyl methacrylate

Cas Number:

80-62-6

Molecular formula:

C5H8O2

IUPAC Name:

methyl 2-methylprop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

Supplier: Röhm GmbH, Darmstadt, Gemany
Batch: 3110033003
Purity: 99.98%
Expiry Date: 12 February 2021
Storage Conditions: Room temperature

Method

Target gene:

hprt

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (each batch is screened)
- Periodically checked for karyotype stability: yes
- Periodically checked for spontaneous mutant frequency: yes (each batch is screened)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 was used as metabolic activation system.
The S9 was prepared and stored according to the currently valid version of the ICCR SOP for rat liver
S9 preparation. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl
(33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4). The protein concentration of the S9 preparation was 31.7 mg/mL (Lot. No.: 200220) in the pre-experiment and the main experiment.

Test concentrations with justification for top dose:

The dose range of the main experiment was set according to data generated in the pre-experiment.
The individual concentrations were spaced by a factor of 2.0.
Experiment I (4 hours):
-S9 mix: 31.3, 62.6, 125.3, 250.5, 501, 1002 μg/mL
+S9 mix: 31.3, 62.6, 125.3, 250.5, 501, 1002 μg/mL μg/mL
The cultures at the lowest concentration with and without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

DMSO, purity ≥99.9
The final concentration of DMSO in the culture medium was 0.5% (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9-mix

Controls 2

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in solution
DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 500
DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency

Rationale for test conditions:

The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic
activation. Test item concentrations between 7.8 μg/mL and 1002 μg/mL (equal to a molar concentration of approximately 10 mM) were used.
No relevant cytotoxic effect, indicated by a relative cloning efficiency of approximately 50% or below
was observed up to the highest concentration with and without metabolic activation.
In the pre-experiment the test medium was checked for precipitation or phase separation at the
beginning and at the end of treatment (4 hours) prior to removal of the test item. No precipitation or
phase separation occurred up to the highest concentration tested.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
a) The mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are an alysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to
assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies
(mean values) obtained for the groups treated with the test item were compared to the solvent control
groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
However, both, biological and statistical significance will be considered together.
Linear Regression Analysis:
without S9 mix: p-value ( calculation based on mean of culture I and II) 0.883
with S9 mix: p-value ( calculation based on mean of culture I and II) 0.758
A t-Test was not performed since all mean mutant frequencies of the groups treated with the test item
were well within the 95% confidence interval of our laboratory’s historical negative control data.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No substantial and dose dependent increase of the mutation frequency was observed in the main experiment

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No relevant cytotoxic effect, indicated by a relative cloning efficiency of approximately 50% or below was observed up to the highest concentration with and without metabolic activation

Vehicle controls validity:

valid

Remarks:

The viability (cloning efficiency II) of the solvent control of the second culture without metabolic activation did not exceed the lower limit of 50%. The data are valid however, as the solvent control of the parallel culture exceeded this limit.

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

valid EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Additional information on results:

The mean mutant frequency obtained for the solvent controls in experiment I was 10.2 mutants per 10E6 cells in the absence of metabolic activation, and 13.0 mutants per 10E6 cells in the presence of metabolic activation. The values were well within the 95% confidence interval of our laboratory’s historical solvent control data and, thus, fulfilled the requirements of the current OECD Guideline 476.
The range of the mutant frequencies (mean values) of the groups treated with the test item was from 8.1 up to 17.4 mutants per 10E6 cells. The values were well within the 95% confidence interval of the laboratory’s historical solvent control data.
The linear regression analysis showed no significant dose dependent trend of the mutation frequency.
Therefore, the criteria for a negative response were met in the presence and absence of metabolic activation.

Any other information on results incl. tables

Main experiment

Summary of Results

				relative cloning efficiency I	relative cell density	rel. adjusted cloning efficiency I	mutant colonies 10E6 cells	95 % confidence interval
	conc. µg/ml	P/ PS	S9 mix
Main experiment / 4 hrs treatment				Mean values of culture I and II
Solvent control with DMSO			-	100	100	100	10.2	3.5 – 31.0
Positive control (EMS)	300.0			98.0	103.8	101.8	319.2	--
Methyl methacrylate	31.3	-	-	97.8	90.7	88.5	#
Methyl methacrylate	62.6	-	-	96.0	103.5	99.4	12.7	3.5 – 31.0
Methyl methacrylate	125.3	-	-	95.0	92.6	88.1	17.3	3.5 – 31.0
Methyl methacrylate	250.5	-	-	97.4	98.4	95.6	10.0	3.5 – 31.0
Methyl methacrylate	501.0	-	-	95.5	82.2	78.3	8.4	3.5 – 31.0
Methyl methacrylate	1002.0	-	-	96.8	81.1	78.5	14.4	3.5 – 31.0
Solvent control with DMSO				100	100	100	13.0	4.2-30.7
Positive control (DMBA)	2.3			67	102.6	69.6	144.0	--
Methyl methacrylate	31.3	-	+	96.7	129.7	125.7	#
Methyl methacrylate	62.6	-	+	95.8	97.2	93.3	8.1	4.2-30.7
Methyl methacrylate	125.3	-	+	88.0	100.9	87.9	8.1	4.2-30.7
Methyl methacrylate	250.5	-	+	99.6	109.8	109.9	11.6	4.2-30.7
Methyl methacrylate	501.0	-	+	77.0	114.0	85.6	17.4	4.2-30.7
Methyl methacrylate	1002.0	-	+	95.3	98.5	93.4	10	4.2-30.7

P / PS = Precipitation / Phase separation visible at the end of treatment

#        culture was not continued as a minimum of only four analysable concentrations is required

 

Applicant's summary and conclusion

Conclusions:

Interpretation of result: negative
In this mutagenicity assay methyl methacrylate did not induce gene mutaions over background at the HPRT locus in V79 cells.

Executive summary:

Methyl methacrylate was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster acc. OECD 476. The treatment period was 4 hours with and without

metabolic activation.

The main experiment was analysed for gene mutation at the following concentrations with and without metabolic activation: 62.6, 125.3, 250.5, 501, 1002 µg/mL

No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.

The mean mutant frequency obtained for the solvent controls was 10.2 mutants per 106 cells in the absence of metabolic activation, and 13.0 mutants per 106 cells in the presence of metabolic activation. The values were well within the 95% confidence interval of our laboratory’s historical solvent control data and, thus, fulfilled the requirements of the current OECD Guideline 476.

The range of the mutant frequencies (mean values) of the groups treated with the test item was from 8.1 up to 17.4 mutants per 106 cells. The values were well within the 95% The linear regression analysis showed no significant dose dependent trend of the mutation frequency.

Therefore, the criteria for a negative response were met in the presence and absence of metabolic activation.

EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, methyl methacrylate is considered to be non-mutagenic in this HPRT assay.