Methyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Methyl methacrylate was not mutagenic in a guideline GLP bacterial reverse mutation test according to OECD test guideline 471 using bacterial strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and E. coli WP2 uvrA with and without metabolic activation and tested up to the required limit of 5000 µg/plate. No precipitation or cytotoxicity occurred. (ICCR_2021_Ames)

Methyl methacrylate was not mutagenic in a guideline GLP In Vitro Mammalian Cell Gene Mutation Test according to OECD test guideline 476 using Chinese Hamster V79 cells with and without metabolic activation and tested up to the limit concentration of 10 mM. No precipitation or cytotoxicity above acceptable levels was observed. (ICCR_2021_HPRT)

Methyl methacrylate was not mutagenic in a guideline GLP In Vitro Mammalian Cell Micronucleus Test according to OECD test guideline 487 using primary human lymphocytes with and without metabolic activation and tested up to the limit concentration of 10 mM. No precipitation or cytotoxicity above acceptable levels was observed. (ICCR_2021_In vitro MNT)

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:

2020-10-06 to 2020-11-06

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)

Version / remarks:

adopted 29 July 2016

Deviations:

no

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

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

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

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

Target gene:

hprt

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

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

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.

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.

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

 

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-11-11 to 2021-01-18

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)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Supplier: Röhm GmbH Darmstadt
Batch: 3110033003
Purity: 9.98 %
Expiry date: 2021-02-12

Target gene:

not applicable (micronucleus test)

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (22 years old) for Experiment I and from a female donor (20 years old) for Experiment II.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 was used as the metabolic activation system.
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 used for this study was 31.7 mg/mL (Lot no. 200220).

Test concentrations with justification for top dose:

With regard to the molecular weight of the test item, 1002 µg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 8.9 to 1002 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
No cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 1002 µg/mL were chosen as top concentration in Experiment II.
Experiment I - 4 h treatment without S9 mix
8.9, 15.5, 27.1, 47.5, 83.1, 145, 254, 445, 668, 1002 µg/ml
Evaluated concentrations: 445, 668, 1002 µg/ml
Experiment II - 20 h treatment without S9 mix
132, 198, 297, 445, 668, 1002 µg/ml
Evaluated concentrations: 445, 668, 1002 µg/ml
Experiment I - 4 h treatment with S9 mix
8.9, 15.5, 27.1, 47.5, 83.1, 145, 254, 445, 668, 1002 µg/ml
Evaluated concentrations: 445, 668, 1002 µg/ml

Vehicle / solvent:

DMSO; dimethyl sulfoxide
Purity: ≥99.9
Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
Experiment I: 4 h with and without metabolic activation followed by 16 h recovery period
Experiment II: 4 h with metabolic activation followed by 16 h recovery period, 20 h without metabolic
activation
- Expression time (cells in growth medium): 20 h (+ cytochalasin B)
- Fixation time (start of exposure up to fixation or harvest of cells): 40 h
SPINDLE INHIBITOR (cytogenetic assays): 4 mg/L cytochalasin B
STAIN (for cytogenetic assays): Giemsa
3
NUMBER OF REPLICATIONS: 2 per experiment
NUMBER OF CELLS EVALUATED: at least 1000 binucleate cells per culture scored for cytogenetic
damage
DETERMINATION OF CYTOTOXICITY
- Method: CBPI (Cytokinesis-block proliferation index) was determined in approximately 500 cells per
culture and cytotoxicity is expressed as % cytostasis

Rationale for test conditions:

The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline
487 for In Vitro Mammalian Cell Micronucleus Test requesting for the top concentration clear toxicity
with cytostasis of 55 ± 5 %, and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 μL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible.
The highest applied concentration in this study (1002 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

Evaluation criteria:

The micronucleus assay is considered acceptable if it meets the following criteria:
- The number of micronuclei found in the negative and solvent controls falls within the range of the laboratory historical control data
- The positive control substances produce significant increases in the number of cells with micronuclei.
A test item can be classified as non-mutagenic if: - the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.

Statistics:

The Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Additional information on results
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: solvent control – 7.92 (exp. I, ); test item 1002 µ/ml – 7.92
> no relevant influence
- Effects of osmolality: solvent control – 396; test item 1002 µ/ml – 395 -> no relevant influence
- Precipitation: no precipitation of the test item in the culture medium observed.
RANGE-FINDING/SCREENING STUDIES:
- pre-experiment with 10 concentrations: 8.9, 15.5, 27.1, 47.5, 83.1, 145, 254, 445, 668, 1002 µg/ml
exposure duration 4 h with and without metabolic activation
- no toxicity observed
HISTORICAL CONTROL DATA:
For the solvent controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments (cf. Annex 1). The calculated 95% control limit of the solvent controls (realized as 95% confidence interval) was applied for the evaluation of acceptability and interpretation of the data (cf. chapter 3.8 and 3.9). Control charts of the corresponding experiments are added as quality control method.
For the positive controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments (cf. Annex 1). The min-max range of the positive controls was applied for the evaluation of acceptability
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiments I and II in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.
OTHER:
In Experiments I and II in the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item. In the presence of S9 mix, however, dose-dependency, tested by trend test was observed. Since none of the values were statistically significantly increased and all values were clearly within the 95% control limit of the historical control data, the finding can be regarded as biologically irrelevant.

Table2            Summary of results

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells	95% Ctrl limit
		in µg/mL	CBPI		in %**	in %
Exposure period 4 h without S9 mix
I	40 h	Solvent control^1	1.79		0.35	0.00 – 1.04
		Positive control^2	1.39	50.6	15.65^S
		445	1.61	22.7	0.30
		668	1.76	4.1	0.30
		1002	1.72	8.6	0.75
Trend test: p-value 0.321
Exposure period 20 h without S9 mix
II	40 h	Solvent control^1	2.02		0.15	0.00 – 0.86
		Positive control^3	1.60	41.1	2.90^S
		445	1.94	8.3	0.05
		668	1.89	13.1	0.10
		1002	1.94	8.7	0.20
Trend test: p-value 0.692
Exposure period 4 h with S9 mix
I	40 h	Solvent control^1	1.65		0.25	0.00 – 1.03
		Positive control^4	1.37	43.5	2.65^S
		445	1.52	20.0	0.35
		668	1.62	4.0	0.40
		1002	1.57	11.7	0.45
Trend test: p-value 0.005^T

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

^SThe number of micronucleated cells is statistically significantly higher than corresponding control values

^TTrend analysis via linear regression is significant (p ˂ 0.05)

¹DMSO                  0.5 % (v/v)
²MMC                   0.8 µg/mL
³Demecolcine           75 ng/mL
⁴CPA                     15.0 µg/mL

Conclusions:

In this micronucleus test methyl methacrylate did not induce micronuclei in human lymphocytes in vitro when tested was tested up to and including the limit concentrations (ca. 10 mM).

Executive summary:

In a mammalian cell micronucleus assay according to OECD guideline 487, adopted 29 July 2016, primary human lymphocyte cultures were exposed to methyl methacrylate in DMSO at concentrations of 0 (control), 445, 668, 1002 µg/ml with and without metabolic activation (S9 mix). Methyl methacrylate was tested up to and including the limit concentration (ca. 10 mM).

No relevant cytotoxicity, indicated by reduced CBPI and described as cytostasis was observed up to and including the highest applied concentration.

Positive controls induced the appropriate response.

There was no evidence of micronucleated cells induced over background.Therefore, methyl methacrylate is considered to be non-clastogenic in this micronucleus test, when tested up to and including the limit concentration.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-30 to 2020-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471: Bacterial
Reverse Mutation Test, corrected June 26, 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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

Target gene:

his, trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
: rat liver
- method of preparation of S9 mix: Phenobarbital/beta-naphthoflavone
- concentration or volume of S9 mix and S9 in the final culture medium : 500 μl
- quality controls of S9: 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. The protein concentration of the
S9 preparation was 33.0 mg/mL (Lot. No.: 030920K) in both experiments
-The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova
Biochem GmbH (35394 Gießen, Germany).

Test concentrations with justification for top dose:

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 occurred 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.

Vehicle / solvent:

DMSO (purity > 99%)
Justification for choice of solvent/vehicle: The solvent was chosen because of its relative nontoxicity for the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine,

Remarks:

TA 1537, TA 98, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-amino anthracene

Remarks:

TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA, with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two
METHOD OF TREATMENT/ EXPOSURE:
4
First experiment: plate incorporation assay
Second experiment: preincubation assa

Evaluation criteria:

According to international guidelines a statistical evaluation of the results is recommended. Howe ver, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered nonmutagenic
in this system. A significant response is described as follows:
A test article is considered mutagenic if in strain TA98, TA 100 and TA 102 the number of reversions
is at least twice as high and in strains TA 1535, TA 1537 it is at least three times higher as compared
to the spontaneous reversion rate. Also, a dosedependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above descr
ibed enhancement factors or not.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Methyl methacrylate at any dose level, 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 the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusions:

negative
In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frame s
hifts in the genome of the strains tested.
Therefore, methyl methacrylate has to be judged as non mutagenic up to 5000 μg/plate in the presence and absence of mammalian metabolic activation according to the Ames test results.

Executive summary:

In a reverse gene mutation assay in bacteria (Ames test), strains TA1535, TA1537, TA98, TA100, and E. coli WP2 uvr A of Salmonella typhimurium were exposed to methyl methacrylate ( 99.98 % at concentrations of 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate in the presence and absence of mammalian metabolic activation S9 -mix. In two independent experiments methyl methacrylate was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II). No precipitation of methyl methacrylate was observed in any tester strain used in experiment I and II (+/- S9 metabolic activation).

No toxic effects occurred in the test groups with and without metabolic activation in both independent experiments.

The plates incubated with the test article showed normal background growth up to 5000μg/plate with and without S9-mix in all strains used. No substantial increases in revertant colony numbers of any

of the five tester strains were observed following treatment with methyl methacrylate at any concentration level, either in the presence or absence of metabolic activation (S9 -mix). There was also no tendency

to higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.

Appropriate reference mutagens were used as positive controls. The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Methyl methacrylate was not mutagenic in any of the in vitro genotoxicity studies in Annex VII or VIII, recently performed according to relevant test guidelines and tested with and without metabolic activation up to limit concentrations and thereby fulfilling the standard information requirements in Annex IX and X without the need for follow-up testing in vivo.

Additionally available earlier in vitro and in vivo genotoxicity studies for methyl methacrylate were summarized and evaluated in the attached publication by Albertini (2017). Occasional findings related to clastogenicity were associated with high toxic doses and experimental limitations resulting in reduced reliability of the data. Overall, methyl methacrylate is considered to have no relevant in vivo genotoxic potential (further information see “Additional information”). Furthermore, methyl methacrylate was not carcinogenic in several guideline carcinogenicity studies. In addition, MMA is rapidly metabolized forming non-mutagenic metabolites.

Investigating the potential for mutagenicity to germ cells, Methyl methacrylate was not mutagenic in vivo according to a valid Rodent Dominant Lethal Test that was performed prior but similar to OECD test guideline 478 in mice (20 animals per treatment group) exposed by inhalation to concentrations of 100 ppm, 1000 ppm and 9000 ppm for 6 h/day during 5 consecutive days. Specific data on toxicity were not reported, however, in the 9000 ppm group 6 out of 20 males died (Anderson, 1976).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Method and results sufficient described,pre guideline study similar to OECD-guideline 478

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Version / remarks:

pre guideline study. OECD 478 was first adopted 1984

Deviations:

no

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Specific details on test material used for the study:

Methylmethacrylate (CAS: 80-62-6)
Purity: not reported

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10-12 w
- Diet: Alderley Park mouse cubes

Route of administration:

inhalation

Vehicle:

dry clean air

Details on exposure:

During exposure, male CD-1 mice were individually housed in chambers made of stainless steel and glass with an internal capacity of three liters. Seven groups of mice, previously shown to be fertile, were treated according to the scheme presented below.
Fertility testing: Prior to the five-day inhalation exposures, male mice were each mated with two virgin adult female mice for five days. After a five-day mating period, the females were transferred to other cages. The females were sacrificed 15 days following the first day of placement with the males and examined for pregnancy. Only males successful in mating were used on the test.
Experimental mating and necropsy: After treatment, male mice were individually housed. Two virgin female mice were placed in each cage. After a five-day mating period, the females were removed and pair-housed. After a two-day rest period, two new virgin female mice were housed with each male for a five-day mating period. This process was repeated until the male mice had been mated for eight weeks. The male mice were then sacrificed and discarded without necropsy. It was assumed the females were fertilized within two to three days after mating pairs were set up. Thirteen days after the fertilization date, each female was sacrificed and examined for pregnancy, living fetuses and early and late fetal development. 

Duration of treatment / exposure:

5 days, 6 hours/day

Frequency of treatment:

Daily

Dose / conc.:

0.405 mg/L air

Remarks:

corresponding to 100 ppm

Dose / conc.:

4.05 mg/L air

Remarks:

corresponding to 1000 ppm

Dose / conc.:

36.45 mg/L air

Remarks:

corresponding to 9000 ppm

No. of animals per sex per dose:

total number of animals: control: 35; test groups: 20; positive controls: 13 (CTX in water), 5 (EMS in water) and (12 HN2 in saline)

Control animals:

yes, concurrent no treatment

Positive control(s):

200 mg cyclophosphamide in water/kg bw once by i.p. injection on day 5; 150 mg ethylmethane sulphonate in water/kg bw orally once a day for 5 days ; 2.5 mg meclorethamine in saline once 

Tissues and cell types examined:

1) total implants/pregnancy; early deaths/pregnancy; and early deaths/total implants/pregnancy. 

Statistics:

A simple 2X2 Chi-square was used to analyze the data. Also, a week-by-week hierarchical analysis of variance was applied. The following three responses on each female were analyzed: 1) total  implants/pregnancy; early deaths/pregnancy; and early deaths/total  implants/pregnancy. For response 2, the Freeman-Tukey Poisson variance stabilizing transformation was used. Non-pregnant females were taken as missing data. Dunnett's t-test was used for multiple comparisons.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mortality was observed in the three dose groups exposed to the test substance. One animal died in the 100-ppm group the week following exposure, one animal died (95% survival) in the 1000-ppm group and six animals died (70% survival rate) in the 9000-ppm group during exposure. Five animals from the cyclophosphamide positive control group died within eight weeks after dosing.  
Fertility successful mating frequency: No effects observed in the MMA-exposed groups. Positive controls showed appropriate reduction in fertility.
Pregnancy frequency: Reduction in the 1000-ppm group in week 6 only was not considered related to MMA toxicity. Positive controls showed a  decrease in frequency.
Total implantations: No effects observed in the MMA-exposed groups. Positive controls showed appropriate reduction in implant numbers.
Early deaths: Percentages of early deaths were not affected in the MMA-exposed groups. Positive controls showed an appropriate increase in the number of early deaths.
Mean number of early deaths: No effects observed in the MMA-exposed  groups. Positive controls showed an appropriate increase in the number of early deaths.
Percentage of total implantations per pregnancy that were early deaths: No effects observed in the MMA-exposed groups.  
Late deaths: No effects were observed in this study.

Mean early death per pregnancy

		Treatment Group
		Methyl methacrylate [ppm]
Period Week	Negative control	100	100	9000	CTX	E.M.S	HN2
Before treatment	0.66	0.39	0.46	0.54	0.71	0.40	0.6
1	0.75	0.72	0.52	0.72	5.53***	5.29***	1.2
2	0.67	0.61	0.61	0.85	3.80***	2.75***	2.2
3	0.78	0.84	1.11	0.87	1.70**	1.00	1.5
4	0.62	0.75	0.61	0.50	0.60	0.86	0.8
5	0.55	0.75	0.40	0.67	0.42	1.11*	0.5
6	0.66	0.65	0.85	0.67	1.00	0.56	0.1
7	0.92	0.74	0.40	0.77	0.83	0.60	0.7
8	0.97	1.00	0.79	0.78	0.93	0.80	0.90

 

Dunnett‘ t-test of transformed values significant at

*           5 %

*           1 %

***       0.1 % level

Percentage of early death / total implants / pregnancy

		Treatment Group
		Methyl methacrylate [ppm]
Period Week	Negative control	100	100	9000	CTX	E.M.S	HN2
Before treatment	5.6	3.7	4.0	4.3	5.8	3.1	9.5
1	6.6	6.0	4.9	9.4	58.3***	61.2***	14.8***
2	5.3	4.8	5.0	8.7	36.5***	21.2***	21.6***
3	7.3	6.7	9.4	7.4	14.8**	13.8	13.1*
4	5.2	6.9	5.3	4.5	4.7	8.5	6.8
5	5.1	6.6	4.0	5.6	3.5	9.5*	4.0
6	5.3	6.4	7.0	6.2	7.7	5.6	0.9
7	7.9	5.9	3.7	8.1	7.9	6.6	6.0
8	7.8	8.8	7.4	6.8	7.4	7.3	6.8

 

Dunnett‘ t-test of transformed values significant at

*           5 %

*           1 %

***       0.1 % level

Conclusions:

Methyl methacrylate was negative in vaild in vivo germ cell study in mice (rodent dominat lethal test).

Executive summary:

Methyl methacrylate was tested in an in vivo pre guideline dominat lethal test in mice. This test evaluates mutation induction, usually at the chromosome level, in sperm by assessing viability of embryos in subsequent matings. Male CD-1 mice were exposed by inhalation to methyl methacrylate at doses of 100, 1000 and 9000 ppm 6 hours/days for five consecutive days. Specific data on toxicity were not given, however, in the 9000 ppm group 6 out of 20 males died.

After exposure the males were mated to females for 5 days.

Following removal, the females were sacrificed 15 days later and products of conception analyzed. No effects were seen as regards number of early embryo death, mean number of early death per pregnancy or pre-implantation egg loss. This test result was negative indicating no in vivo germ cell mutagenesis. The cyclophosphamid control gave clearly positive results under the conditions of the test.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Method and results sufficient described, similar to OECD-guideline 475.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

pre guideline study. Guideline study, guideline first adopted 1984

Deviations:

yes

Remarks:

The conditions of OECD TG 475 include: the collection of BM at two different times after single treatment; the analysis of min 200 metaphases/ animal. Here, the BM was collected only once after single treatment; max 50 cells/ animal were analysed.

GLP compliance:

no

Type of assay:

chromosome aberration assay

Specific details on test material used for the study:

Supplied by ICI;
Stabilizer: 11 ppm hydroquinone

Species:

rat

Strain:

other: Alderley Park

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Alderley Park
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 150-200 g
- Housing: individually
- Diet (e.g. ad libitum): Alderley Park rat cubes
- Water (e.g. ad libitum): unspecified

Route of administration:

inhalation

Vehicle:

unchanged

Details on exposure:

Atmosphere of various concentrations of test substance and conrol were generated by a controlled fluid-feed atomiser technique (Gage 1959)
Atmospheres were monitored by infra-red spectrophotometer (Wilks Moran 1A)

Duration of treatment / exposure:

single treatment: 2 hrs
repeated treatment: five hrs a day for 5 consecutive days

Frequency of treatment:

single treatment: once
repeated treatment: daily for 5 days

Post exposure period:

Animals were sacrificed 24 hr following the last exposure period.

Dose / conc.:

0.4 mg/L air

Remarks:

corresponding to 100 ppm

Dose / conc.:

4.1 mg/L air

Remarks:

corresponding to 1000 ppm

Dose / conc.:

36.9 mg/L air

Remarks:

corresponding to 9000 ppm

No. of animals per sex per dose:

single treatment: 2-4 rats per group or 5 rats per group
repeated treatment: 7 rats per group.

Control animals:

yes, concurrent no treatment

Positive control(s):

10, 750 and 7500 ppm benzene

Tissues and cell types examined:

bone marrow samples were collected and processed

Details of tissue and slide preparation:

Animals were sacrificed 24 hours after expsosure
Preparation of bone marrows according to the method of Sugiyama (1971) but with slight modifications:
- i.p. injection of colchinine two hours prior to sacrifice to arrest dividing cells in metaphase
-killing rats by an overdose of Fuothane
- after femurs were removed from animals bone marrow was harvested by aspiartion with Hanks' Basis Salt solution
-cells tearted with hpotinic solution followed by fixation in glacial acetic acid:methanol, 1:3
-Slides were prepared by air-drying and stained with Giemsa
- 50 cells from each animal were examined

Following abnormaties were assigend:
- chromatid and chromosome gaps
- chromatid breaks
- fragments
- other complex abnormalties

Statistics:

The data were transformed using a variance stabilizing transformation; the data were analysed using an analysis of variance and a one sided students t test was used on the transformed data

Sex:

male

Genotoxicity:

ambiguous

Toxicity:

not specified

Vehicle controls validity:

other: air was vehivle and negative control

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The study was negative for chromosomal aberration frequencies when gaps were excluded as usual.
When the data from the two 2 hr exposure experiments were combined and gaps were included, 1000 and 9000 ppm MMA groups were significantly different from controls; there was evidence of a dose response relationship.
The group exposed to 9000 ppm for five repeated exposures was significantly different from controls; there was evidence of a dose response relationship.

Combined abnormalities of the two single treatment assays

 

Treatment	% abnormal cells (out of 50 cells)
Ctrl	5.1
100 ppm MMA	5.5
1000ppm MMA	8.0*
9000 ppm MMA	9.4**
10 ppm B	10.3**
750 ppm B	18.0***
7500 ppm B	26.5***

 B=Benzene

Abnormalities of the repeated treatment assay

 

Treatment	% abnormal cells (out of 50cells)
Ctrl	3.1
100 ppm MMA	1.4
1000ppm MMA	6.3
9000 ppm MMA	6.9*#
10 ppm B	7.4**
750 ppm B	10.0***
7500 ppm B	14.0***

 # Statistically significant, however, compared to the control in the single-treatment segment (5.1 % ab.) the increase is biologically unimportant and does not comprise a positive effect.

Mean % abnormalities (excluding gaps)

 

Treatment	Single treatment A	Single treatment B	Repeated treatment
Ctrl	1.0	1.2	0.3
100 ppm MMA	0.7	0.8	0.3
1000ppm MMA	3.3	2.0	0.3
9000 ppm MMA	1.0	2.0	1.2
10 ppm B	5.0	1.6	2.0*
750 ppm B	8.0*	4.0	2.0*
7500 ppm B	15.3**	11.6*	3.7*

 

*    = significance level: 5%

**  = significance level: 1%

*** = significance level: 0.1%

Conclusions:

No increase in chromosome aberrations excl. gaps. An allegedly significant increase in chromosome aberrations incl. gaps is due to an - in comparison - low control value and biologically unimportant compared to other control values. In addition, the biological significance of chromosome gaps is unclear and, as an isolated finding, would not be regarded as a positive test result.

Executive summary:

Methyl methacrylate was tested in a pre guideline cytogenetic bone marrow test in rats which were exposed by inhalation (ICI, 1976). The test was performed by single application for 2 hours (7 animals per dose group) and for repeated application (7 animals per dose group) for 5 hours on 5 consecutive days. Exposure concentrations were 100, 400, 700 and 1000 ppm in both tests. Positive control was 7500 ppm benzene.

As far as possible 50 metaphases were analysed per animal.

When the data from the two 2 hr exposure experiments were combined and gaps were included, 1000 and 9000 ppm MMA groups were significantly different from controls; there was  evidence of a dose response relationship.

The group exposed to 9000 ppm for five repeated exposures was significantly different from controls; there was evidence of a dose response relationship.

The study was negative for chromosomal aberration frequencies when gaps were excluded as usual.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12.01.1977 - 16.01.1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Method and results sufficiently described, with significant technical problems

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

The conditions of OECD TG 475 include: the collection of BM at two different times after single treatment; the analysis of min 200 metaphases/ animal. Here, the BM was collected only once after single treatment; max 50 cells/ animal were analysed.

GLP compliance:

no

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Unit, Alderley Park, Cheshire
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 150-200 g
- Housing: four/cage
- Diet (e.g. ad libitum): Alderley Park rat cubes
- Water (e.g. ad libitum): unspecified


ENVIRONMENTAL CONDITIONS
- Air changes (per hr): minimum 8
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation

Vehicle:

unchanged

Details on exposure:

whole body

Duration of treatment / exposure:

single treatment: 2 hours
repeated treatment: 5 hours

Frequency of treatment:

repeated treatment: daily for 5 days

Post exposure period:

24 h after the last exposure

Dose / conc.:

0.4 mg/L air

Remarks:

corresponding to 100 ppm

Dose / conc.:

1.6 mg/L air

Remarks:

corresponding to 400 ppm

Dose / conc.:

2.8 mg/L air

Remarks:

corresponding to 700 ppm

Dose / conc.:

4 mg/L air

Remarks:

corresponding to 1000 ppm

No. of animals per sex per dose:

12 animals in the negative control group; 8 animals in each of the treated and positive control groups.

Control animals:

yes, concurrent no treatment

Positive control(s):

Benzene, 7500 ppm

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Animals were sacrificed 24 hours after expsosure
Preparation of bone marrows according to the method of Sugiyama (1971) but with slight modifications:
- i.p. injection of colchinine two hours prior to sacrifice to arrest dividing cells in metaphase
-killing rats by an overdose of Fuothane
- after femurs were removed from animals bone marrow was harvested by aspiartion with Hanks' Basis Salt solution
-cells tearted with hpotinic solution followed by fixation in glacial acetic acid:methanol, 1:3
-Slides were prepared by air-drying and stained with Giemsa
- 50 cells from each animal were examined

Following abnormaties were assigend:
- chromatid and chromosome gaps
- chromatid breaks
- fragments
- other complex abnormalties

Statistics:

Proportion of cells with any abnormalities or proportion of cells with any abnormalities other than gaps: Analysis of Variance after transformation of the original data using a double arcsine transformation.
Proportion of rats which had any cells with any abnormality other than gaps: One-sided Fischers Exact test.

Sex:

male

Genotoxicity:

ambiguous

Toxicity:

no effects

Vehicle controls validity:

other: air was vehivle and negative control

Negative controls validity:

valid

Positive controls validity:

valid

The maximum concentration tested caused significant reductions in mitotic activity in the bone marrow of the
exposed animals. Small and non-dose related increases in the percentages of cells with chromosomal aberrations were
observed in the animals exposed to MMA. However, these increases were almost exclusively due to gap-type aberrations and when these were excluded from the data small increases were only observed at 400 ppm. Such small 
increases, observed at lower, but not at higher concentrations tested, are not considered to be 
biologically significant.

For further details, see attached document including 11 tables.

Executive summary:

Methyl methacrylate was tested in a pre guideline cytogenetic bone marrow test in rats which were exposed by inhalation (ICI, 1976). The test was performed by single application for 2 hours (5 animals per dose group) and for repeated application (7 animals per dose group) for 5 hours on 5 consecutive days. Exposure concentrations were 100, 1000 and 9000 ppm in both tests. Positive control was benzene.

The study demstratates some methological problems: Analysis of 50 metaphases was not possible for 10 out of 27 animals in the acute and 10 out 26 in the subacute test.

MMA may cause weak, but statistically significant, increases in chromosome damage in rat at some exposure levels at the sampling times investigated by comparison with the negative control values both after single and multiple exposures. However, there is no constistent dose response and the effects are of an indeterminate toxicological significance in view of the overall negative toxicological profile of the substance.

A clear conclusion cannot be drawn this study.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Similar to OECD-guideline 474, all relevant study details available

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

The conditions of OECD 474 include: the collection of BM at two different times after single treatment; the scoring of at least 4000 immature erythrocytes/ animal. Here, the BM was collected only once; only 2000 erythrocytes were evaluated per animal.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Specific details on test material used for the study:

purity: > 99 %

Species:

mouse

Strain:

other: ddy

Sex:

male

Route of administration:

oral: gavage

Vehicle:

olive oil, 25 ml/kg

Duration of treatment / exposure:

4 doses

Frequency of treatment:

3 doses: once, 24 h before terminal sacrifice
1 dose: 4 split doses every 24 h, the last one 24 h before terminal sacrifice, total duration 5 d

Post exposure period:

24 h

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

1 130 mg/kg bw/day (nominal)

Dose / conc.:

2 260 mg/kg bw/day (nominal)

Dose / conc.:

4 520 mg/kg bw/day (nominal)

Remarks:

Dose corresponds to 50% of LD50

Remarks:

4 x 1130 mg/kgbw per dose

No. of animals per sex per dose:

6 (repeated treatment: 5)

Control animals:

yes, concurrent vehicle

Positive control(s):

3 mg Mitomycin C, single dose by i.p. administration 24 h prior to preparation

Tissues and cell types examined:

Sampling time for bone marrow: 3 single doses - 24 h post-administration; for repeated administration: 5 days after first administration.

Statistics:

according to Kastenbaum/Bowman

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

at all doses, single and repeated

Vehicle controls validity:

valid

Positive controls validity:

valid

The substance has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. Olive oil (25 ml/kg) was used as the solvent control and mitomycin C (3 mg/kg, i.p.) as the positive control. 2000 erythrocytes were evaluated per animal (12000/10000 per dose). No increase in micronucleated polychromatic erythrocytes was observed at any dose, while an induction of micronuclei was seen in the positive control. MMA was not mutagenic in vivo under test conditions.

Conclusions:

Result: negative
Methyl methacrylate has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. MMA was not mutagenic in vivo under test conditions.

Executive summary:

Methyl methacrylate has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. Olive oil (25 ml/kg) was used as the solvent control and mitomycin C (3 mg/kg, i.p.) as the positive control. 2000 erythrocytes were evaluated per animal (12000/10000 per dose). No increase in micronucleated polychromatic erythrocytes was observed at any dose, while an induction of micronuclei was seen in the positive control.  The substance has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. Olive oil (25 ml/kg) was used as the solvent control and mitomycin C (3 mg/kg, i.p.) as the positive control. 2000 erythrocytes were evaluated per animal (12000/10000 per dose). No increase in micronucleated polychromatic erythrocytes was observed at any dose, while an induction of micronuclei was seen in the positive control. MMA was not mutagenic in vivo under test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Methyl methacrylate was not mutagenic in any of the in vitro genotoxicity studies in Annex VII or VIII, recently performed according to relevant test guidelines and tested with and without metabolic activation up to limit concentrations and thereby fulfilling the standard information requirements in Annex IX and X without the need for follow-up testing in vivo.

Additionally available earlier in vitro and in vivo genotoxicity studies for methyl methacrylate are briefly summarized below.

Gene mutation in bacteria

Methyl methacrylate was not mutagenic in further bacterial reverse mutation tests (Zeiger, 1987; Schweikl, 1994; Waegemaekers, 1984). The selection of bacterial strains was not fully according to current guideline requirements but the results support the absence of mutagenicity in bacteria for MMA.

Gene mutation in mammalian cells

In a pre-guideline mouse lymphoma assay, methyl methacrylate was not mutagenic in the absence of metabolic activation when tested in the range of 3.91 - 150 nl/ml up to the limit of cytotoxicity (Cifone, 1981). In the presence of metabolic activation, methyl methacrylate at concentrations of 100 - 300 nl/ml (0.9 -2.7 mM) significantly increased the frequency of tk +/- mutations in a dose dependent manner, however, clear effects were observed only at doses with high toxicity below 20% relative growth. Colony size was not mentioned in the report.

Another pre-guideline mouse lymphoma assay was done with MMA at concentrations of 1000 – 3000 µg/ml (10-30 mM) only in the absence of metabolic activation (Moore, 1988). The concentration range tested is above the limit concentration of 10 mM according to current guideline, if not limited by cytotoxicity. The weakly positive test results (small colonies, indicative of potential clastogenicity) showing no clear dose dependency, a high number of background events and only seen at 10 – 30 % relative survival are therefore considered to be of limited reliability.

Methyl methacrylate was further tested in the National Toxicology Program (NTP) in a pre-guideline mouse lymphoma assay with and without metabolic activation (S9). Myhr et al. (1990) reviewed the data reporting MMA was positive at concentrations of 14 mM without S9 but the results were inconclusive in the presence of S9. Cell survival data were not provided. The majority of the data were small colony, indicating slow growth and clastogenicity.

Methyl methacrylate was tested in a HPRT test (Schweikl, 1998) in the absence of metabolic activation at concentrations of 10 – 30 mM. Excessive toxicity was observed at 30 mM. Only a weak increase of mutations at borderline levels of cytotoxicity was observed at 20 mM, representing a concentration above the limit concentration of 10 mM according to current test guideline. At 10 mM results were within control data. The result of this study is therefore seen ambiguous and of limited validity.

Cytogenicity in vitro

Methyl methacrylate induced chromosome aberrations in CHO cells at concentrations (lowest effective dose [LED]) of 15 mM in the absence of S9 and 50 mM (weak positive) in the presence of S9 (Anderson et al., 1990). Cell survival was not reported. By contrast, when tested in V79 cells, there was no clear dose-response increase in micronuclei with MMA up to 2.0 mM in the absence of S9 (Schweikl et al., 2001 ). An increase in micronuclei did occur in this study, but only at a clearly toxic (reduced cell numbers on slides) MMA concentration of 3.0 mM.

In vivo genotoxicity studies

An in vivo bone marrow micronucleus assay was performed with mice (Hachiya et al., 1981). In an acute test MMA was given by gavage in doses ranging from 1,130 to 4,520 mg/kg, in a subacute assay, daily doses of 1,130 mg/kg were given on 4 consecutive days. All groups consisted of 6 animals; sampling was done 24 h after (last) administration. The percentage of reticulocytes from all bone marrow cells was not affected; data on general toxicity were not given. There was no increase in the frequency of micronucleated polychromatic erythrocytes related to MMA exposure.

Methyl methacrylate was investigated for in vivo cytogenetic effects in a study of inhalation exposures in rats at a range of concentrations up to 9000 ppm either once for 2-h or for five days (5 h/day) (Anderson, 1976). Chromosome aberrations were assessed in bone marrow cells. The authors judged the study to be negative for the single dose and equivocal for the multiple dose portions as elevations over control were small and without a dose-related response. This study was followed by another inhalation study employing the same dosing schedule with MMA at exposure levels up to 1000 ppm (Anderson et al., 1979). The authors again concluded that the single dose portion was negative for chromosome aberration in bone marrow cells but that the multiple-dose portion was equivocally positive. Details of the second study, which presumably apply also to the first, include a harvest time at 24 h, inclusion of chromosome "gaps" in the analysis and the scoring of 50 cells per animal. Current OECD guidelines specify 200 cells. Gaps are no longer considered to be biologically significant. Furthermore, there is no mention of laboratory historical negative control levels for chromosome aberration, something that also is at odds with current guidelines. Although exclusion of gaps would probably have resulted in the multiple-dose portions of these studies also being reported as negative, these early studies do not meet current OECD guidelines.

Investigating the potential for mutagenicity to germ cells, Methyl methacrylate was not mutagenic in vivo according to a valid Rodent Dominant Lethal Test that was performed prior but similar to OECD test guideline 478 in mice (20 animals per treatment group) exposed by inhalation to concentrations of 100 ppm, 1000 ppm and 9000 ppm for 6 h/day during 5 consecutive days. Specific data on toxicity were not reported, however, in the 9000 ppm group 6 out of 20 males died. (Anderson, 1976)

Additional considerations on mutagenicity

The reported key in vitro genotoxicity studies investigate potential mutagenicity through direct DNA reactivity of the test substance without metabolic conversion as tested in the absence of a metabolic activation system. This information provides an evaluation of the reactivity on tissues of first site of contact in vivo at respective routes of exposure. Methyl methacrylate did not show any mutagenic potential in the absence of a metabolic activation system in the standard information requirement in vitro genotoxicity studies.

In addition, no mutagenic potential was observed in the presence of a metabolic activation system in those studies. Methyl methacrylate is rapidly metabolized in the body with a half-life in blood of approximately 4.4 minutes (see “Toxicokinetics”). It is therefore extremely unlikely that MMA as intact ester reaches the germ cells. The resulting metabolites methacrylic acid and methanol are not classified for mutagenicity according to CLP.

Furthermore, methyl methacrylate has been shown to be non-carcinogenic in several guideline carcinogenicity studies by inhalation or oral exposure (drinking water) in rats, mice or hamster (NTP, 1986; Lomax/Rohm, 1992; Lomax, 1997; Borzelleca, 1964). This clearly establishes that MMA does not possess a toxicologically relevant mutagenic potential in somatic cells. As ECHA’s endpoint specific guidance (2017[2]) indicates that “there is considerable evidence of a positive correlation between the mutagenicity of substances in vivo and their carcinogenicity in long-term studies with animals.” it scientifically justified that MMAs lack of carcinogenicity also demonstrates a lack of in vivo genotoxicity in somatic cells.

A comprehensive summary and evaluation of available genotoxicity studies for the group of lower alkyl methacrylates, including methyl methacrylate, and methacrylic acid has been published by Albertini (2017). Overall, almost universally negative results were obtained for reverse mutations in bacteria. Tk-/- mutations in L5178Y in mouse lymphoma cells were detected primarily as slow growing mutants - a phenotype associated with the mutations being due to large scale alterations. These in vitro mutational changes are usually seen only at high dose toxic concentrations - higher than those achieved in vivo and above currently accepted limit concentrations in vitro. Therefore, several of the mutation studies do not meet current guidelines. Despite the partly inconsistent findings in vitro, there is no convincing evidence to demonstrate genotoxic effects in vivo in any test organism. All results in sub-mammalian species were reported as negative. The weight of evidence in mammals is also negative, with the scattering of equivocal/positive claims by authors of early studies likely resulting from scoring chromosome changes that are no longer thought to reflect aberrations (i.e. gaps). Furthermore, few of the early studies would be considered valid by OECD guidelines.

Based on the evaluation of pre-existing genotoxicity data for methyl methacrylate and considering the consistently negative results from the recent standard information requirement in vitro studies it is therefore concluded that methyl methacrylate has no relevant mutagenic potential.

Justification for classification or non-classification

Methyl methacrylate was not mutagenic in any of the in vitro genotoxicity studies in Annex VII or VIII, recently performed according to relevant test guidelines and was tested with and without metabolic activation up to limit concentrations. Thereby fulfilling the standard information requirements in Annex IX and X without the need for follow-up testing in vivo.

Additionally taking into account the lack of carcinogenicity, the rapid metabolism forming non-mutagenic metabolites and the overall evaluation of pre-existing genotoxicity studies, methyl methacrylate is considered to have no relevant in vivo genotoxic potential.

The lack of mutagenicity to germ cells was demonstrated in a rodent dominant lethal test.

Therefore, MMA does not meet the criteria to be classified for its mutagenic potential according to CLP, 1272/2008/EC and UN-GHS requirements, respectively.