Methacrylic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For the assessment of the mutagenic potential of methacrylic acid, ECHA guidance R.7a: Endpoint specific guidance is followed. According to Table R.7.7–1 REACH information requirements for mutagenicity, three reliable in vitro studies are available: a reverse gene mutation assays in bacteria (Ames test), a mammalian cell gene mutation assay (HPRT test) and an in vitro micronucleus test in human lymphocytes.

 

Bacterial reverse gene mutation assays

In a reverse gene mutation assay in bacteria (ICCR 2021a) according to OECD guideline 471, adopted 26 June, 2020, strains TA 1535, TA 1537, TA 98, TA 100 of S. typhimurium and E. coli WP2 were exposed to methacrylic acid in water at concentrations of 3; 10; 33; 100; 333; 1000; 2500; and 5000µg/plate in a plate incorporation assay and 33, 100, 333, 1000, 2500 and 5000.0 µg/plate in a pre-incubation assay in the presence and absence of mammalian metabolic activation (S9 mix). No precipitation of the test item occurred up to the highest investigated dose.

Methacrylic acid was tested up to cytotoxic concentrations. There was no evidence of induced mutant colonies over background.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. Methacrylic acid is not mutagenic in a reverse mutation assay acc. OECD 471.

 

Mammalian cell gene mutation assay

Methacrylic acid was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster acc. OECD 476 (ICCR 2021b). The treatment period was 4 hours with and without metabolic activation.

The main experiment was analyzed for gene mutation at the following concentrations with and without metabolic activation: 26.9; 53.8; 107.6; 215.3; 420.5; 861.0 µg/ml.

A relevant cytotoxic effect indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) occurred at 861.0 µg/mL (relative adjusted cloning efficiency I of 15.8 %) with metabolic activation. Moderate cytotoxic effect indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) occurred at 420.5 µg/mL in the absence of metabolic activation.

The mean mutant frequency obtained for the solvent controls in experiment I was 7.0 mutants per 10e6 cells in the absence of metabolic activation, and 8.0 mutants per 106 cells in the presence of metabolic activation. The values were well within the 95% confidence interval of the 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 7.3 up to 20.9 mutants per 106 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.

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 methacrylic acid did not induce gene mutations at the HPRT locus in V79 cells and is considered to be non-mutagenic in this HPRT assay.

 

Mammalian cell micronucleus test

In a mammalian cell micronucleus assay according to OECD guideline 487 (adopted 29 July 2016) primary human lymphocyte cultures were exposed to methacrylic acid (99.86 %) in water with and without metabolic activation (S9 mix; ICCR 2021c).

The following concentrations were evaluated (calculations were not adjusted to purity):

Experiment I:
4 h exposure, 16 h recovery, fixation time 40 h, without S9: 281, 492, 861 µg/mL
4 h exposure, 16 h recovery, fixation time 40 h, with S9: 281, 492, 861 µg/mL

Experiment II:
20 h exposure, fixation time 40 h, without S9: 281, 492, 861 µg/mL
In Experiment I and II in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.
In Experiment I and II in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that methacrylic acid did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes and is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-11-18 to 2021-02-09

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, Gemany
Batch: R0004-66
Purity: 99.86%
Expiry Date: 12 February 2021
Storage Conditions: Room temperature

Target gene:

not applicable (micronucleus test)

Species / strain / cell type:

lymphocytes: human

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 (23 years old) for Experiment I and from a male don
or (30 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, 861 µg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 5.6 to 861 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, 861 µg/mL were chosen as top treatment concentration for Experiment II.
Experiment I - 4 h treatment without S9 mix
5.6, 9.8, 17.1, 30.0, 52.5, 91.8, 161, 281, 492, 861 µg/ml
Evaluated concentrations: 281, 492, 861 µg/ml
Experiment II - 20 h treatment without S9 mix
52.5, 91.8, 161, 281, 492, 861 µg/ml
Evaluated concentrations: 281, 492, 861 µg/ml
Experiment I - 4 h treatment with S9 mix
Evaluated concentrations: 281, 492, 861 µg/ml

Vehicle / solvent:

Culture medium with 10.0 % deionised water
Justification for choice of solvent/vehicle:
The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation
Purity: 97.0 – 103.0 %
Dissolved in: Saline (0.9 % NaCl [w/v])
Concentration: 17.5 µg/mL

Untreated negative controls:

no

True negative controls:

yes

Remarks:

Culture medium with 10 % deinonised water

Positive controls:

yes

Positive control substance:

mitomycin C

other:

Remarks:

Without metabolic activation
Name: MMC; mitomycin C (pulse treatment)
Purity: 98 %
Dissolved in: Deionised water
Concentration: 1.0 µg/mL
Name: Demecolcine (continuous treatment)
Purity: ≥ 98 %
Dissolved in: Deionised water
Concentration: 100 ng/mL

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 (861 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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Additional information on results
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: solvent control – 7.37 (exp. I, ); test item 861 μ/ml – 6.58 (without S9 mix)
- Effects of osmolality: solvent control – 293; test item 861 μ/ml – 297 -> no relevant influence
- Precipitation: no precipitation of the test item in the culture medium observed.
RANGE-FINDING/SCREENING STUDIES:
- pre-experiment with 10 concentrations: 5.6, 9.8, 17.1, 30.0, 52.5, 91.8, 161, 281, 492, 861 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. 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. 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. The min-max range of the positive controls was applied for the evaluation of acceptability
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I and II in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.
OTHER:
In Experiment I and II in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.
Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Exp.	Preparation interval	Test item concentration in µg/mL	Proliferation index CBPI	Cytostasis in %*	Micronucleated cells in %**	95% Ctrl limit in %
Exposure period 4 h without S9 mix
I	40 h	Solvent control^1	1.74		0.40	0.00 – 1.04
		Positive control^2	1.34	54.4	12.60^S
		281	1.68	7.6	0.55
		492	1.68	8.1	0.35
		861	1.66	10.7	0.30
Trend test: p-value 0.417
Exposure period 20 h without S9 mix
II	40 h	Solvent control^1	2.05		0.40
		Positive control^3	1.59	44.3	3.95^S	0.00 – 0.86
		281	1.98	6.7	0.35
		492	1.92	12.9	0.10
		861	1.82	21.6	0.20
Trend test: p-value 0.272
Exposure period 4 h with S9 mix
I	40 h	Solvent control^1	1.54		0.45	0.00 – 1.03
		Positive control^4	1.24	55.9	3.30^S
		281	1.53	2.8	0.50
		492	1.58	n.c.	0.40
		861	1.55	n.c.	0.40
Trend test: p-value 0.363
*       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 ^S       The number of micronucleated cells is statistically significantly higher than corresponding control values ^TTrend analysis via linear regression is significant (p ˂ 0.05) ^1       DMSO            0.5 % (v/v) ^2MMC               0.8 µg/mL ^3Demecolcine     75 ng/mL ^4CPA               15.0 µg/mL

Conclusions:

In this micronucleus test methacrylic acid did not induce micronuclei in human lymphocytes in vit
ro 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 methacrylic acid in culture medium with 10.0 % deionised water at concentrations of 0 (control), 281, 492, 861 µg/ml with and without metabolic activation (S9 mix). Methacrylic acid 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, methacrylic acid is considered to be non-clastogenic in this micronucleus test, when tested up to and including the limit concentration.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-17 to 2020-10-07

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: R0004-66
Purity: 99.86%
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: 26.9, 53.8, 107.6, 215.3, 420.5, 861.0 μg/mL
+S9 mix: 26.9, 53.8, 107.6, 215.3, 420.5, 861.0 μg/mL
The maximum test item concentration of the pre-experiment and the main experiment (861.0 µg/mL) was equal to a molar concentration of about 10 mM.

Main experiment:
Experiment II (4 hours):
-S9 mix: 26.9, 53.8, 107.6, 215.3, 420.5, 861.0 μg/mL
+S9 mix: 26.9, 53.8, 107.6, 215.3, 420.5, 861.0 μg/mL

Vehicle / solvent:

deionized water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Positive controls:

yes

Positive control substance:

other: 7,12-dimethylbenz(a)anthracene

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 6.7 µg/mL and 861.0 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 analysable 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) 0.994
with S9 mix: p-value ( calculation based on mean of culture I and II) 0.507
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 relevant and reproducible increase in mutant colony numbers/10e6 cells was observed in the main experiment up to the maximum concentration.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Relevant cytotoxic effects indicated by an adjusted cloning efficiency I below 50% occurred at 861.0 µg/mL with metabolic activation and at 420.5 µg/mL 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:

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 7.0 mutants per 106 cells in the absence of metabolic activation, and 8.0 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 7.3 up to 20.9 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.	P/	S9
	µg/ml	PS	mix
Main experiment / 4 hrs treatment				Mean values of culture I and II
Solvent control with water			-	100	100	100	7.0	3.5 – 31.0
Positive control (EMS)	300.0			85.9	68.5	58.7	173.6	--
Methacrylic acid	26.9	-	-	98.6	72.7	71.6	11.0	3.5 – 31.0
Methacrylic acid	53.8	-	-	97.2	74.2	72.1	9.7	3.5 – 31.0
Methacrylic acid	107.6	-	-	95.6	62.4	59.8	20.9	3.5 – 31.0
Methacrylic acid	215.3	-	-	94.4	65.6	61.5	12.4	3.5 – 31.0
Methacrylic acid	420.5	-	-	90.6	51.1	46.1	10.5	3.5 – 31.0
Methacrylic acid	861.0	-	-	95.6	53.1	50.8	11.4	3.5 – 31.0
Solvent control with water				100	100	100	8.0	4.2-30.7
Positive control (DMBA)	2.3			96.8	104.5	100	154.5	--
Methacrylic acid	26.9	-	+	105.4	101.5	105.3	7.3	4.2-30.7
Methacrylic acid	53.8	-	+	100.5	102.7	100.3	9.2	4.2-30.7
Methacrylic acid	107.6	-	+	102.7	103.7	104.4	8.3	4.2-30.7
Methacrylic acid	215.3	-	+	96.9	97.6	94.2	16.9	4.2-30.7
Methacrylic acid	420.5	-	+	70.7	94.7	66.6	12.5	4.2-30.7
Methacrylic acid	861.0	-	+	17.0	97.3	15.8	10.3	4.2-30.7

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

Conclusions:

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

Executive summary:

Methacrylic acid 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 analyzed for gene mutation at the following concentrations with and without metabolic activation: 26.9; 53.8; 107.6; 215.3; 420.5; 861.0 µg/ml.

A relevant cytotoxic effect indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) occurred at 861.0 µg/mL (relative adjusted cloning efficiency I of 15.8 %) with metabolic activation. Moderate cytotoxic effect indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) occurred at 420.5 µg/mL in the absence of metabolic activation.

The mean mutant frequency obtained for the solvent controls in experiment I was 7.0 mutants per 106 cells in the absence of metabolic activation, and 8.0 mutants per 106 cells in the presence of metabolic activation. The values were well within the 95% confidence interval of the 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 7.3 up to 20.9 mutants per 106 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.

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 methacrylic acid did not induce gene mutations at the HPRT locus in V79 cells and is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-10-02 to 2020-10-14

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: R0004-66
Purity: 99.86%
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:

- Vehicle(s)/solvent(s) used: deionized water
Justification for choice of solvent/vehicle: The solvent was chosen because of its relative nontoxicity
for the bacteria.


- Justification for percentage of solvent in the final culture medium:

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 mis

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:

TEST ITEM PREPARATION: Since an acidic pH value of methacrylic acid in water was obtained in the stock solution, it was neutralised with NaOH 2M. The pH value after neutralization was 7 in both experiments.
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
First experiment: plate incorporation assay
Second experiment: preincubation assay

Evaluation criteria:

EVALUATION OF RESULTS
According to international guidelines a statistical evaluation of the results is recommended. However, 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 non-mutagenic 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 described enhancement factors or not.

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 specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

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 shifts in the genome of the strains tested.
Therefore, methacrylic acid 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 methacrylic acid ( 99.86 % 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 methacrylic acid 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 methacrylic acid 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 5000 μg/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 methacrylic acid 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

No genetic toxicity studies in vivo are available.

In vivo genetic toxicity studies are not required under REACH regulation as the required in vitro genetic toxicity studies (gene mutation in bacteria, mutagenicity test in vitro and cytogenetic test) in vitro are available which were conducted according to OECD guidelines and no genetic toxicity has been observed in any of the three studies (Table R.7.7–1 REACH information requirements for mutagenicity).

 

 

Additional information

Supporting information is available in an Ames test where only 4 strains have been tested (Haworth, 1983). Methacrylic acid was not mutagenic in Salmonella typhimurium strains TA1537, TA1538, TA98, TA100 with and without metabolic activation.

Justification for classification or non-classification

Methacrylic acid was negative in three reliable in vitro studies: a reverse gene mutation assays in bacteria (Ames test) acc. OED 471, a mammalian cell gene mutation assay (HPRT test) acc. OED 476 and an in vitro micronucleus test in human lymphocytes, OECD 487. All test are valid and sufficient for classification.

Therefore, methacrylic acid has not to be classified for its mutagenic potential according to CLP (1272/2008/EEC) and UN-GHS requirements.