2-methoxyethyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse gene mutation assay: negative (read-across from the close analgue ETMA)

Mammalian cell gene mutation assay: negative (read-across from the metabolite 2-methoxyethanol and the analogues ester methyl methacrylate)

In vitro chromosome aberration test: positive (read-across from the metabolites 2-methoxyethanol, and methacrylic acid and the analgues ester methyl methacrylate

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
• they are manufactured from similar or identical precursors under similar conditions
• they share structural similarities with common functional groups: methacrylate esters
• the metabolism pathway leads to comparable products (methacrylic acid and short chain alcohol).

Therefore, read-across from the existing (eco)toxicity studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see crossreference “Justification for read-across”

3. ANALOGUE APPROACH JUSTIFICATION
see crossreference “Justification for read-across”

4. DATA MATRIX
see crossreference “Justification for read-across”

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The substance did not induce mutant colonies over background. ETMA was tested up to limit concentrations of 5000 µg/plate.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances are both metabolites of the target substance and fast metabolism occurs.
Therefore, read-across from the existing toxicity studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached “Justification for read-across”

3. ANALOGUE APPROACH JUSTIFICATION
see attached “Justification for read-across”

4. DATA MATRIX
see attached “Justification for read-across”

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Target gene:

TK

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

weakly positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The substance is negative without S9 but weakly positive in the presence of induced rat liver S9.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances are both metabolites of the target substance and fast metabolism occurs.
Therefore, read-across from the existing toxicity studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached “Justification for read-across”

3. ANALOGUE APPROACH JUSTIFICATION
see attached “Justification for read-across”

4. DATA MATRIX
see attached “Justification for read-across”

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Sister chromatid exchange and chromosome aberrations in chinese hamster ovary cells were positive with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

negative (read-across from 2-methoxyethanol and methyl methacrylate)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances are both metabolites of the target substance and fast metabolism occurs.
Therefore, read-across from the existing toxicity studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached “Justification for read-across”

3. ANALOGUE APPROACH JUSTIFICATION
see attached “Justification for read-across”

4. DATA MATRIX
see attached “Justification for read-across”

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Species:

mouse

Strain:

CD-1

Sex:

male

Route of administration:

inhalation

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

negative

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances are both metabolites of the target substance and fast metabolism occurs.
Therefore, read-across from the existing toxicity studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached “Justification for read-across”

3. ANALOGUE APPROACH JUSTIFICATION
see attached “Justification for read-across”

4. DATA MATRIX
see attached “Justification for read-across”

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Species:

mouse

Strain:

CD-1

Sex:

male/female

Route of administration:

intraperitoneal

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

The intraperitoneal injection of the test substance at different concentrations (single doses) in male and female CD-1 mice did not induce polychromatic erythrocytes in the bone marrow of the animals. The in vivo micronucleus assay was therefore negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No experimental data on MTMA are available for the assessment of genetic toxicity. However, studies are available for the source substances metabolites 2-methoxyethanol, and methacrylic acid, and the analgues ester methyl methacrylate. A detailed justification for read-across is attached to IUCLID section 13.

 

Hypothesis for the analogue approach

The read across hypothesis relies on the observation that alkyl esters are rapidly hydrolysed by carboxylesterase enzymes within the body to release methacrylic acid (MAA) and free alcohol. Local effects, including genotoxicity and sensitisation, if they occur are likely to be due to electrophilic reactivity of the parent ester 2 -methoxyethyl methacrylate (MTMA). Due to the short half-life of the parent ester within the body systemic exposure to parent ester is extremely unlikely so the observed systemic toxicity profile is determined by the systemic toxicity profile of the primary metabolites Methacrylic acid (MAA) and 2-methoxyethanol.

 

This read-across hypothesis corresponds to scenario 1 – biotransformation to common compounds – of the read-across assessment framework) i.e. properties of the target substance are predicted to be quantitatively equal to those of the source substance. Namely, the metabolites Methacrylic acid and 2-methoxyethanol predict the toxicological properties of the parent compound MTMA.

 

Based on the available experimental data, including data from acute toxicity and genotoxicity studies, the read-across hypothesis is supported by close structural analogy and similar toxicological profile of the substances.

 

Toxicokinetics

AE 1.1 Formation of common (identical) compound(s)

The focus of this AE is on the scientific explanation and documentation on how the (bio)transformation from source and target substances to the common compound(s) occur. It will be shown that biotransformation from parent ester to primary metabolite occurs rapidly within the body and that the ensuing metabolism of these primary metabolites is well understood thereby providing a high confidence in the assertion that the metabolites alone influence systemic toxicity alone.

 

After oral or inhalation administration, methacrylate esters are expected to be rapidly absorbed via all routes and distributed. Dermal absorption of esters is extensive only with occlusion of the site. Heylings (2013) used a QSPeR model for whole human skin based on that described by Potts and Guy (1992) to predict the dermal penetration rate of a large number of methacrylate esters, including MTMA (Heylings, 2013). For MTMA a low rate of dermal penetration is predicted (1.366µg/cm²/h).

Toxicokinetics seem to be similar in man and experimental animals. MMA and other short chain alkyl-methacrylate esters are initially hydrolyzed by non-specific carboxylesterases to methacrylic acid and the structurally corresponding alcohol in several tissues, including but not limited to liver, olfactory epithelium, stratum corneum and blood. This has been shown for linear alkyl esters, several ether methacrylates, diesters as well as cycloalkyl and -aryl esters (Jones 2002, DOW 2013, McCarthy and Witz, 1997). Because of the structural similarity of MTMA to the other esters rapid hydrolysis is expected in the order of minutes.

Methacrylic acid (MAA) is subsequently cleared predominantly via the liver (valine pathway and the TCA (Tricarboxylic Acid) cycle).

 

The carboxylesterases are a group of non-specific enzymes that are widely distributed throughout the body and are known to show high activity within many tissues and organs, including the liver, blood, GI tract, nasal epithelium and skin. Those organs and tissues that play an important role and/or contribute substantially to the primary metabolism of the short-chain, volatile, alkyl-methacrylate esters are the tissues at the primary point of exposure, namely the nasal epithelia and the skin, and systemically, the liver and blood.

 

2-methoxyethanol is mainly metabolized to methoxyacetic acid and excreted via the urine (Mebus et al, 1992; Miller RR, 1987).

 

Alternative(minor) pathway: GSH Conjugation

Methacrylate esters can conjugate with glutathione (GSH) in vitro, although they show a low reactivity, since the addition of a nucleophile at the double bond is hindered by the alpha-methyl side-group (Cronin, 2012, Freidig et al. 1999). Hence, ester hydrolysis is considered to be the major metabolic pathway for alkyl-methacrylate esters, with GSH conjugation only playing a minor role in their metabolism, and then possibly only when very high tissue concentrations are achieved.

 

The fast hydrolysis observed for other Methacrylic acid esters is predicted to occur also for MTMA. Thus, following systemic exposure to MTMA the organisms will be mainly exposed to the metabolites Methacrylic acid and 2-methoxyethanol.

 

On this basis the systemic biological targets for the common compound(s)(AE 1.2) and the exposure of these systemic biological target(s) to the common compound(s) (AE 1.3) will be the same for MTMA as they are for the primary metabolites.

 

Furthermore, since carboxylesterases are widely distributed throughout the body and the half-life of the parent ester is very short the impact of parent compound (AE 1.4) is unlikely to be significant other than at the site of initial contact. Indeed, local hydrolysis at the site of contact is likely to be very rapid thereby minimising exposure to parent ester even at local targets. Since the source and target compounds are monoconstituents of high purity there are no impurities worthy of consideration. Finally, since the hydrolysis of the parent ester to Methacrylic acid and 2-methoxyethanol is equimolar and does not involve the formation of non-common compounds (AE 1.5) (including possible intermediates) their possible impact on the property under consideration does not have been considered.

 

Experimental data

No experimental data are available for the target substance MTMA. However, based on the proposed hypothesis read-across from the metabolites of MTMA methacrylic acid and 2-methoxyethanol is proposed. Additionally, Methyl methacrylate is used as a surrogate for the target substance MTMA to address the reactivity of the ester. As the alkyl chain does not hinder the nucleophilic attack, similar Michael Addition rates are predicted for all Methyl methacrylate and MTMA.

 

In vitro studies

Ames test

In a bacterial reverse mutation assay using Salmonella strains TA97a, TA98, TA100 and TA102 2-methoxyethanol tested up to 10 mg/plate did not produce evidence of mutation with or without exogenous metabolic activation by rat liver extracts.

In a bacterial reverse mutation assay, Salmonella strains TA97, TA98, TA100 and TA1535 were tested using 2-methoxyethanol. Doses up to 10 mg/plate did not produce evidence of mutation with or without exogenous metabolic activation by rat or hamster liver extracts.

 

The metabolites of MTMA methacrylic acid and 2-methoxyethanol, as well as methyl methacrylate and ETMA were not mutagenic in the Ames test, as well. This result further supports the read-across for the other genotoxicity studies.

 

Mammalian cell gene mutation assays

In an in vitro gene mutation study of Chinese hamster ovary K1-BH4 clones, 2-methoxyethanol tested at concentrations up to 887 mM did not induce mutation at cytotoxic concentrations with or without exogenous metabolic activation.

 

Methyl methacrylate was weakly positive a mouse lymphoma (TK) assay in presence and negative in absence of S-9 mix. Without S-9 mix doses up to 100 nl/ml were tested, higher doses led to total toxicity. With S-9 mix methyl methacrylate was positive in the dose range 100 nl/ml to 250 nl/ml, however, clear effects were observed only at doses with high toxicity below 20% relative growth.

In a HPRT assay with and without metabolic activation, methyl methacrylate was weakly positive in V79 cells (Schweikl et al. 1998). Without metabolic activation, the mutant frequencies in the tested concentrations of 10 and 20 mM were 6 and 16 per million surviving cells, while in the control treatment 3 mutants per 10e6 surviving cells were observed. The cell numbers of the low and high dose treatment were 71 and 49% of the control, respectively. Data from the trial with metabolic activation were not reported in detail.

In a further lymphoma assay which was only run without S-9 mix, weak effects were obtained for doses producing high toxicity (Moore et al., 1988). According to the authors, 2000 μg/ml was positive in both experiments (92 and 98 mutants per 106 survivors vs. 54 and 68 in the negative controls), relative survival was approximately 20% and 30%; in one experiment the highest dose of 499 μg/ml induced 143 mutants at 10% relative survival; in the second experiment the highest dose of 3100 μg/ml induced 220 mutants with 11% relative survival. The vast majority of induced colonies were small ones (indicating that the genetic effect was derived from clastogenicity and not from gene mutations). 

 

Based on read-across, the target substance MTMA is considered to be not mutagenic but likely clastogenic in an in vitro mammalian cell gene mutation test.

 

Chromosome aberration tests

A cytogenetic study was conducted to assess the genotoxic potential of 2-methoxyethanol using CHO cell lines and human lymphocytes. The results showed that human lymphocytes treated with 10-30 mM MALD (methoxyacetaldehyde; metabolite of 2-methoxyethanol) for 1 h or 0.05-0.5 mM MALD for 24 h induced significant dose-dependent increase of sister-chromatid exchanges (SCE) (p < 0.05). It also induced significant dose-dependent increase (p < 0.05) of chromosome aberrations in human lymphocytes (10-40 mM treated for 1 h, or 0.05-2.5 mM for 24 h) and in both CHO cell lines (1.25-20 mM for 3 h). Treatment of these cells with the parent compound, 2-methoxyethanol did not induce chromosome aberrations nor SCE unless very high doses of the chemical were used. In conclusion, these results indicate that MALD is clastogenic to different cell types.

 

Methyl methacrylate showed the potential for induction of mutagenic effects, esp. clastogenicity; however, this potential seems to be limited to high doses with strong toxic effects.

 

Methyl methacrylate is used as a surrogate for the target substance MTMA to address the reactivity of the ester. As the alkyl chain does not hinder the nucleophilic attack, similar Michael Addition rates are predicted for all Methyl methacrylate and MTMA.

Based on read-across, the target substance MTMA should be considered to have clastogenic potential in vitro.

 

In vivo studies

Due to ambiguous in vitro results, additional in vivo data are provided for 2-methoxyethanol and Methyl methacrylate.

In a chromosome aberration study in mice, a single treatment of 2-methoxyethanol did not induce chromosomal aberrations in the bone marrow of mice treated by gavage (at doses up to 1900 mg/kg) or by intravenous injection.

In a dominant lethal study in mice, no effects were observed on total implants or early deaths following a single gavage treatment with 2-Methoxyethanol at doses up to 1500 mg/kg.

Two chromosomal aberration tests were conducted by Anderson et al. (1976, 1979) investigating the effect of inhalation exposure to methyl methacrylate. A clear conclusion could not be drawn from these studies. Hachiya et al. (1982) reported on a negative bone marrow micronucleus assay with mice. Overall, it may be concluded from mammalian cell culture assays that the metabolites 2-methoxyethanol and Methacrylic acid have the potential to be high-toxicity clastogens (i. e. induction of chromosomal aberrations is bound to highly toxic doses). Therefore, the in vivo data are used to finally assess the genotoxic potential and it is concluded that neither 2-methoxyethanol nor Methacrylic acid are mutagenic in vivo.

 

Based on the available data on the source substances 2-methoxyethanol, methacrylic acid and methyl methacrylate, MTMA is not mutagenic in bacteria and in mammalian cells. Moreover, while chromosomal aberrations have been detected in vitro, no clastogenicity was observed in vivo.

 

There are no data gaps for this endpoint. There is no reason to believe that the negative results would not be relevant to humans.  

Justification for classification or non-classification

Based on the available data, MTMA does not need to be classified for mutagenicity according to the criteria given in regulation (EC) 1272/2008. Thus, no labelling is required.