2-ethoxyethyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse gene mutation assay: negative

Mammalian cell gene mutation assay: negative (read-across from 2-ethoxyethanol and methyl methacrylate)

In vitro chromosome aberration test: positive (read-across from 2 -ethoxyethanol, methacrylic acid and methyl methacrylate)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Remarks:

used to justify read-across for other endpoints

Adequacy of study:

supporting study

Study period:

1994-12-20 to 1995-01-13

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:

revised draft document of May 1994

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 July 1992

Deviations:

no

GLP compliance:

yes

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

Test concentrations with justification for top dose:

preliminary toxicity test: 0, 10, 100, 500, 1000, 2500 and 5000 µg/plate
mutagenicity tests: 0, 312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

other: 2-anthramine, Danthron

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); second test with metabolic activation: preincubation

DURATION
- Preincubation period: 60 min (second test with metabolic activation)
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: background bacterial lawn

Evaluation criteria:

The following criteria were used as an aid for determining a positive response:
- a reproducible and significant dose relationship
and/or
- a reproducible and significant increase (i.e. a doubling in the number of revertants for at least one of the tested strains when compared to that of the controls) for at least one of the doses

A test substance is considered as non-mutagenic in this test system if the above two criteria are not fully met.
Biological and statistical significances were considered during the evaluation.

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

COMPARISON WITH HISTORICAL CONTROL DATA:
controls were within historical range

ADDITIONAL INFORMATION ON CYTOTOXICITY:
cytotoxicity was observed in strain TA100 at concentrations of 1250 µg/plate and higher without metabolic activation and in strain TA1537 at a concentration of 5000 µg/plate without metabolic activation, but only in one experiment; in the preincubation test cytotoxicity was
observed in strains TA1535 and TA100 at a concentration of 5000 μg/plate with metabolic activation;
no cytotoxicity observed up to limit dose in all other strains

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, revised draft document of May 1994, S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains were exposed to ETMA (99.94% a.i.) in DMSO at concentrations of 0, 312.5, 625, 1250, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The test was performed as plate incorporation assay; the second experiment with metabolic activation was performed as pre-incubation test with 60 minutes pre-incubation. ETMA was tested up to limit concentrations (5000 µg/plate).

Cytotoxicity was observed in strain TA100 at concentrations of 1250 µg/plate and higher without metabolic activation and in strain TA1537 at a concentration of 5000 µg/plate without metabolic activation, but only in one experiment. In the preincubation test cytotoxicity was observed in strains TA1535 and TA100 at a concentration of 5000 µg/plate with metabolic activation. No cytotoxicity observed up to limit dose in all other strains. The positive controls induced the appropriate responses in the corresponding strains. 

There was no evidence of induced mutant colonies over background. 

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 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-ethoxyethanol 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: 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

A bacterial reverse gene mutation assay is available for ETMA. For the target substance ETMA, an Ames test is available. Further studies (mammalian cell gene mutation assay, cytogenicity assays, in vivo tests on clastogenicity) are not available for the target substance ETMA. However, based on the proposed hypothesis read across to the metabolites methacrylic acid and 2-ethoxyethanol is proposed. A detailed justification for read-across is attached to IUCLID section 13. Due to the expected short half-life, no significant exposure to the parent compound ETMA is expected, but only exposure to the metabolites. To address any residual concern for nucleophilic reactivity of the parent ester, read across to Methyl methacrylate, which is the methyl ester of methacrylic acid is proposed since it has a comprehensive dataset including genotoxicity data.

 

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-Ethoxyethyl methacrylate (ETMA). 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-Ethoxyethanol.

 

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

 

The read across hypothesis relies on two modes of action:

1)   Local effects mediated by electrophilic reactivity of the parent ester. Hydrolysis occurs at the site of this ester bond so is a detoxification process with respect to nucleophilic action, and

2)   Systemic effects mediated by the metabolites of hydrolysis of the alkyl ester bond by carboxylesterase enzymes within the body, namely, methacrylic acid (MAA) and free alcohol.

 

Structural similarities:

ETMA is an ester of Methacrylic acid and 2-Ethoxyethanol. It is produced by esterification of Methacrylic acid and 2-Ethoxyethanol. Hydrolysis of the ester bond of ETMA produces Methacrylic acid (2-Methylprop-2-enoic acid) and 2-Ethoxyethanol on an equimolar basis. Therefore, there is close structural similarity of the source and target substances.

 

Structural differences:

The only structural difference between the source and target substances is the absence of the ester bond. There is some uncertainty as to the extent to which the electrophilic reactivity of the parent ester could contribute to systemic toxicity. The short half-life of the parent ester within the body means that the ester bond and any associated electrophilic reactivity of the parent ester is short lived and unlikely to be present to any appreciable extent within the systemic circulation. It is therefore unlikely to contribute to the observed systemic toxicity profile.

 

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 ETMA (Heylings, 2013). For ETMA a low rate of dermal penetration is predicted (8.261 µg/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 ETMA 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-ethoxyethanol is mainly metabolized toethoxyacetic acid and ethylene glycol. Exhalation of unmetabolized glycol ether is a minor route of elimination (Medinsky MA et al., 1990).

 

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 ETMA. Thus, following systemic exposure to ETMA the organisms will be mainly exposed to the metabolites Methacrylic acid and 2-Ethoxyethanol.

 

On this basis the systemicbiological 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 ETMA 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 Methacrylicacid and 2-Ethoxyethanol is equimolar and does not involve the formation ofnon-common compounds (AE 1.5) (including possible intermediates) their possible impact on the property under consideration does not have been considered.

 

Experimental data

In vitro studies

Ames test

In a reverse gene mutation assay in bacteria (S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102) ETMA was tested up to limit concentration of 5000 µg/plate in the presence and absence of mammalian metabolic activation. There was no evidence of induced mutant colonies over background. Thus, ETMA was not mutagenic in the Ames test.

The metabolites methacrylic acid and 2-ethoxyethanol, as well as methyl methacrylate 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 a mammalian cell gene mutation assay in mouse lymphoma L5178Y cells (TK assay) 2-ethoxyethanol was negative without S9 but was judged to be weakly positive in two of three trials conducted in the presence of induced rat liver S9.

 

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 ETMA is considered to be not mutagenic but likely clastogenic in an in vitro mammalian cell gene mutation test.

 

Chromosome aberration tests

2-ethoxyethanol showed a positive response in the sister chromatid exchange assay and in the chromosome aberration assay of cloned chinese hamster ovary cells (CGO-W-B1). Aberrations were only induced without the metabolic activation (S9 liver homogenate from male Sprague-Dawley rats), whereas SCEs were observed with and without the metabolic activation.

 

The intraperitoneal injection of 2-ethoxyethanol 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.

 

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 ETMA 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 ETMA.

Based on read-across, the target substance ETMA 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-Ethoxyethanol and Methyl methacrylate. 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-Ethoxyethanol 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-ethoxyethanol nor Methacrylic acid are mutagenic in vivo.

Based on the available data on ETMA and the source substances 2-ethoxyethanol, methacrylic acid and methyl methacrylate, ETMA 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, ETMA does not need to be classified for mutagenicity according to the criteria given in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC. Thus, no labelling is required.