2-methoxyethyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Repr. 1 B

NOAEL (fertility effects) = 20 mg/kg bw/d (read-across from metabolite 2 -methoxyethanol)

Link to relevant study records

Reference

Endpoint:

fertility, other

Remarks:

subchronic toxicity study with additional assessment of reproduction toxicity

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

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: Adverse effects on sperm and male reproductive organs

Critical effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: number of live pups per litter and proportion of pups born alive decreased significantly

Critical effects observed:

not specified

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Reduced number of live pups per litter

Critical effects observed:

not specified

Reproductive effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No experimental data on MTMA are available for the assessment of fertility effects. However, studies are available for the metabolites 2-methoxyethanol and methacrylic acid. 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.

 

Data availability

No experimental data are available for the target substance MTMA. However, based on the proposed hypothesis read-across from the metabolites methacrylic acid and 2-methoxyethanol is proposed.

 

"Reproductive Assessment by Continuous Breeding" (RACB) protocol was originally designed using mice as the test species. The purpose of the present study was to develop a RACB protocol in CD Sprague-Dawley rats. 2-methoxyethanol, a known reproductive toxicant, was used as the test article.

 

Reproductive Toxicity of 2-methoxyethanol in Sprague-Dawley Rats, Litter Two:

Dose levels selected were 0.01, 0.03, and 0.10%, administered via drinking water. In a modification of the standard protocol, male and female rats ~20 pairs per treatment group, 40 pairs of control animals) were cohabited for approximately 6 weeks, separated to allow delivery, nursing and weaning of the second litter, then re- cohabited for approximately nine more weeks. The weaned second litter was used for F1 reproductive testing. The control and 0.03% F0 pairs were also utilized for a crossover mating trial to determine the affected sex.

Only one litter was born in the 0.10% dose group, and no pups were available for F1 testing. At 0.03% level of EGMME, number of live pups per litter and proportion of pups born alive decreased significantly, both in F0 and F1 testing. In the crossover mating, proportion of pups born alive decreased significantly in the 0.03% male X control female group. Declining control F0 fertility and productivity noted in this study indicate that the standard RACB design (weaning last rather than second litter) is better suited to use with rats.

Reproductive Toxicity of 2-methoxyethanol in Sprague-Dawley Rats, Litter Five:

Dose levels selected were 0.006, 0.012, and 0.024%, administered via drinking water. Male and female rats (20 pairs per treatment group, 40 pairs of control animals) were continuously exposed for a 7-day pre-cohabitation period and 112-day cohabitation (Task 2).

While there was no decrease in average litter size with increased dose, the number of live male pups per litter and the total number of pups per litter were decreased in the 0.024% group. Both absolute and adjusted live pup weight were increased in all EGMME groups, but this was not dose-related. During the cross-over mating to determine the affected sex, there were fewer live male pups born to the 0.024% male X control female pairs. During the mating trial for the second generation, fewer male and total pups were delivered in the high-dose group, and both absolute and adjusted pup weight were increased in the middle and high dose groups. Low control fertility (63% fertile) during the cross-over mating is a cause for concern, however suggested protocol changes should alleviate some of this problem.

The NOAEL of 2-methoxyethanol was 0.01% in drinking water (11 mg/kg bw/d) in this study.

 

2-Methoxyethanol when administered to male rabbits for 12 weeks via drinking water produced a marked inhibition of normal spermatogenesis. The effect is marked with a NOAEL of 12.5 mg/kg and a LOAEL of 25 mg/kg. The effect is very specific with no clear effects on sperm morphology and no effect on the ability of the sperm that remain to fertilize a female rabbit. No other adverse effects that could be attributed to treatment were observed at the LOAEL.

 

In a 90 d inhalation study with methacrylic acid histopathology of the sexual organs and additional fertility parameters were investigated. Up to 350 ppm (1232 mg/m³) no effects were seen on gross pathology including organ weights, histopathology, sperm motility or sperm morphology.

 

The metabolite mainly determining reproductive toxicity is 2-methoxyethanol. Thus, the overall NOAEL for the endpoint toxicity to reproduction (fertility effects) is based on the NOAEL of 11 mg/kg bw/d obtained in a study according to the Continuous breeding protocol. The NOAEL of MTMA for effects to fertility is 20 mg/kg bw/d (extrapolated based on molecular weight).

 

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

Effects on developmental toxicity

Description of key information

Repr. 1 B

NOAEL (embryotoxicity) = 20 mg/kg bw/d (read-across from the metabolite 2-methoxyethanol)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

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: supporting information

Species:

rat

Remarks on result:

not measured/tested

Abnormalities:

not examined

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: number of live pups per litter and proportion of pups born alive decreased significantly

Abnormalities:

not examined

Developmental effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No experimental data on MTMA are available for the assessment of developmental toxicity. However, studies are available for the metabolites 2-methoxyethanol and methacrylic acid. 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.

 

Data availability

No experimental data are available for the target substance MTMA. However, based on the proposed hypothesis read-across from the metabolites methacrylic acid and 2-methoxyethanol is proposed.

 

"Reproductive Assessment by Continuous Breeding" (RACB) protocol was originally designed using mice as the test species. The purpose of the present study was to develop a RACB protocol in CD Sprague-Dawley rats. 2-methoxyethanol, a known reproductive toxicant, was used as the test article.

 

Reproductive Toxicity of 2-methoxyethanol in Sprague-Dawley Rats, Litter Two:

Dose levels selected were 0.01, 0.03, and 0.10%, administered via drinking water. In a modification of the standard protocol, male and female rats ~20 pairs per treatment group, 40 pairs of control animals) were cohabited for approximately 6 weeks, separated to allow delivery, nursing and weaning of the second litter, then re- cohabited for approximately nine more weeks. The weaned second litter was used for F1 reproductive testing. The control and 0.03% F0 pairs were also utilized for a crossover mating trial to determine the affected sex.

Only one litter was born in the 0.10% dose group, and no pups were available for F1 testing. At 0.03% level of EGMME, number of live pups per litter and proportion of pups born alive decreased significantly, both in F0 and F1 testing. In the crossover mating, proportion of pups born alive decreased significantly in the 0.03% male X control female group. Declining control F0 fertility and productivity noted in this study indicate that the standard RACB design (weaning last rather than second litter) is better suited to use with rats.

Reproductive Toxicity of 2-methoxyethanol in Sprague-Dawley Rats, Litter Five:

Dose levels selected were 0.006, 0.012, and 0.024%, administered via drinking water. Male and female rats (20 pairs per treatment group, 40 pairs of control animals) were continuously exposed for a 7-day pre-cohabitation period and 112-day cohabitation (Task 2).

While there was no decrease in average litter size with increased dose, the number of live male pups per litter and the total number of pups per litter were decreased in the 0.024% group. Both absolute and adjusted live pup weight were increased in all EGMME groups, but this was not dose-related. During the cross-over mating to determine the affected sex, there were fewer live male pups born to the 0.024% male X control female pairs. During the mating trial for the second generation, fewer male and total pups were delivered in the high-dose group, and both absolute and adjusted pup weight were increased in the middle and high dose groups. Low control fertility (63% fertile) during the cross-over mating is a cause for concern, however suggested protocol changes should alleviate some of this problem.

The NOAEL of 2-methoxyethanol was 0.01% in drinking water (11 mg/kg bw/d) in this study.

 

No significant increase in embryo/fetal lethality or fetal malformations were observed after exposure to methacrylic acid. While maternal toxicity was observed, methacrylic acid caused no evidence of developmental toxicity up to 300 ppm.

 

The metabolite mainly determining developmental toxicity is 2-methoxyethanol. Thus, the overall NOAEL for the endpoint toxicity to reproduction (developmental toxicity) is based on the NOAEL of 11 mg/kg bw/d obtained in a study according to the Continuous breeding protocol. The NOAEL of MTMA for effects to fertility is 20 mg/kg bw/d (extrapolated based on molecular weight).

 

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

Justification for classification or non-classification

Based on the available data, MTMA is classified as Repr. 1B (H360FD) according to the criteria given in regulation (EC) 1272/2008