Ethyl methacrylate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

A series of in vitro and PBPK models were used to determine and predict  the skin absorption and metabolism of a series of methacrylate monomers.  Initial studies were conducted using the rat epidermal membrane model.

GLP compliance:

not specified

Species:

other: rat and human

Strain:

Fischer 344

Sex:

not specified

Details on study design:

The rat whole-skin studies were conducted by collecting skin samples from  the flanks of Fischer 344 rats and placing a prepared sample in a  diffusion cell chamber. The receptor fluid was physiological saline. A  sample (100 µl/cm2) of methacrylic acid (MAA), methyl  methacrylate (MMA),  n-butyl methacrylate (nBMA), octyl methacrylate (OMA) or lauryl  methacrylate (LMA) was applied to the skin sample for up to 48 hours. The  amount of parent ester and MAA were measured in the receptor fluid. MAA  was also tested in a sample of human skin.

Toxicokinetic parameters:

half-life 1st:

Toxicokinetic parameters:

half-life 2nd:

Toxicokinetic parameters:

half-life 3rd:

The results of the whole-skin penetration studies and the model predictions for

other methacrylate esters are presented in the table.

Table: Summary of peak rates of absorption of MAA and alkyl-methacrylate  esters 

through whole rat skin:

 Ester      Ester    MAA      Period    % Absorp. Dose/(hours)                             

Peak        Peak     Peak    Applied length of exposure
 -----------------------------------------------------------
MAA                     4584     5 - 8        70%/24 hr  
MMA         360          108     2.5 - 24     11.3%/24 hr
EMA                      190**                              
i-BMA                     56**                              
n-BMA                     41     2 - 10       0.4%/10 hr
HMA                       20**                              
2EHMA                      9**                              
OMA                       10     8 - 24       0.24%/24 hr
LMA                       12     8 - 24       0.26%/24 hr
-----------------------------------------------------------

Ester Peak = rate of appearance of the parent ester (µg/cm2/hr) 
MAA Peak = rate of appearance of the hydrolysis product, MAA (µg/cm2/hr)
Period Peak Absorp. = Time (hours) after application for peak absorption
% Applied Dose = total % absorbed
** Predicted rates of MAA from model estimates.

Conclusions:

Due to the slower absorption, methacrylate esters of molecular weight equal to or greater than EMA (114 a.m.u) are metabolized during penetration of the skin and are not expected to enter the circulation as the parent ester.

Executive summary:

In a valid scientific study, due to the slower absorption, methacrylate esters of molecular weight equal to or greater than EMA (114 a.m.u) are metabolized during penetration of the skin and are not expected to enter the circulation as the parent ester.

Reference 2

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

A series of in vitro and in vivo studies with a series of methacrylates were used to develop PBPK models  that accurately predict the metabolism  and fate of these monomers.

GLP compliance:

not specified

Species:

rat

Strain:

not specified

Sex:

not specified

Toxicokinetic parameters:

half-life 1st:

Toxicokinetic parameters:

half-life 2nd:

Toxicokinetic parameters:

half-life 3rd:

Metabolites identified:

not measured

The studies confirmed that alkyl-methacrylate esters are rapidly hydrolyzed by ubiquitous carboxylesterases. First pass (local) hydrolysis of the parent ester has been shown to be significant  for all routes of exposure. In vivo measurements of rat liver indicated  this organ has the greatest esterase activity.  Similar measurements for  

skin microsomes indicated approximately 20-fold lower activity than for liver. However, this activity was substantial and capable of almost complete first-pass metabolism of the alkyl-methacrylates. For example,  no parent ester penetrated whole rat skin in vitro for n-butyl  methacrylate, octyl methacrylate or lauryl methacrylate tested  experimentally with only methacrylic acid identified in the receiving  fluid. In addition, model predictions indicate that esters of ethyl  methacrylate or larger would be completely hydrolyzed before entering the circulation via skin absorption. This  pattern is consistent with a lower rate of absorption for these esters  such that the rate is within the metabolic capacity of the skin. Parent  ester also was hydrolyzed by S9 fractions from nasal epithelium and was predicted to be effectively hydrolyzed following inhalation exposure. These studies showed that any systemically absorbed parent ester will be  effectively removed during the first pass through the liver (CL as % LBF,  see table). In addition, removal of methacrylic acid from the blood also  occurs rapidly (T50%; see table).  

Table:
Rate constants for ester hydrolysis by rat-liver microsomes and predicted  systemic fate kinetics for methacrylates following i.v. administration:
 Ester    Vmax       Km        CL    T50%    Cmax    Tmax
----------------------------------------------------------
EMA       699.2     106.2    99.5%    4.5    12.0     1.8
----------------------------------------------------------
Vmax (nM/min/mg) and Km (µM) from rat-liver microsome (100 µg/ml)determinations;  
CL = clearance as % removed from liver blood flow, T50% = Body  elimination 

time (min) for 50% parent ester, Cmax = maximum concentration  (mg/L) of MAA 

in blood, Tmax = time (min) to peak MAA concentration in  blood from model predictions.

Conclusions:

In conclusion, the in vivo and in vitro investigations as well as the PBPK models developed from the data showed that alkyl-methacrylate esters are rapidly absorbed and are hydrolyzed at exceptionally high rates to methacrylic acid by high capacity, ubiquitous carboxylesterases. Further, the removal of the hydrolysis product, methacrylic acid, also is very rapid (minutes).

Executive summary:

In a valid sientific study, the data showed that alkyl-methacrylate esters are rapidly absorbed and are hydrolyzed at exceptionally high rates to methacrylic acid by high capacity, ubiquitous carboxylesterases. Further, the removal of the hydrolysis product, methacrylic acid, also is very rapid (minutes).

Reference 3

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

A physiologically based pharmacokinetic model has been formulated to predict the pharmacokinetics and systemic disposition of alkylmethacrylate esters in rats and humans.

GLP compliance:

not specified

Species:

other: rat and human

Strain:

other: Wistar/Fischer F344/ not applicable

Sex:

male

Details on test animals or test system and environmental conditions:

Epidermal membrane absorption studies
Skin was used from male rats of the Wistar-derived strain (supplied by Charles River UK Ltd, Margate, Kent, UK.) aged 28 days ± 2 days

Whole skin absorption studies
Skin was taken from male Fisher F344 (supplied by Harlan Olac) rats weighing between 200 and 250 g.

Human epidermal membrane absorption studies
Extraneous tissue was removed from human abdominal whole skin samples obtained post mortem in accordance with local ethical guidelines

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Duration of exposure:

up to 48 h

Doses:

100 µL/cm2

No. of animals per group:

3 (human: 2)

Dose:

100 µL/cm2

Parameter:

percentage

Absorption:

ca. 55 %

Remarks on result:

other: 16 h

Remarks:

rat epidermis; linear absorption for 8 h

Dose:

100 µL/m2

Parameter:

percentage

Absorption:

ca. 10 %

Remarks on result:

other: 4-24 h

Remarks:

human epidermis; linear absorption for >= 24 h

Rat epidermis

The fastest rate of absorption of MMA through rat epidermal membrane was recorded as being 5688 μg*cm-2*hr-1 and this occurred between 2 and 8 hrs following application of the chemical. The rate slowed considerably after 8 hours, falling to virtually zero by 16 hours. Nearly half (45.5%) of the donor reservoir had been depleted by 8 hours with 55% of the chemical appearing in the receptor chamber by 16 hours. The rate plateaus after eight hours, which is indicative of the donor reservoir being depleted.

 

Human epidermis

Methyl methacrylate absorbed at a peak rate of 453 μg*cm-2*hr-1, between 4 and 24 hrs, with 10.2% of the applied dose having been absorbed during this time. There appears to be a lag time with the rate of absorption between 0-4 hrs calculated to be 259 μg cm-2 hr-1. The rates of absorption through human epidermis are considerably slower than those measured for MMA through rat epidermis.

 

Whole rat skin

Of the alkyl-methacrylate esters whose rate of absorption through whole rat skin was investigated, methyl methacrylate is the most rapidly absorbed chemical. Carboxylesterases present in the viable tissue mediate the hydrolysis of these esters, producing the acid metabolite, together with the structurally corresponding alcohol. In contrast to the larger esters, MMA is not completely hydrolysed during the absorption process; this is substantiated by the appearance of both the parent ester and the metabolite MAA in the receptor fluid. Appearance of both chemicals in the receptor fluid can be explained by MMA possessing a rate of absorption that is higher than the rate with which it is hydrolysed.

The peak rate of appearance of MMA, which occurred between 2.5-24 hrs was calculated to be 360 μg*cm-2*hr-1. This compares to a peak rate of appearance for the metabolite MAA, which occurred between 4-24 hrs and was calculated as 108 μg cm-2 hr-1. Of the original dose applied to the whole skin, 8.7% appeared as MMA in the receptor chamber, while 2.6% appeared as MAA. Therefore in total, 11.3% of the ester was depleted from the donor reservoir.

---

The results of the whole-skin penetration studies and the model predictions for 

other methacrylate esters are  presented in the table.

Summary of the peak rates of absorption of MAA & alkyl-methacrylate esters through whole rat and human skin

Substance	Molecular volume	Rat whole rat				Human whole skin
		Peak rate of appearance (µg*cm-2*h-1)+- SEM		Period of peak absorption rate (hours)	% age of applied dose absorbed over x hours	Rate of absorption of ester/MAA (μg*cm-2 *hr-1)
		Ester	MAA
MAA	78.96		4584+-344	5-8	70%/24	327
MMA	93.1978	360+-20.9	108+-4.59	2.5-24	11.3%/24	33.4**
EMA	107.436		190**			13.6**
iBMA	135.646		56**			4**
nBMA	135.856		40+-9.4	2-10	0.4%/10	2.9**
6HMA	164.277		20**			1.4**
2EHMA	191.66		9**			0.6**
OMA	192.696		10.3+-0.65	8-24	0.24%/24	0.7**
12LMA	249.536		11.8+-2.11	8-24	0.26%/24	0.8**

The values in normal type were obtained experimentally, whilst those in italics are predicted values.

** Values are predicted rates of appearance of total chemical including parent ester and metabolite

Conclusions:

The fastest rate of absorption of MMA through rat epidermal membrane was recorded as being 5688 μg /cm²/hr and this occurred between 2 and 8 hrs following application of the chemical. Absorption through human epidermal membrane was slower with a peak rate of 453 μg/cm²/hr, between 4 and 24 hrs, with 10.2% of the applied dose having been absorbed during this time. There appears to be a lag time with the rate of absorption between 0-4 hrs calculated to be 259 μg /cm²/hr.

Penetration of alkyl methacrylate through whole rat skin was slower than through separated epidermal membrane. MMA was the most rapidly absorbed chemical of the alkyl methacrylates studied. Carboxylesterases present in the viable tissue mediate the hydrolysis of these esters, producing the acid metabolite, together with the structurally corresponding alcohol. In contrast to the larger esters, MMA is not completely hydrolysed during the absorption process; this is substantiated by the appearance of both the parent ester and the metabolite MAA in the receptor fluid. Appearance of both chemicals in the receptor fluid can be explained by MMA possessing a rate of absorption that is higher than the rate with which it is hydrolysed.
The peak rate of appearance of MMA, which occurred between 2.5-24 hrs was calculated to be 360 μg /cm²/hr. This compares to a peak rate of appearance for the metabolite MAA, which occurred between 4-24 hrs and was calculated as 108 μg /cm²/hr. Of the original dose applied to the whole skin, 8.7% appeared as MMA in the receptor chamber, while 2.6% appeared as MAA. Therefore in total, 11.3% of the ester was depleted from the donor reservoir.

Executive summary:

The in vivo and in vitro investigations as well as the PBPK models developed from the data showed that alkyl-methacrylate esters are rapidly absorbed and are hydrolyzed at exceptionally high rates to methacrylic acid by high capacity, ubiquitous carboxylesterases. Further, the removal of the hydrolysis product, methacrylic acid, also is very rapid (minutes).

Description of key information

For EMA, there are in vitro toxicokinetic data, in vitro/ in silico data on dermal absorption and in silico data on GSH reactivity available.

In addition, there are extensive data available for the methyl ester (MMA) and this has been reviewed in the EU Risk Assessment (EuRA) (2002). Sufficient data is available to confirm applicability of this data across all members of the category and this has been reviewed in the OECD SIAR (2009). Data on MAA, the common metabolite, has been reviewed in the EU Risk Assessment (2002).

Key value for chemical safety assessment

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100

Additional information

Category assessment according to ECHA’s Read Across Assessment Framework (RAAF)

This endpoint contains comprehensive data for the rationale of this category approach, namely the chemical reactivity of the esters (see subchapter “Conjugation”) and the common primary metabolic pathway (see subchapter “Metabolism”; see chapter1.1of the Category Document). These aspects can be either qualitatively categorized as “same type of effect” (i.e. scenario 4 or 6 according to RAAF; chemical reactivity of the category members) or as “(Bio) transformation to common compound(s)” (i.e. scenario 3 or 5 according to RAAF; common primary metabolic pathway; see chapter1.2of the Category Document). Relevant common and specific assessment elements of these scenarios are listed inTable1of the Category Document.

Moreover, these data serves as basis for the category assessment in many higher tier endpoint in this category document.The endpoint specific “scientific assessment option” of the read across is “acceptable with high confidence” for the all category substances.

 

Data availability

There are extensive data available for the C1 methyl ester (MMA) and this has been reviewed in the EU Risk Assessment (2002). Sufficient data is available to confirm applicability of this data across all members of the C1-C8 methacrylate category and, in the case of C2 to C8 esters, this has been reviewed in the OECD SIAR (2009). Data on MAA, the common acid metabolite, has been reviewed in the EU Risk Assessment (2002) while data on the corresponding alcohol metabolites has been reviewed by the OECD SIAR process between 2001 and 2005 (n-butyl alcohol, 2001; ethanol, methanol and isobutanol, 2004; 2-ethyl hexanol, 2005). The following text relies on these reviews.

 

Absorption - general

All members of the category can be absorbed following ingestion with the shorter esters being absorbed more rapidly through the skin than the longer esters. The volatile esters in the category are efficiently scrubbed from the inhaled air in the upper respiratory tract after inhalation.

MMA was reviewed under the EU Risk Assessment Program while the other esters were addressed under the OECD SIDS program, but this did not address absorption. The EU Risk Assessment on MMA concluded that; “after oral or inhalation administration, methyl methacrylate is rapidly absorbed and distributed. In vitroskin absorption studies in human skin indicate that methyl methacrylate can be absorbed through human skin, absorption being enhanced under occluded conditions. However, only a very small amount of the applied dose (0.56%) penetrated the skin under unoccluded conditions (presumably due to evaporation of the ester from the skin surface (CEFIC, 1993)). After inhalation exposure to rats 10 to 20% of the substance is deposited in the upper respiratory tract where it is metabolized (by non-specific esterases to the acid, MAA (Morris, 1992)).”

Absorption - dermal

Jones (2002) studied the permeability of separated rat and human skin to alkyl methacrylate esters as part of a PhD thesis on development of a PBPK model to predict the pharmacokinetics and toxicity of methacrylate esters. Data on MMA, nBMA and 2-EHMA indicate that rat skin is more permeable to the absorption of these esters than human skin and there is a steep decline in the rate of penetration across the category from MMA to the larger ester, 2-EHMA. The rate of penetration of EMA was predicted to be between that of MMA and nBMA and that of iBMA comparable to nBMA.

Table: Summary of the results for the peak rates of absorption of alkyl methacrylate esters through rat & human epidermis(adapted from Jones, 2002)

Sub-stance	Rat epidermis	Rat epidermis	Rat epidermis	Human epidermis	Human epidermis	Human epidermis
	Peak rate of absorption (μg/cm²/hr) ±SEM	Period of peak absorption rate (hours)	% age of applied dose absorbed over x hours	Peak rate of absorption (μg/cm²/hr) ±SEM	Period of peak absorption rate (hours)	% age of applied dose absorbed over x hours
MMA	5888±223	2-8	46% / 16h	453±44.5	4-24	10% / 24h
EMA	4421	-	-	253	-	-
i-BMA	1418	-	-	80	-	-
n-BMA	1540±69	0-6	18% / 24h	76.7±9.8	0-24	2% / 24h
HMA	147	-	-	25	-	-
2EHMA	234±4.8	0-30	7.8% / 30h	22.7.7±3.7	3-24	0.6% / 24h
OMA	159±15	0-24	-	7.8	-	-

 Key: The values in normal type were obtained experimentally, whilst those in italics, are predicted values based on statistical analysis (single exponential fit) of the experimental data

The in vitro measured data of Jones is very comparable to the predictions made later by Heylings who used a QSPeR model for whole human skinbased on that described by Potts and Guy(1992) to predict the dermal penetration rate of a large number of methacrylate esters, including EMA (see table below). Quantitatively the trend of decreased skin penetration rate across the Lower Alkyl (C1-C8) Methacrylates category was confirmed.

Table: QSAR prediction of absorption of methacrylate esters through whole human skin (extract from Heylings, 2013)

Substance	Molecular Weight	Log P	Predicted Flux (μg/cm^2/h)	Relative Dermal Absorption
EMA	114.14	1.87	36.132	Moderate

 

 

Primary Metabolism

Taken from the EU Risk Assessment on MMA; “Activities of local tissue esterases of the nasal epithelial cells appear to be lower in man than in rodents (Green, 1996 later published as Mainwaring, 2001). Toxicokinetics seem to be similar in man and experimental animal. After arthroplasty using methyl methacrylate-based cements, exhalation of unchanged ester occurs to a greater extent than after i.v., i.p. or oral administration. After oral or parenteral administration methyl methacrylate is further metabolised by physiological pathways with the majority of the administered dose being exhaled as CO2(Bratt and Hathway, 1977; ICI, 1977a). Conjugation with GSH or NPSH plays a minor role in methyl methacrylate metabolism and only occurs at high tissue concentrations (McCarthy and Witz, 1991; Elovaara et al., 1983)”.

Taken from the OECD SIAR: “Other short chain alkyl-methacrylate esters, like MMA, are initially hydrolyzed by non-specific carboxylesterases to methacrylic acid and the structurally corresponding alcohol in several tissues (ECETOC 1995, 1996b).

 

Figure 1: Carboxylesterase mediated hydrolysis of a methacrylate ester to MAA and the structurally corresponding alcohol: see category document

 

Kinetics data have been reported for the hydrolysis of two lower alkyl methacrylates including EMA; see following table; McCarthy and Witz, 1997). The studied substances showed comparable hydrolysis rates in vitro.

Table: Hydrolysis of Acrylate Esters by Porcine Liver Carboxylesterase in vitro (extract from McCarthy and Witz., 1997)

Substance	Km (mM)	Vmax (nmol/min)
Ethyl methacrylate (EMA)	159±90	5.2±2.5

 

Studies completed after the MMA RA have confirmed that all lower alkyl methacrylate esters are rapidly hydrolysed by ubiquitous carboxylesterases (see table below; adapted from Jones; 2002). First pass (local) hydrolysis of the parent ester has been shown to be significant for all routes of exposure. For example, no parent ester can be measured systemically following skin exposure to EMA and larger esters, as the lower rate of absorption for these esters is within the metabolic capacity of the skin (Jones, 2002). Parent ester will also be effectively hydrolysed within the G.I. tract and within the tissues of the upper respiratory tract (particularly the olfactory tissue). Systemically absorbed parent ester will be effectively removed during the first pass through the liver (%LBF; see table below;) resulting in their relatively rapid elimination from the body (T_50%; see table below;).

 

Table: Rate Constants for ester hydrolysis by rat-liver microsomes and predicted systemic fate kinetics following i.v. administration (adapted from Jones; 2002)

Substance	Rat liver microsomes (100mg ml^-1)	Rat liver microsomes (100mg ml^-1)	CL (%LBF)	T50%(min)	Cmax(MAA) (mg L^-1)	Tmax(MAA) (min)
	Vmax(nM min^-1mg^-1)	Km    (mM)
EMA	699.2	106.2	99.5%	4.5	12.0	1.8

Fate kinetics determined using the “well-stirred” model; CL%LBF – Clearance as percentage removed from liver blood flow i.e. first pass clearance; T_50%- time taken for 50% of parent ester to have been eliminated from the body; C_max– maximum concentration of MAA in circulating blood; T_max– time in minutes to peak MAA concentration in blood “Jones, 2002”.

 

Subsequent metabolism of the primary metabolites within the body

Again, taken from the OECD SIAR:Methacrylic acid and the corresponding alcohol are subsequently cleared predominantly via the liver (valine pathway and the TCA (TriCarboxylic Acid) cycle, respectively). The carboxylesterasesare 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 (Satoh & Hosokawa, 1998; Junge & Krish, 1975; Bogdanffy et al., 1987; Frederick et al., 1994). 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.

In terms of MAA, the common metabolite for these esters a comparison of measured blood concentration data after i. v. administration of 10 and 20 mg/kg MAA was made and a simulation based on a one-compartment model shows good agreement (Jones, 2002). Based on that information, the following kinetic parameters were determined from a simultaneous fit of the in vivo data to a one-compartment model with non-linear elimination (Vss = 0.039 L/SRW; Vmax = 19.8 mg/hr x SRW; Km = 20.3 mg/L; SRW: standard rat weight = 250 g) the half-life of MAA in blood was calculated as 1.7 min.

Interms of the corresponding alcohol metabolite for these esters, they are all subsequently rapidly metabolised, primarily in the liver, by oxidative pathways involvingaldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), cytochrome P450 (CYP2E1), and catalase enzymes to the corresponding aldehyde and acid before ultimately being converted to CO₂. In the case of the more volatile alcohols a significant portion is excreted un-metabolised in urine and/or exhaled air.

For ethanol (CAS 64-17-5), the alcohol metabolite of EMA, the above mentioned metabolic steps are confirmed in OECD SIDS (2004). The second step (acetaldehyde converted to acetate) is generally rapid so that intermediate aldehyde concentrations are usually very low. However, polymorphism is seen in the acetaldehyde dehydrogenase enzyme meaning that some ethnic groupings are less well adapted to metabolise this intermediate with resultant higher concentrations, although they still remain low. The main pathway for ethanol metabolism proceeds via alcohol dehydrogenase. Other pathways for ethanol oxidation have been described but at exposures relevant to occupational and consumer exposure during manufacture and use of ethanol containing products, the alcohol dehydrogenase metabolic route in the liver dominates and does not become saturated so that other pathways are not considered as relevant here. The maximum rate of metabolism is 100 - 125 mg/kg body weight/hour (Ellenhorn & Barceloux, 1988). Ethanol is metabolised principally by the liver, which accounts for 92-95% of capacity with minor amounts metabolised in other tissues such as the kidney and lung (Crabb et al., 1987).

 

Conjugation

The C=C double bond of methacrylate esters makes these chemicals potential Michael acceptors capable of electrophilic attack of protein and other cellular macromolecules. This is the mode of action through which a wide range of toxicities including allergic contact dermatitis is thought to be mediated. This reactivity also means, however, that methacrylate esters are capable of conjugating with cellular glutathione (GSH). Quantitative structure activity relationships (QSAR) have been developed for the reaction with glutathione based upon the local charge-limited electrophilicity index ω^q(Schwöbel et al., 2010) and the¹³C chemical shift, of theβ-Carbon atom (Fujisawa and Kadoma, 2012) i.e. the unsubstituted end of the double bond in these molecules. The most recent of these models from 2012 is claimed to have a reliability factor (r²) of 0.99 for the prediction of the rate of the Michael addition reaction with GSH for a range of acrylate and methacrylate monomers. This publication includes calculation of the rate constants for EMA and n-BMA. This model was used by Tindale to calculate the rates for the remaining esters in the C1-C8 category (Tindale, 2015).The rate of reaction is low for all members of the C1-C8 methacrylate category (seeTable16) whether based upon the local charge-limited electrophilicity index ω^q(Schwöbel et al., 2010, Cronin, 2012, 2015), or¹³C chemical shift, of the β-Carbon atom (Fujisawa and Kadoma, 2012; Tindale, 2015)when compared with other esters and unsaturated ketones and aldehydes (McCarthy & Witz, 1991).This is becausethe addition of a nucleophile at the double bond is hindered by the alpha-methyl side-group in the case of the methacrylates (McCarthy & Witz, 1991; McCarthy et al., 1994). A minor impact is also exerted by the +I-effect of the alcohol subgroup, but the incremental impact on electrophilicity rapidly decreases with increasing alcohol chain length. Therefore for direct electrophilic reactions the alcohol group will only have a minor, rather monotonic influence with increasing chain length. This is reflected in the very similar GSH reactivity constants for all members of the category. On this basis, 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. Furthermore, since there is no structural alert for Michael addition reactivity in the case of MAA (Cronin, 2012) or the alcohol metabolites, hydrolysis of the ester is essentially a detoxification process with regard to this MoA. Schultz et al. (2005; in Ebbrell et al. 2016) measured relative high methacrylate concentrations required to deplete GSH byspectrophotometry, leading to low log values and supporting the assessment of low reactivity of these substances.

Table: GSH reactivity of methacrylate esters

Substance	GSH reactivity*   ( meas.) logKGSH	GSH reactivity* (predicted.) logKGSH	GSH reactivity assessment**	GSH reactivity*** (predicted.) logKGSH	GSH depletion (meas.)**** - log RC50 average
EMA	-1.24	-0.88	slight	-0.79	-1.53
Metabolites
MAA			No alert found

*as quoted by Schwöbel et al., 2010
**as calculated by Cronin, 2012 using the method of Schwöbel et al., 2010
***as calculated by Tindale, 2015 according to the method of(Fujisawa and Kadoma, 2012)

**** as quoted by Ebbrell et al. 2016: RC50 is defined as the concentration of reactive chemical required to deplete GSH by 50% in 120 min. Average RC50 values are given for chemicals with multiple measurements. RC50 values were provided by TW Schultz (2005), obtained using a previously published spectrophotometric peptide depletion assay.

Trends

Particularly in the case of skin absorption there is trend of decreasing rate of absorption of the parent ester C1-C8with increasing ester chain length. This trend is also likely to extend to some extent to absorption across all barrier membranes including the gut and respiratory epithelium. All esters are rapidly hydrolysed within the body whether in local tissues, the blood or ultimately within the liver by non-specific esterases. There is an observed trend towards increasing half-life of the parent ester with increasing ester chain length. The common primary metabolite, MAA, as well as the corresponding alcohol primary metabolites are cleared rapidly in all cases.

Summary and discussion on toxicokinetics

Methacrylate esters are absorbed to a greater or lesser extent by all routes and are subsequently rapidly hydrolysed by carboxylesterases to methacrylic acid (MAA) and the respective alcohol. Clearance of the parent ester from the body is in the order of minutes for the short chain esters to tens of minutes for the longer esters. The primary metabolites, MAA and the corresponding alcohol, are also subsequently cleared rapidly from bloodby standard physiological pathways, with the majority of the administered dose being exhaled as CO2. In the case of MAA the systemic half-life in rats is only 1.7 minutes. On the basis of the rapid metabolism and short half-lives a systemic accumulation of the esters and their metabolites is not expected.

Local effects resulting from the hydrolysis of the ester to the irritant and corrosive metabolite MAA are only observed following inhalation exposure in rodents and this has been shown to be due to the localised concentration of high levels of non-specific esterases in the Bowman’s glands of the nasal olfactory tissues. This combination of highly efficient mechanisms of absorption/deposition and localised enzyme activity does not occur in the case of the dermal and oral routes so localised tissue corrosion would not be expected to occur. In summarising the available PBPK data on MMA SCOEL concluded that “ExtensivePBPK modelling work has predicted that on kinetic grounds for a given level of exposure to MMA human nasal olfactory epithelium will be at least 3 times less sensitive than that of rats to the toxicity of MMA” (SCOEL, 2005).Local effects are not anticipated as a result of the localised concentration of the corresponding alcohol since the alcohols themselves do not produce local effects.

Overall there is a high level of confidence in the toxicokinetic and toxicodynamic assessment for these chemicals based upon in vitro and in vivo studies in rodents and human tissues. This is further supported by clear trends across the category consistent with predicted trends based on recognised QSAR based models. In terms of the overall relevance of the findings in animals to humans there is a high degree of confidence since the same toxicokinetic/dynamic processes are known to occur in humans. In the case of dermal exposure there is robust in vitro data based upon measurements in animal and human skin supported by an established QSAR model that shows that dermal absorption, and therefore risk, of these esters is lower in humans than in rodents. In the case of inhalation exposure well recognised morphological and physiological differences between rodents and humans have been confirmed for the methyl ester to indicate a lower sensitivity of humans than rodents to the local effects in the upper respiratory tract. This is consistent with findings in limited studies in clinical volunteers and by cross-sectional studies in workers with long-term exposure to concentrations of MMA vapour well in excess of the effect concentration in rodents. There is a high level of confidence that the findings for MMA can be equally applied to the ethyl ester. This is based upon the close structural similarity and physico-chemical properties supported by robust PBPK observations and effect data in animals.

Overall, therefore, it is considered that the available toxicodynamic data is sufficiently adequate, particularly since this is consistent with the general trend within the category and the predicted limited opportunity for exposure. In conclusion therefore, there is a high level of confidence in the toxicokinetic assessment for the category of C1-C8 methacrylate esters.