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Endpoint:
basic toxicokinetics in vivo
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
Objective of study:
metabolism
toxicokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
This non-GLP pharmacokinetic study of both HEMA and HPMA in rats via intravenous (IV) administration was conducted to evaluate the potential quick hydrolysis of both HEMA and HPMA in vivo.
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
Fischer 344/DuCrj
Details on species / strain selection:
F344/DuCrl rats were selected because of their use in previous toxicological studies for the two test materials (HEMA and HPMA). Rats are a suitable species for the analysis of metabolism of chemicals in vivo. The F344/DuCrl rats are also suitable due to the availability of historical background data, and the reliability of the commercial supplier.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Kingston, New York)
- Age at study initiation: 9 weeks
- Weight at study initiation: 191-208 g
- Housing: all animals were single housed in glass Roth-type metabolism cages for
acclimation purposes.
- Diet (e.g. ad libitum): LabDiet Certified Rodent Diet #5002 (PMI Nutrition International,
St. Louis, Missouri) in pelleted form. Feed was provided ad libitum.
- Water (e.g. ad libitum): Municipal water was supplied to all study animals ad libitum throughout the study.
- Acclimation period: Upon arrival, all animals were acclimated to the laboratory for at least two days prior to the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-26°C
- Humidity (%): 50% with a range of 38-78%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intravenous
Vehicle:
physiological saline
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Appropriate amounts of HEMA or HPMA were added to sterile saline to obtain the appropriate dose of 5 mg/kg bw using aseptic techniques. The amount of dose solution administered was targeted at ~2.5 mL/kg bw and injected over a minimum of 45 seconds which corresponded to injection rates ranging from 0.7 to 0.8 mL/minute based on the averaged body weight of 0.2 kg.
Duration and frequency of treatment / exposure:
once
Dose / conc.:
5 mg/kg bw/day
No. of animals per sex per dose:
2 males
Control animals:
no
Positive control:
no
Details on study design:
- Dose selection rationale: A dose level of 5 mg/kg body weight (bw) for HEMA and 5 mg/kg bw for HPMA was used in this study. These two dose levels are equimolar based on the study design.
Details on dosing and sampling:
TOXICOKINETIC STUDY
- Tissues and body fluids sampled (delete / add / specify): bloodpling:
- Time and frequency of sampling: 5-10-30-60-180 min
- From how many animals: 2, not pooled
- Method type(s) for identification: GC-MS
- Limits of detection and quantification: LOQ: 45.0 ng/mL (HEMA)
Statistics:
Descriptive statistics were used, i.e., mean ± standard deviation, when applicable. All calculations in the database were conducted using Microsoft Excel (Microsoft Corporation, Redmond, Washington) spreadsheets and databases in full precision mode
(15 digits of accuracy). Certain pharmacokinetic parameters were calculated for blood, including AUC (area-under-the-curve), using a pharmacokinetic computer modeling program PK Solutions (v.2.0.6., Summit Research Services, Montrose, Colorado).
Type:
metabolism
Results:
Rapid hydrolysis after intavenous administration in rats
Key result
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: ca. 1 min
Remarks:
mean

mean T1/2 around 1 min (0.84 and 1.06 min for each animal, respectively)

Conclusions:
After i.v. administration in rats, HEMA quickly hydrolyses in the order of a few minutes. The estimated half-lives for HEMAwas near 1 minute, indicating that the current study results support the assumption that HEMA was quickly hydrolyzed to its primary metabolites.
Executive summary:

To support the REACH registration for hydroxyethyl methacrylate (HEMA) and hydroxypropyl methacrylate (HPMA), a Read-across approach can be applied if test materials can be quickly hydrolyzed to the methacrylic acid and the corresponding alcohols (glycols) in vivo. This non-GLP pharmacokinetic study of both HEMA and HPMA in rats via intravenous administration was conducted to evaluate the potential

quick hydrolysis of both HEMA and HPMA in vivo.

Two male rats per compound were intravenously administered HEMA or HPMA individually at 5.0 mg/kg dose level with saline as the dose vehicle. After dose administration, blood samples (200 μl) were collected at 5, 10, 30, 60, and 180 minutes into individual glass vials containing ethyl acetate (600 μL) acidified with 1% formic acid. After vortexing, the levels of HEMA and HPMA in the blood samples were

quantitatively analyzed by GC/MS-MS.

The results showed that levels of both HEMA and HPMA dropped rapidly after administration and were not quantifiable by 60 minutes with limit of quantitation (LOQ) of 48.8 ng/mL (HPMA) and 45.0 ng/mL (HEMA). The estimated half-lives for HEMA and HPMA were less than or near 1 minute, indicating that the current study results support the assumption that both HEMA and HPMA were quickly hydrolyzed after intravenous administration in rats.

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report which meets basic scientific principles
Qualifier:
no guideline available
Principles of method if other than guideline:
Determination of in vitro hydrolysis rates of methacrylate esters; determination of half-lifes in rat liver microsomes and whole rat blood. determination of Km and Vmax values for ester hydrolysis in rat liver microsomes; these values were used for PBPK modeling to simulate in vivo blood concentrations
GLP compliance:
yes
Radiolabelling:
no
Species:
other: rat liver microsomes and rat blood
Vehicle:
DMSO
Duration and frequency of treatment / exposure:
phase I: 120 min (samples collected at 0, 2, 5, 15, 30, 60 and 120 minutes)
phase II: 5 min (samples collected at 0 and 5 minutes)
Remarks:
Doses / Concentrations:
phase I: 0.25 mM
phase II: 0.05, 0.1 and 5.0 mM
No. of animals per sex per dose:
not applicable; in vitro test
Control animals:
other: not applicable; in vitro test
Positive control:
Methyl methacrylate
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Method type(s) for identification: liquid chromatography separation with accurate mass quadrupole/time-of-flight mass spectrometry detection (LC/QTOF-MS) to quantitate methacrylic acid concentrations
- Limits of detection and quantification: LLQ (phase I) = 0.0117 mM methacrylic acid; LLQ (phase II) = 0.00509 mM methacrylic acid
Type:
metabolism
Results:
the ester was rapidly converted to MAA in whole rat blood (Phase I) and rat liver microsomes (Phase II): half life 4.62 min (liver microsomes) / 99 min (blood). In phase II hydrolyses exp. 1 mol of MAA was produced for every mol of HEMA

Negative controls in the rat liver microsome experiments included incubations with heat-inactivated microsomes, no microsomes and no NADPH. Removal of NADPH made little or no difference in hydrolysis rates. Heat inactivation significantly reduced hydrolysis rates, and absence of microsomes resulted in no hydrolysis. 

HEMA was rapidly converted to MAA in whole rat blood and rat liver microsomes with hydrolysis half-lives of 4.62 min (liver microsomes) and 99 min (blood).

Vmax (in vitro) = 111 nmol/min/mg

Vmax (in vivo) = 39 mg/hr/g liver

Km (in vitro) = 889 µM

Km (in vivo) = 116 mg/L

PBPK modelling showed rapid hydrolysis of HEMA.

Conclusions:
Interpretation of results (migrated information): other: The metabolism data and modelling results show that HEMA would be rapidly hydrolysed in the rat.
The metabolism data and modelling results show that HEMA would be rapidly hydrolysed in the rat.
Executive summary:

This in vitro metabolism study was conducted to investigate in vitro hydrolysis rates of HEMA. Half-lifes were determined in rat liver microsomes and whole rat blood. Further experiments were conducted to determine Km and Vmax values for ester hydrolysis in rat liver microsomes. These values were used for PBPK modelling.

HEMA was rapidly converted to MAA in whole rat blood and rat liver microsomes with hydrolysis half-lives of 4.62 min (liver microsomes) and 99 min (blood).

Vmax (in vitro) = 111 nmol/min/mg

Vmax (in vivo) = 39 mg/hr/g liver

Km (in vitro) = 889 µM

Km (in vivo) = 116 mg/L

In summary, the metabolism data and modelling results show that HEMA would be rapidly hydrolysed in the rat.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Documentation sufficient for assessment
Objective of study:
other: toxicokinetics and distribution
Principles of method if other than guideline:
14C-HEMA was administered to mice via gastric tube or subcutaneous. The clearance of 14C-HEMA and 14C content were determined by measuring the 14C activity.
GLP compliance:
not specified
Radiolabelling:
yes
Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 20-25 g
- Fasting period before study: 12 hours prior to experiment
- Housing: housed separately
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days prior to each experiment

Route of administration:
other: subcutaneous injection and oral gastric tube
Vehicle:
physiological saline
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: HEAMA dissolved in 0.9% NaCl solution labelled with tracer.
- Volume 10 uL/g bw

Duration and frequency of treatment / exposure:
single exposure
Remarks:
Doses / Concentrations:
20 umol/kg bw HEMA dissolved in 0.9% NaCl solution
- Volume: 10 uL/g bw
No. of animals per sex per dose:
56
Control animals:
yes, concurrent vehicle
Details on study design:
In these experiments a HEMA dose level was chosen exceeding the dose levels expected in humans exposed to (co)monomers from a single composite restoration.
Details on dosing and sampling:
Experiment 1: Organs, organ-wall and content of organs as well as blood were taken immediately. Tested organs were: liver, kidney, blood, skin, brain, heart, spleen, lung, muscle (from 3 areas), testis, eyes, bone, wall of stomach, content of stomach, wall of small intestine, content of small intestine, wall of large intestine, and content of large intestine. Organs were immediately washed with 2 X 10 ml distilled H2O and then the tissues were weighed and homogenized. Tissues were dissolved in TEAH.

Experiment 2: exhaled air was captured during the total experimental period Urine and feces were collected at 0.5, 1, 2, 6, 12 and 24 h after the beginning of the experiment. Organs, organ-wall and content of organs, blood, urine and feces were taken immediately. Organs were taken for analysis as described for the first in vivo experiment.

The clearance of 14C-HEMA and the 14C content in organs, wall and content of organs, blood, urine, feces and exhaled air were determined by measuring the 14Cactivity. It was measured with TEAH in aqueous solution with Omni-Szintisol. The 14C-activity was determined in a 2500 TR liquid scintillation analyzer (Canberra-Packard, Dreieich, Germany).
Statistics:
In order to calculate the amount of 14C-activity in blood, skin and muscle, a total mass of 6.7, 20, and 40% of body weight was assumed for the respective organ. The data are presented as means of the 14C-percentage of the administered 14C-HEMA dose +/- SEM.
Details on distribution in tissues:
After oral application in the first in vivo experiment 14C was found to be 62% of the applied 14C-HEMA dose in the entire mouse 5 min after application. Highest 14C contents were found in the content of the stomach and in thewall of stomach (19.8% and 10.5%, respectively) followed by liver (5.1%), blood (3.3%), brain and lung (0.2%). 14C was found to about 38% in the entire mouse 15 min after application. After 24 h the elimination of 14C was nearly complete (0.5% 14C was found in the entire mouse). The plasma half-life period of 14C-HEMA is lower than 10 min. After subcutaneous application in the first in vivo experiment 14C was found to be 43% of the applied 14C-HEMA dose in the entire mouse 5 min after application. Highest 14C contents were found in muscle (18.4%) followed by blood (5.7%), skin (5.6%), injection area (3.7%) and liver (2.7%) (Table 2). 15 min after application 14C was found to about 39% in the entire mouse. After 24 h the elimination of 14C was nearly complete (<1% 14C was found in the entire mouse). The plasma half-life period of 14C-HEMA is lower than 10 min.


Similar 14C contents were found in organs, organ-wall and content of organs at corresponding time intervals after subcutaneous and oral application, compared to the 14C contents after subcutaneous and oral application in mice in the first in vivo experiment.
Details on excretion:
In mice, in the first in vivo experiment, the total 14C recovery (organs+wall and content of organs) decreased from 62% 5 min after oral application to 0.5% 24 h after oral application. The total 14C recovery decreased from 43% 5 min after subcutaneous application to 0.9% 24 h after subcutaneous application. A second set of experiment was performed to capture urine, feces and exhaled air from mice which received HEMA (+14C-HEMA). After oral application in the second in vivo experiment mice excreted 14C to about 7% of the applied 14C-HEMA dose via urine and to about 23% via feces within 24 h. Between the second and the sixth hour after application 14C was excreted to about 4.1% via urine. Calculated from this 14C content in the urine at the dose administered, a HEMA and/or metabolite concentration of about 20.5 nM in the spontaneous urine can be estimated. Mice exhaled 14C as 14C O2 to about 62% within 24 h. 14C was exhaled as 14C O2 to about 59% even within 1 h by mice. The total 14C recovery (feces +urine+ organs+walls and contents of organs+wash water +þ14CO2) increased up to 94% up to 24 h.

After subcutaneous application in the second in vivo experiment mice excreted 14C to about 14% of the applied 14C-HEMA dose via urine and to about 12% via feces within 24 h. Between the second and the sixth hour after application 14C was excreted to about 9.6% via urine. Calculated from this 14C content in the urine at the dose administered, a HEMA and/or metabolite concentration of about 48 nM in the spontaneous urine can be estimated. Mice exhaled 14C as 14CO2 to about 67% within 24 h. The total 14C recovery (feces +urine+ organs+walls and contents of organs+wash water+ 14CO2) increased up to 95% up to 24 h.
Conclusions:
The show that uptake of HEMA from the stomach and small intestine in mice was rapid, that distribution throughout the body with both routes of administration was broad. An enrichment of HEMA is unlikely, because 14C clearance from the body appeared to be essentially complete one day following gastric or subcutaneous application. Peak tissue levels of HEMA were substantially below those known to be cytotoxic.
Executive summary:

2-Hydroxyethyl methacrylate (HEMA) is one of the major components released from dental resin-based composites. In this study uptake, distribution, and excretion of 14C-HEMA applied via gastric tube or subcutaneous administration at dose levels well above those encountered in dental care were examined in mice to test the hypothesis that HEMA can reach cytotoxic levels in mammalian tissues. 14C-HEMA was taken up rapidly from the stomach and intestines after gastric administration and was widely distributed in the body following administration by each route. Most 14C was excreted within one day as 14CO2. Two metabolic pathways of 14C-HEMA can be described. The peak HEMA levels in all tissues examined after 24 h were lower than known toxic levels.

Endpoint:
dermal absorption
Type of information:
other: QSPR
Remarks:
Based on an established human skin model by Potts and Guy (Potts RO and Guy RH (1992). Predicting Skin Permeability. Pharm. Res. 9(5): 663-669)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Generally accepted calculation method
Principles of method if other than guideline:
The physicochemical parameters of MW, Log P and saturated aqueous solubility have been used in the evaluation of 56 methacrylate compounds. An output of predicted steady-state flux was calculated using the principles defined in the Potts and Guy prediction model. (Potts RO and Guy RH (1992). Predicting Skin Permeability. Pharm. Res. 9(5): 663- 669)
GLP compliance:
no
Species:
other: human skin model
Details on test animals and environmental conditions:
not applicable; in silico modelling
Type of coverage:
other: not applicable; in silico modelling
No. of animals per group:
not applicable; in silico modelling
Absorption in different matrices:
predicted flux 151.3 μg/cm²/h; the relative dermal absorption is high

Based on a molecular weight of 130.1 g/mol and a g Kow of 0.42, the predicted flux of HPMA is 151.3 μg/cm²/h; the relative dermal absorption is high.

Conclusions:
The dermal absorption of HEMA is predicted to be high; the predicted flux is 151.3 μg/cm²/h.
Executive summary:
The dermal absorption (steady-state flux) of HEMA has been estimated by calculation using the principles defined in the Potts and Guy prediction model.

Based on a molecular weight of 130.1 g/mol and a logKow of 0.42, the predicted flux of HEMA is 151.3μg/cm²/h; the relative dermal absorption is high.

Description of key information

Reliable experimental data exists to demonstrate that HEMA is rapidly metabolized by a ubiquitous metabolic pathway within the body. HEMA is metabolised to ethylene glycol and methacrylic acid. There is a high level of confidence based upon experimental data showing that the half-live of HEMA is in the order of a few minutes.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

Data on the toxicokinetics of HEMA are available. These data show that HEMA is hydrolyzed by carboxylesterases to MAA and the respective alcohol, EG. Further, studies in guinea pigs and in mice with HEMA indicate that most of the administered material is rapidly cleared from the body following metabolism to CO2. These results are consistent with the conclusion that HEMA is rapidly metabolized by tissue esterases to the respective glycol ethylene glycol (EG) and methacrylic acid (MAA). Subsequently, both glycol and methacrylic acid are further oxidized before excretion.

 

ADME and Ester Hydrolysis

Methacrylate esters in general

For the understanding of the toxikokinetic of the hydroxyalkyl-methacrylate esters HEMA and HPMA it is important to understand the general metabolism of methacrylate esters in mammals. For MMA and other short-chain alkyl-methacrylate esters extensive toxicokinetic data are available. These data have been reviewed and summarized in the EU Risk Assessment for MMA as well as the OECD SIAR for short-chain alkyl-methacrylate esters. A brief summary follows:

After oral or inhalation administration, methacrylate esters are expected to be rapidly absorbed and distributed. Dermal absorption of esters is extensive only with occlusion of the site. 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. Methacrylic acid (MAA) and the corresponding alcohol are subsequently cleared predominantly via the liver (valine pathway and the TCA (Tricarboxylic Acid) cycle, respectively). 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.

 

HC=C(CH3)H-C=OOR + H2O ---(carboxylesterase)--->  HC=C(CH3)H-C=OOH  +  R-OH

 

Figure: Ester hydrolysis by carboxylesterases, “R” is a placeholder for any alkyl or hydroxyalkyl group  

 

As supporting evidence, metabolism and half-live data from a range of lower alkyl-methacrylate esters are shown here which indicate that this category of methacrylate esters of comparable molecular weight (HEMA MW 130.1; HPMA MW 144.2) are rapidly hydrolysed by ubiquitous carboxylesterases. First pass (local) hydrolysis of the parent ester has been shown to be significant for all routes of exposure (see Category Justification, chapter 5.2).

 

HEMA

A non-GLP toxicokinetic study of both HEMA and HPMA in rats via intravenous administration was conducted to evaluate the potential quick hydrolysis of both HEMA and HPMA in vivo (Dow, 2017).

Two male rats were intravenously administered HEMA individually at 5.0 mg/kg dose level with saline as the dose vehicle. After dose administration, blood samples (200 μl) were collected at 5, 10, 30, 60, and 180 minutes into individual glass vials containing ethyl acetate (600 μL) acidified with 1% formic acid. After vortexing, the levels of HEMA in the blood samples were quantitatively analyzed by gas chromatography tandem mass spectrometry (GC/MS-MS).

The results showed that HEMA dropped rapidly after administration and were not quantifiable by 60 minutes with limit of quantitation (LOQ) of 45.0 ng/mL. The estimated half-live for HEMA was around one minute (i.e. 0.84 and 1.06 minutes for the two testged animals), indicating that the current study results support the assumption that HEMA was quickly hydrolyzed after intravenous administration in rats.

A comparable conclusion has been made in an earlier study which used a combination ofin vitroester hydrolysis experiments with liver microsomes and whole rat blood and a PBPK model, slightly modified from the model developed by Jones (2002) in a study with lower alkyl methacrylates (Dow, 2013). This study was conducted to investigate in vitro hydrolysis rates of methacrylate esters for which limited data exist. Seven methacrylate esters, including 2-hydroxyethyl methacrylate (HEMA) were chosen for experimental determination of metabolism rates in whole rat blood and rat liver enzymes at a single substrate concentration. All seven methacrylates were quickly hydrolyzed to methacrylic acid (MAA) and the corresponding alcohol in both whole rat blood and rat liver microsomes. The half-life of HEMA after incubation with rat liver microsomes was 4.62 minutes, in whole rat blood 99 minutes. Based on the in vivo hydrolysis rates, the PBPK model predicted the following rates for rat liver:Vmax= 39 mg/hr/g liver and Km= 116 mg/L. The corresponding half-lives in vivo(T1/2α =0.040 hr / 2.4 min – distribution and metabolism; T1/2β =0.077 hr / 4.62 min – metabolism phase)are very short and consistent with thein vivodata above on rats (Dow 2017) and below on mice (Durner 2009).

Durner et al (2009) measured the absorption, distribution and toxicokinetics of HEMA in mice following oral and subcutaneous injection routes.In this study, uptake, distribution, and excretion of 14C-HEMA applied via gastric tube or subcutaneous administration at dose levels well above those potentially encountered in dental care were examined in mice to test the hypothesis that HEMA can reach cytotoxic levels in mammalian tissues. Each mouse received HEMA (20 mmol/kg bw [104 mg/kg/25 g mouse], dissolved in 0.9% NaCl solution), labeled with a tracer dose of radioactive14C-HEMA 0.7 kBq/g bw) either by subcutaneous injection beneath the shoulder skin or via gastric tube. The clearance of14C-HEMA and the14C content in organs, wall and content of organs, blood, urine, feces and exhaled air were determined by measuring the14C activity. In a separate experiment, each mouse was kept in a closed chamber with controlled air flow. The exhaled air was captured during the total experimental period by flowing through 7 bottles, one behind the other, filled with 250 ml ice-cold 5 N NaOH.14CO2 was captured as14C Na2CO3and the total 14C-activity determined. Urine and feces were collected at 0.5, 1, 2, 6, 12 and 24 h after the beginning of the experiment.

14C-HEMA was taken up rapidly from the stomach and intestines after gastric administration and was widely distributed in the body following administration by each route. After oral application in the first in vivo experiment14C was found to be 62% of the applied14C-HEMA dose in the entire mouse 5 min after application. Highest14C contents were found in the content of the stomach and in the wall of stomach (19.8% and 10.5%, respectively) followed by liver (5.1%), blood (3.3%), brain and lung (0.2%). After subcutaneous application in the first in vivo experiment 14C was found to be 43% of the applied14C-HEMA dose in the entire mouse 5 min after application. Highest14C contents were found in muscle (18.4%) followed by blood (5.7%), skin (5.6%), injection area (3.7%) and liver (2.7%).

Most14C was excreted within one day as14CO2. After 24 h the elimination of14C was nearly complete (0.5% - 1%14C was found in the entire mouse) by either route of administration. The plasma half-life period of14C-HEMA was estimated to be lower than 10 min.

After oral application in the second in vivo experiment mice excreted14C equivalent to 7% of the applied14C-HEMA dose via urine and to about 23% via feces within 24 h. Mice exhaled14C as14CO2 equivalent to about 62% of the applied dose within 24 h.14C was exhaled as14CO2 equivalent to 59% of the applied dose even within 1 h by mice. The total14C recovery increased to 94% by 24 h following oral dosing.

After subcutaneous application in the second in vivo experiment mice excreted14C equivalent to about 14% of the applied14C-HEMA dose via urine and to about 12% via feces within 24 h. Mice exhaled14C as14CO2 equivalent to about 67% of the injected dose within 24 h. The total14C recovery increased to 95% up by 24 h.

The authors concluded that the metabolism of HEMA in vivo in mice is so rapid that the concentration of HEMA is only in the nanomolar range, indicating that the toxic levels for mammalian cells [micromolar to millimolar range] will not be reached. It is therefore unlikely that HEMA released from dental materials could have any systemic toxic effects. The study did not support the hypothesis, that HEMA reaches toxic levels in body tissue.

Reichl et al (2002) investigated the metabolism and toxicokinetics of HEMA in guinea pigs using14C-HEMA labeled on the carboxyl carbon and administered by either oral or subcutaneous routes. Low fecal14C levels (about 2% of the dose) and urinary levels of about 15% after 24 h were noted with either route of administration. Direct measurement of exhaled radiolabeled CO2showed that about 70% of the dose left the body via the lungs. Clearance from most tissues following gastric and intradermal administration was essentially complete within one day. The authors postulated the existence of reactive oxygenated intermediates based on the ratio of radiolabeled pyruvate to malate in bile; however, no direct evidence for these postulated structures was presented.

 

Conjugation

The reactivity towards glutathion of more than 50 methacrylates and other chemicals with related structures has been estimated with a QSPR model by Cronin (2012). For both hydroxyalkyl methacrylates it is predicted that they are slightly reactive towardsglutathione(GSH). This is consistent with experimental data by Freidig et al. (1999) who investigated and compared the reactivity with glutathion of a series of acrylate and methacrylate esters.

Methacrylate esters can conjugate with 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. 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.

 

Absorption

Heylings (2013) 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 the hydroxyalkyl methacrylates (Table below).

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

Substance

Molecular

Weight

Log P

Predicted Flux

(μg/cm2/h)

Relative Dermal

Absorption

HEMA

130.1

0.42

151.293

High

HPMA

144.2

0.97

150.827

High

 

As relatively small, hydrophilic esters the hydroxyalkyl methacrylates are predicted to be rapidly absorbed. However, as indicated by the studies above, they will be subject to hydrolysis by local esterases in the stratum corneum.

 

MAA

For 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 was performed based on a one-compartment model. This shows good agreement with the measured data in vivo (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.

 

EG

As described in the ATSDR review (2010), “Ethylene glycol is converted to glycolaldehyde by nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase. Subsequent reduction of NAD results in the formation of lactic acid from pyruvate. Glycolaldehyde has a brief half-life and is rapidly converted to glycolic acid (and to a lesser extent glyoxal) by aldehyde dehydrogenase and aldehyde oxidase, respectively. Glycolic acid is oxidized to glyoxylic acid by glycolic acid oxidase or lactic dehydrogenase. Glyoxylic acid can be metabolized to formate, glycine, or malate, all of which may be further broken down to generate respiratory CO2, or to oxalic acid, which is excreted in the urine. In excess, oxalic acid can form calcium oxalate crystals. Rate-limiting steps in the metabolism of ethylene glycol include the initial formation of glycolaldehyde and the conversion of glycolic acid to glyoxylic acid, both of which are saturable processes. The conversion of glycolic acid to glyoxylic acid is the most rate-limiting step in ethylene glycol metabolism.

 

A flow diagram with the metabolic pathways of EG can be found as an attachment to this document.

 

The metabolism of Ethylene glycol, namely the saturation, has a substantial impact on renal and developmental effects in animals and is thus picked up in chapters 5.7 (Repeated Dose Toxicity) and 5.10 (Toxicity for Reproduction).

 

Conclusions

Methacrylate esters are readily absorbed by all routes and rapidly hydrolyzed by carboxylesterases to methacrylic acid (MAA) and the respective alcohol, ethylene glycol. Clearance of the parent ester from the body is in the order of minutes. The primary methacrylic metabolite, MAA, is subsequently cleared rapidly from blood and, as indicated by studies with MMA, this metabolism is by standard physiological pathways, with the majority of the administered dose being exhaled as CO2. The metabolism of EG, the primary glycolic metabolite of HEMA is also well understood as based on standard physiological pathways. CO2, or oxalic acid, which is excreted in the urine, are the ultimate metabolites. The similarity of metabolism between HEMA and MMA also implies that systemic toxicity data on MMA and other lower chain alkyl methacrylic acid esters are relevant to the potential toxicity profile of HEMA. Thus, following absorption of MMA or HEMA into the body the metabolic disposition of the two materials are likely to be similar differing substantively only on the alcohol/glycol moiety released and toxicity data for MMA may also be of relevance to HEMA.  

Local effects (irritation) resulting from the hydrolysis of the ester to MAA are only observed following inhalation exposure and this has been shown to be due to the localised concentration of non-specific esterases in nasal olfactory tissues. In summarising the available PBPK data on MMA SCOEL concluded that “Extensive PBPK 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). For HEMA, however, similar to EHMA, the lower alkyl methacrylate category member with the highest molecular weight and low vapour pressure, it is unlikely that this is a relevant mode of action, since the vapour pressure is too low so that toxic, local MAA levels cannot be reached in the respective tissues.  

Studies indicate that HEMA may react both non-enzymatically and enzymatically with glutathione and that N-acetylcysteine may antagonize the cytotoxicity and genotoxicity of HEMA (and by extension HPMA) in vitro. These effects however occur only at very high, millimolar concentrations and is consistent with the prediction of slight reactivity with GSH based upon QSAR considerations of their structures (Cronin 2012). As weak electrophiles with only transient presence within the body they will be unlikely to contribute significantly, as compared with the primary metabolites, to the profile of systemic toxicity observed after repeated dosing. In this regard, the systemic toxicity of MAA is non-specific and common to both esters, whereas the glycols and their subsequent metabolism are likely responsible for any differences observed in the toxicity profile of the parent ester in vivo. Since the systemic toxicity of EG is more marked than that of PG this is likely responsible for the more marked toxicity observed with HEMA compared with HPMA upon repeated dosing.

Summary and discussion on toxicokinetics

The read across hypothesis and the satisfaction of the higher tier data requirements for HEMA relies on the observation that HEMA is metabolized very rapidly within the body to their respective alcohol (EG) and methacrylic acid (MAA) and as a consequence repeated dose systemic toxicity reflect the combined toxicities of the primary metabolites, which have already been studied extensively. Toxicokinetics therefore is a key element in the mode of action for many endpoints as well as in the read across hypothesis.

Experimental data exists to demonstrate that HEMA is metabolized by a ubiquitous metabolic pathway for all esters, including methacrylates within the body. HEMA is metabolised to EG and methacrylic acid. There is a high level of confidence based upon experimental data showing that the half-live of HEMA is in the order of a few minutes (Dow, 2017). Supporting evidence for the expimental data on HEMA is coming from data on a number of structurally related methacrylate esters in the same range of molecular weight and polarity (Jones, 2002; Dow, 2013).