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Reference
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Determination of in vitro hydrolysis rates of methacrylate esters, i.e. 1,3-BDDMA & 1,4-BDDMA; determination of half-lifes, elimination rates and intrinsic clearance in rat liver microsomes and whole rat blood.
GLP compliance:
yes
Radiolabelling:
no
Species:
other: rat liver microsomes and rat blood
Strain:
Fischer 344
Vehicle:
DMSO
Duration and frequency of treatment / exposure:
120 min (samples collected at 0, 2, 5, 15, 30, 60 and 120 minutes)
Dose / conc.:
0.25 other: mM
Remarks:
Whole blood
Dose / conc.:
0.229 other: mM
Remarks:
Liver Microsomes
No. of animals per sex per dose / concentration:
not applicable; in vitro test
Control animals:
other: not applicable; in vitro test
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/ESI/QTOF-MS) to quantitate methacrylic acid concentrations
Statistics:
Descriptive statistics were used, i.e., mean ± standard deviation. All calculations in the database were conducted using Microsoft Excel (Microsoft Corporation, Redmond, Washington) spreadsheets and database was set to full precision mode (15 digits of accuracy).
Type:
metabolism
Results:
The ester was rapidly converted to MAA in whole rat blood (12.4 min) and rat liver microsomes (3.55 min).
Metabolites identified:
yes
Details on metabolites:
Methacrylic acid

1,3 -BDDMA was rapidly converted to MAA in whole rat blood and rat liver microsomes with hydrolysis half-lives of 3.55 min (liver microsomes) and 12.4 min (blood).

Based on the half-live values, the intrinsic clearance rate Clintand elimination rate kevalues for 1,3-BDDMA in rat liver microsomal incubation conditions, were calculated as 116 µl/min/mg and 0.195 min-1, respectively.

Based on the half-live values, the intrinsic clearance rate Clintand elimination rate kevalues for 1,3-BDDMA in rat whole blood incubation conditions, were calculated as 112 µl/min/mg and 0.0559 min-1, respectively.

Conclusions:
The metabolism data and modelling results show that 1,3-BDDMA would be rapidly hydrolysed in the rat.
Executive summary:

Overall, the current study results show that both 1,3-BDDMA and1,4-BDDMA were quickly hydrolyzed in both rat blood and rat liver microsomes. In blood, the hydrolysis half-life of 1,3-BDDMA was about two times longer (slower hydrolysis) than 1,4-BDDMA; however, in rat liver microsomes, 1,3-BDDMA and 1,4-BDDMA have a similarly short half-life (~ 3.5 min). Based on these study results, it is expected that both 1,3-BDDMA and 1,4-BDDMA will be rapidly hydrolyzed to MAA in vivo and therefore, both compounds would be expected to have similar toxicity potential.

Description of key information

 1,3-BDDMA is likely to be absorbed by all routes. Due to the low vapour pressure, the dermal route is the primary route of exposure, since inhalation is unlikely. The dermal absorption rate however is calculated to be low. The ester is rapidly hydrolysed by carboxylesterases within a few minutes to methacrylic acid (MAA) and the respective alcohol, 1,3-Butanediol. 1,3-Butanediol will be transformed to beta-hydroxybutyrate and, subsequently, to acetoacetate. Metabolites can be degraded to Acetyl-CoA which can be broken down to generate respiratory CO2. MAA is metabolized to succinic acid which will be degraded through the tricarboxylic acid cycle and primarily excreted as CO2. Based on physicochemical properties, no potential for bioaccumulation is to be expected. 

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
50
Absorption rate - dermal (%):
50
Absorption rate - inhalation (%):
100

Additional information

Oral absorption

The physicochemical properties of 1,3-BDDMA (log P = 3.1; read-across from 1,4-BDDMA) and the molecular weight of 226,27 g/mol are in a range suggestive of absorption from the gastro-intestinal tract subsequent to oral ingestion (ECHA guidance 7c, 2017). For chemical safety assessment an oral absorption rate of 50% is assumed as a worst case default value (ECHA R.8 guidance, 2012).

 

Dermal absorption

Based on a QSAR Prediction of Dermal Absorption (extract from Heylings JR, 2013) 1,3-BDDMA is predicted on the basis of their molecular weight and lipophilicity to have a relatively low ability to be absorbed through the skin. The predicted flux was 2.895μg/cm²/h. However, for chemical safety assessment, a dermal absorption rate of 50% is assumed as a worst case default value (ECHA R.8 guidance, 2012).

 

Inhalative absorption

Due to the low vapour pressure of 1,3-BDDMA (0.1 Pa at 20°C; read-across from 1,4-BDDMA), exposure via inhalation is unlikely. For chemical safety assessment an inhalative absorption rate of 100% is assumed as a worst case default value (R8 guidance, 2012)

 

Distribution

As a small, water-soluble molecule with a logP > 0, a wide distribution can be expected (ECHA guidance 7c, 2017). No information on potential target organs is available.

 

Metabolism of Methacrylic esters

Di- and mono-ester hydrolysis

Ester hydrolysis has been established as the primary step in the metabolism of methacrylate esters. In the case of diol di-methacrylate esters the first step would be hydrolysis of one of the ester bonds to produce the corresponding mono-ester followed by subsequent hydrolysis of the second ester bond to produce methacrylic acid (MAA) and the corresponding alcohol 1,3-BDDMA. The metabolic pathway is shown in the category document.

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 (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. For multifunctional methacrylates mostly the same would be the case except, because of the much lower vapour pressure, inhalation is generally not a relevant route of exposure and there is a very low likelihood of a relevant exposure of the nasal epithelium to vapour.

Kinetics data have been reported for the hydrolysis of two multifunctional methacrylates (EGDMA and TREGDMA) by porcine liver carboxylesterase in vitro. For comparison reasons, the results from two lower alkyl methacrylates (EMA and BMA), are also presented in the table below (McCarthy and Witz, 1997). The four studied substances showed comparable hydrolysis ratesin vitro.

Table: Hydrolysis of Acrylate Esters by Porcine Liver Carboxylesterasein vitro (extract from McCarthy and Witz., 1997); supporting data from lower alkyl methacrylates in italics

Ester

Km (mM)

Vmax (nmol/min)

Tetraethyleneglycol dimethacrylate (TREGDMA)

39±15*

2.9±1.0

Ethyleneglycol dimethacrylate (EGDMA)

64±24*

6.9±2.4

Ethyl methacrylate (EMA)

159±90

5.2±2.5

n-Butyl methacrylate (n-BMA)

72±28*

1.8±0.6

*Significantly different (p < 0.05) from ethyl acrylate

 

A recent study, designed to extend an earlier work on lower alkyl methacrylates (Jones 2002, see below) to higher and more complex methacrylate esters, studied the in vitro metabolism of higher and more complex methacrylate esters in rat blood and liver microsomes. This study included three esters of the multifunctional methacrylate category (TREGDMA, EGDMA and 1,4-BDDMA) (DOW, 2013). The results of those studies are summarized in below table.

Table: Elimination Rates, Intrinsic Clearance and Half-life in Rat Liver Microsomes and Whole Rat Blood for Five Methacrylate Esters at 0.25 mM Substrate Concentration (DOW, 2013); data from supporting substances in grey.

 

  Liver Microsomes

  Liver Microsomes

Liver Microsomes

  Whole Blood

  Whole Blood

Whole Blood

Molecule

Clint

(μl/min/mg)

ke

Half‐Life (min)

Clint(μl/min)

ke

Half‐Life (min)

TREGDMA

116

0.23

3.01

219

0.12

5.68

EGDMA

142

0.28

2.45

796

0.44

1.56

1,4-BDDMA

78

0.16

4.46

304

0.17

4.10

MMA

1192

2.38

0.29

19

0.01

63.00

HEMA

74

0.15

4.62

12

0.01

99.00

ke: elimination rate

Clint: intrinsic clearance (ke x volume of incubation / mg/mL microsomal protein)

MMA: Methyl methacrylate (supporting substance); HEMA: Hydroxyethyl methacrylate (supporting substance)

All studied methacrylate esters were rapidly converted to MAA in whole rat blood and rat liver microsomes. Hydrolysis half-lives of the studied category members were in the order of minutes for blood and liver microsomes, respectively.

The incubations in whole rat blood and rat liver microsomes were performed on three separate days with MMA included as a positive control on each day. Rat liver microsome hydrolysis rates for the positive control (MMA) were somewhat variable between days. This was likely due to the rapidity of hydrolysis of MMA. Often, measurable levels of MAA were present even in the zero minute samples and the substrate was completely hydrolyzed by 2 minutes. This made it difficult to accurately calculate hydrolysis rates for MMA in these experiments. However, generally the calculated rates were similar to rates for hydrolysis for MMA reported previously (Jones, 2002; Mainwaring et al., 2001) and confirmed that the in vitro test systems were enzymatically active for each day of incubation experiments. The other studied methacrylates exhibited rat liver microsome hydrolysis rates approximately 10 fold lower than MMA. From its very rapid degradation to MAA, MMA can be understood as suitable donor substance for MAA as common primary metabolite of all category members.

 

A second extension of the metabolism study has been performed in 2017 comparing the metabolic rates of 1,3- and 1,4-BDDMA. This study indicated that the two isomers were indeed very similar, while the metabolic rates of the linear diol ester (1,4-BDDMA) appeared to be slightly higher than those of the branched isomer (1,3-BDDMA; DOW, 2017; see following table).

Table: Elimination Rates, Intrinsic Clearance and Half-life in Rat Liver Microsomes and Whole Rat Blood; Satellite Study Comparison 1,3-BDDMAand 1,4-BDDMA (DOW, 2017)

 

Liver Microsomes

Liver Microsomes

Liver Microsomes

Whole Blood

Whole Blood

Whole Blood

Molecule

Clint
(μl/min/mg)

ke

Half‐Life (min)

Clint (μl/min)

ke

Half‐Life (min)

1,3-BDDMA

116

0.195

3.55

112

0.0559

12.4

1,4-BDDMA

119

0.199

3.48

246

0.123

5.63

Supporting information on Alkyl methacrylates

The above mentioned EMA and n-BMA were also studied in an elaborate series of in vitro studies on carboxylesterase activity with 7 alkyl methacrylates ranging from methyl methacrylate to octyl methacrylate (with increasing ester size) (Jones, 2002). This was used to establish a PB-PK model of in vivo clearance for several tissues (blood, liver, skin and nasal epithelium) from rats and humans, which showed that methacrylate mono-esters are rapidly hydrolyzed by carboxylesterases to methacrylic acid (MAA) and the respective alcohol. The validity of the model was verified with targeted in vivo experiments. Whilst there was a trend of increasing half-life of alkyl methacrylates with increasing chain length (up to octyl), clearance of the parent ester from the body was always in the order of minutes.

Although the absolute rate measurements obtained by Jones differ slightly to those determined by McCarthy and Witz, presumably due to differences in experimental conditions such as protein content etc., the rates obtained for the two lower alkyl methacrylates (EMA and BMA) can be used to draw parallels between the work of the two researchers indicating that the kinetics for the hydrolysis of EGDMA and TREGDMA fall within the range observed by Jones for lower alkyl methacrylates. On this basis the parent ester would be expected to have a short systemic half–life within the body being effectively cleared from the blood within the first or second pass through the liver.

Hydrolysis of the di- and monoester would yield the common metabolite MAA and the respective alcohol.

 

Subsequent metabolism

Methacrylic acid (MAA, CAS 79-41-4)

From the available extensive toxicokinetic data on lower alkyl methacrylates it has been established that the common primary metabolite methacrylic acid is subsequently cleared predominantly via the liver (valine pathway and the TCA (TriCarboxylic Acid) cycle, respectively; ECB, 2002; OECD SIAR, 2009). Methyl methacrylate (MMA) is rapidly degraded in the body to MAA and can thus be understood as metabolite donor for MAA. The metabolic pathway is shown in the category document.

 

1,3-Butandiol (1,3-BD; CAS 107-88-0 (racemat))

Due to structural analogy to 1,4-BD, analogous metabolic pathways are assumed for 1,3-BD. This is confirmed by the detection of ß-hydroxybutyrate (analogous substance to γ-hydroxybutyric acid inFigure 5) and acetoacetate in rats, pigs and chicks after feeding with 1,3-BD (Nahapetian 1971; Rosmos et al. 1975; Tobin et al. 1975; Desrochers et al. 1992). In rabbits, negliable glucuronic acid conjugation was observed after gavage application of 4 mmol/kg bw (Gessner et al., 1960).

The metabolic pathway is shown in the category document.

Glutathione reactivity

A QSAR model for the methacrylates in the category predicts only slight reactivity with glutathione for the category members and no reactivity for the primary metabolite of the methacrylic moiety, methacrylic acid (Cronin, 2012).

Table: QSAR prediction of GSH reactivity (Protein Binding Potency; Cronin, 2012)

Abbreviation

SMILES

Molecular Weight

Log P

Sat. Water Sol. (µg/mL)

Protein Binding Potency

1,3-BDDMA

CC(CCOC(=O)C(=C)C)OC(=O)C(=C)C

226.27

3.1

(1,4- BDDMA)

243

(1,4- BDDMA)

Slightly reactive

 

Studies with methacrylates in vitro confirm low reactivity with GSH, in particular compared to the corresponding acrylates, and have proposed that this is due to steric hindrance of the addition of a nucleophile at the double bond by the alpha-methyl side-group (McCarthy & Witz, 1991, McCarthy et al., 1994, Tanii and Hashimoto, 1982). For example, HEMA as the primary metabolite of EGDMA affected intracellular GSH depletion only in concentrations of 5 or 10 mM in two different human cell types (Chang et al. 2005).

 

Table: Apparent Second-Order Rate Constants for the Reaction of Glutathione with Methacrylate Esters (extract from McCarthy et al., 1994); data from the supporting substances in grey

Ester

App. 2ndorder rate const.

Kapp[L/mol/min]

Tetraethyleneglycol dimethacrylate (TREGDMA)

1.45±1.0

(0.725±0087)*

Ethyleneglycol dimethacrylate (EGDMA)

0.83±0.12

(0.406±0.059)*

Methyl methacrylate (MMA)

0.325±0.059

Ethyl methacrylate (EMA)

0.139±0.022

Butyl methacrylate (BMA)

No appreciable reaction rate

*Bifunctional esters calculated as two independent esters.

 

In vivo data on category members are absent. However, in an inhalation study with MMA at an overtly cytotoxic exposure level off 566 ppm and absolute deposition rates of 35-42μg/min under unidirectional flow, a 20% lowering of nasal non-protein sulfhydryl (NPSH) content was observed, indicative of direct protein reactivity, whereas methacrylic acid exposures had no effect, even at higher delivered dose rates. Around the local (nasal) LOAEL, at an exposure concentration of 109 ppm, MMA had no effect on nasal NPSH levels (Morris and Frederick, 1995). 

Hence, ester hydrolysis is considered to be the major metabolic pathway for alkyl and multifunctional methacrylate esters, with GSH conjugation only playing a minor role in their metabolism.

 

Excretion

As the ester will not survive first pass metabolism in the liver, excretion of the parent compound is of no relevance. The primary metabolite, MAA, is cleared rapidly from blood by standard physiological pathways, with the majority of the administered dose being exhaled as CO2.

 

In summary, the metabolism data and modelling results show that 1,3-BDDMA would be rapidly hydrolysed in the rat.

Human information

There are no relevant toxicokinetic data for the category substances in humans.

For lower alkyl methacrylates there is information indicating that skin absorption rates are lower in human skin compared to rat skin, while for MMA it has been demonstrated that human fate kinetics is similar to those in rats (Jones, 2002).

 

Summary and discussion on toxicokinetics

Methacrylate esters are absorbed by all routes while the dermal absorption is limited with the larger members of the category. Due to the low vapour pressure of the category substances, the dermal route is the primary route of exposure, since inhalation is unlikely. The rate of dermal absorption decreases with increasing ester chain length. All esters are rapidly hydrolyzed by carboxylesterases to methacrylic acid (MAA) and the respective alcohol. In the case of di- and triesters the apparent rate of hydrolysis is highest for the parent ester, but this likely reflects the higher number of hydrolysable target sites instead as opposed to any greater specific activity. Ester hydrolysis can occur in local tissues at the site of contact as well as in blood and other organs by non-specific carboxylesterases. By far the highest enzyme activity has been shown in liver microsomes indicating that the parent ester will be fully metabolized in the liver. Clearance of the parent ester from the body is in the order of minutes. There is a trend towards increasing half-life of the ester in blood with increasing ester chain length, however, none of the esters will survive first pass metabolism in the liver to any significant extent. The primary methacrylic metabolite, MAA, is subsequently cleared rapidly from blood by standard physiological pathways, with the majority of the administered dose being exhaled as CO2. The respective alcohol moieties will undergo further metabolism in the liver.

 

1,3-BDDMA will rapidly be hydrolyzed by unspecific carboxyl esterases in the liver into methacrylic acid and 1,3-BD1,3-Butanediol will be transformed to beta-hydroxybutyrate and, subsequently, to acetoacetate. Metabolites can be degraded to Acetyl-CoA which can be broken down to generate respiratory CO2. MAA is metabolized to succinic acid which will be degraded through the tricarboxylic acid cycle and primarily excreted as CO2. 

 

 

Compliance to REACh requirements

The information requirement is covered with reliable in vitro studies on the primary metabolism, reliable in vitro/ in vivo studies on the metabolism of the methacrylic metabolite MAA as well as reliable publication data on the metabolism of the alcohol metabolite 1,3-BD. All mentioned sources are reliable (Reliability 1 or 2) so that the category/ read across approach can be performed with a high level of confidence. 

 

References

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Chang H-H. et al. (2005). Stimulation of glutathione depletion, ROS production and cell cycle arrest of dental pulp cells and gingival epithelial cells by HEMA. Biomaterials 26, 745-753

Desrochers et al. (1992). Metabolism of R- and S-1,3-butanediol in perfused livers from meal-fed and starved rats. Biochem. J. (285) 647-653

European Chemicals Bureau (2002). European Union - Risk Assessment Report on Methyl methacrylate. European Union - Risk Assessment Report, Vol. 22

Frederick C. B., Udinsky J. R., Finch L (1994).The regional hydrolysis of ethyl acrylate to acrylic acid in the rat nasal cavity. Toxicology letters, 70: 49-56.

Jones O (2002). Using physiologically based pharmacokinetic modelling to predict the pharmacokinetics and toxicity of methacrylate esters. A Thesis submitted to Univ. of Manchester for the degree of Doctor of Philosophy.

Junge W, Krisch K (1975) The carboxylesterases/amidases of mammalian liver and their possible significance. Critical Reviews in Food Science and Nutrition, 371-434

Gessner PK, ParkeDV, Williams RT (1960). Studies in Detoxication. 80. The metabolism of glycols. Biochemical Journal, 74: 1-5

Mainwaring G, Foster JR, Lund V, Green T (2001). Methyl methacrylate toxicity in rat nasal epithelium: Studies of the mechanism of action and comparisons between species. Toxicology 158: 109-118

McCarthy TJ, Hayes EP, Schwartz CS, Witz G (1994) The relationship of selected acrylate esters toward Glutathion and Deoxyribonucleosides in vitro: Structure-activity relationships. Fundam. Appl. Toxicol. 22: 543-548

McCarthy TJ, Witz G (1997).Structure-activity relationships in the hydrolysis of acrylate and methacrylate esters by carboxylesterase in vitro. Toxicology 116: 153-158. Owner company: Published.

NTP (1996) Summary Report on the Metabolism, Disposition, and Toxicity of 1,4-Butanediol, (CAS No. 110-63-4). Toxicity Report Series No. 54, NIH Publication 96-3932S

Morris JB, Frederick CB (1995). Upper respiratory tract uptake of acrylate ester and acid vapors. Inhalation Toxicology 7: 557-574

Nahapetian, A. (1971). Metabolism in Vivo of 1,3-Butanediol in the Rat. Thesis for doctor of science degree, Massachusetts Institute of Technology; cited in U.S. FDA (1997): Food Additives Permitted for Direct Addition to Food for Human Consumption; 1,3-Butylene Glycol; Fed Reg 62, Nr. 92 (21 CFR Part 172)

OECD (2009). SIDS/SIAP/SIAR Category Short-chain Alkyl Methacrylates

Rosmos DR, Belo PS and Leveille GA (1975). Federation Proc. 34, 2186; cited in Joint FAO/WHO Expert Committee on Food Additives: BUTANE-1,3-DIOL. http://www.inchem.org/documents/jecfa/jecmono/v14je03.htm

Satoh T, Hosokawa M (1998). The Mammalian carboxylesterases: From models to functions. Annual Review of Pharmacology and Toxicology 38, 257-288. Medicine and Biology 283, 333-335

Tanii H., Hashimoto K.(1982); Structure-Toxicity Relationship of Acrylates and Methacrylates; Toxicol. Lett. 11: 125-129

Tobin, R. B. et al. (1975). Nutritional and Metabolic Studies in Humans With 1,3- Butanediol. Federation Proceedings, 34:2171-2176, 1975; cited in U.S. FDA (1997): Food Additives Permitted for Direct Addition to Food for Human Consumption; 1,3-Butylene Glycol; Fed Reg 62, Nr. 92 (21 CFR Part 172)