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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Objective of study:
other: Metabolism rate, Km and Vmax determinations.
Principles of method if other than guideline:
- Principle of test:
This study was conducted to investigate in vitro hydrolysis and glutathione conjugation rates of methyl acrylate (MA). Methyl Acrylate was part of a group of materials chosen for initial experimental determination of metabolism rates in rat liver microsomes and whole rat blood at a single substrate concentration (500 µM). Additionally, Km and Vmax determinations were made by performing incubations utilizing various concentrations (32.25, 62.5, 125, 250, and 500 µM) at a single rat liver microsomal protein concentration of 0.1 mg/mL or 0.5 mg/mL. After rates were determined, a third set of incubations were performed to determine the degree of glutathione conjugation in the presence of glutathione transferases (GST).

- Short description of test conditions:
This study was conducted in two stages. The first stage (Stage I) comprised of test material incubation in two different matrices, either liver microsomes or whole blood from F344 rats. Whole blood was obtained from Bioreclamation, LLC (Hicksville, New York, USA) and liver microsomes were obtained from Sekisui Xenotech, LCC (Kansas City, Kansas, USA). The second phase of the study (Stage II) explored glutathione activity via incubation of the test material with tritiated glutathione/glutathione in the presence of glutathione transferase with subsequent chemical analysis.

- Parameters analysed / observed:
Determination of metabolism rate in rat liver microsomes and whole rat blood at a single substrate concentration (500 µM) were made. Additionally, Km and Vmax determinations were made by performing incubations utilizing various concentrations (32.25, 62.5, 125, 250, and 500 µM) at a single rat liver microsomal protein concentration of 0.1 mg/mL or 0.5 mg/mL. After rates were determined, a third set of incubations were performed to determine the degree of glutathione conjugation in the presence of glutathione transferases (GST).
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl acrylate
EC Number:
202-500-6
EC Name:
Methyl acrylate
Cas Number:
96-33-3
Molecular formula:
C4H6O2
IUPAC Name:
methyl acrylate
Specific details on test material used for the study:
Material Name: Methyl Acrylate
Chemical Name: 2-Propenoic acid methyl ester
Lot/Reference/Batch Number: SHBH6895
Purity/Characterization (Method of Analysis and Reference): The non-GLP purity of the test material was determined to be 99.7% by gas chromatography (GC) with identification by Infrared (IR) spectroscopy (Grady, 2016).
Test Material Stability Under Storage Conditions: The test material MA, lot SHBH6895, was not tested for neat test material stability.

Administration / exposure

Statistics:
Descriptive statistics were used, i.e., mean ± standard deviation, or relative standard deviation (standard deviation/mean). All calculations were conducted using Microsoft Excel spreadsheets in full precision mode (15 digits of accuracy). The half-life value of methyl acrylate and the rate of hydrolysis to AA in whole rat blood and rat liver microsomes in Stage IA were determined using GraphPad Prism version 5.03 (GraphPad Software, La Jolla, CA, USA). The percent remaining of MA were fit to one-phase exponential decay curves to determine ke (min^-1) and Clint (µL/min/mg). Calculation of Km and Vmax for the AA formation from acrylate in rat liver microsmes (Stage IB) was also performed using GraphPad Prism v5.03 according to the Michaelis-Menten Kinetic Model.

Results and discussion

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
In rat liver microsomes, Methyl Acrylate hydrolyzed to form the metabolite acrylic acid (AA) with a half-life of less than 8.5 minutes. In whole rat blood, MA quickly metabolized, shown by a significant and (nearly) complete loss of the parent acrylate. Based on the measured concentrations of the remaning parent acrylate, the half-life value for MA was less than 12 minutes in rat blood.  MA formed a single GSH conjugate in the presence of GST. 

Any other information on results incl. tables

Methyl acrylate (MA) was chosen for initial experimental determination of metabolism rates in rat liver microsomes and whole rat blood at a single substrate concentration (500 µM). Additionally, Kmand Vmaxdeterminations were made by performing incubations utilizing various concentrations (32.25, 62.5, 125, 250, and 500 µM) at a single rat liver microsomal protein concentration of 0.1 mg/mL or 0.5 mg/mL. After rates were determined, a third set of incubations were performed to determine the degree of glutathione conjugation to all acrylates in the presence of glutathione transferases (GST).

In rat liver microsomes, MA hydrolyzed to form the metabolite acrylic acid (AA). MA has a half-life of less than 8.5 minutes.

In whole rat blood, MA quickly metabolized, shown by a significant and (nearly) complete loss of the parent acrylate. However, unlike the rat liver microsomal incubations, the concentrations of the formed AA were significantly lower. In fact, except the background levels of AA, no significant AA was observed in all blood incubations of MA. Thus, despite significant loss in parent acrylate, there is not a corresponding formation of AA in blood. Based on the measured concentrations of the remaning parent acrylate, the half-life value for MA was less than 12 minutes in rat blood. 

In the serial substrate concentrations (31.25, 62.5, 125, 250, and 500 µM) of Methyl acrylate, further experiments were conducted to determine Kmand Vmaxvalues for MA hydrolysis to AA in rat liver microsomes.

MA formed GSH conjugates in the presence of GST. The rate of formation of the GSH conjugate was 1.67 nmol/mg protein/min. 

Overall, these in vitro metabolism results imply that MA can be quickly metabolized through hydrolysis to AA and/or glutathione conjugation in vivo.

Applicant's summary and conclusion

Conclusions:
In rat liver microsomes, MA was hydrolyzed to form the metabolite acrylic acid (AA). MA has a half-life of less than 8.5 minutes.
In whole rat blood, MA is metabolized. However, unlike the rat liver microsomal incubations, the concentrations of the acrylate calculated from the formed AA was significantly lower than the concentrations of the remaining corresponding acrylate. Thus, despite significant loss in parent acrylate, there is not a corresponding formation of AA in blood. In fact, AA was not quantifiable from blood incubations of MA. Based on the measured concentrations of the remaning parent acrylate and the concentrations of the parent acrylate calculated from the formed AA, the half-life value for MA is less than 12 minutes in rat blood.
In the serial substrate concentrations (32.25, 62.5, 125, 250, and 500 µM) of MA, further experiments were conducted to determine Kmand Vmaxvalues for MA hydrolysis to AA in rat liver microsomes. Kmand Vmaxvalues were determined for MA. The measured Kmand Vmaxvalues were 2002 (µM) and 512 (µM) and 216 (nmol/min/mg), respectively.
MA formed GSH conjugates in the presence of GST. The rate of formation of these GSH conjugates was 1.67 nmol/mg protein/min. 
Overall, these in vitro metabolism results indicate that MA can be quickly metabolized primarily through hydrolysis to AA and/or glutathione conjugation in vivo.
Executive summary:

This study was conducted to investigate in vitro hydrolysis and glutathione conjugation rates of Methyl acrylate (MA). Methyl acrylate was chosen for initial experimental determination of metabolism rates in rat liver microsomes and whole rat blood at a single substrate concentration (500 µM). Additionally, Kmand Vmaxdeterminations were made by performing incubations utilizing various concentrations (32.25, 62.5, 125, 250, and 500 µM) at a single rat liver microsomal protein concentration of 0.1 mg/mL or 0.5 mg/mL. After rates were determined, a third set of incubations were performed to determine the degree of glutathione conjugation to MA in the presence of glutathione transferases (GST). In rat liver microsomes, MA was hydrolyzed to form the metabolite acrylic acid (AA). MA has a half-life of less than 8.5 minutes.

In whole rat blood, MA is metabolized. However, unlike the rat liver microsomal incubations, the concentrations of the acrylate calculated from the formed AA was significantly lower than the concentrations of the remaining corresponding acrylate. Thus, despite significant loss in parent acrylate, there is not a corresponding formation of AA in blood. In fact, AA was not quantifiable from blood incubations of MA. Based on the measured concentrations of the remaning parent acrylate and the concentrations of the parent acrylate calculated from the formed AA, the half-life value for MA is less than 12 minutes in rat blood.

In the serial substrate concentrations (32.25, 62.5, 125, 250, and 500 µM) of MA, further experiments were conducted to determine Kmand Vmaxvalues for MA hydrolysis to AA in rat liver microsomes. Kmand Vmaxvalues were determined for MA. The measured Kmand Vmaxvalues were 2002 (µM) and 512 (µM) and 216 (nmol/min/mg), respectively.

MA formed GSH conjugates in the presence of GST. The rate of formation of these GSH conjugates was 1.67 nmol/mg protein/min. 

Overall, these in vitro metabolism results indicate that MA can be quickly metabolized primarily through hydrolysis to AA and/or glutathione conjugation in vivo.