2-(2-butoxyethoxy)ethyl methacrylate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

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:

2021

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Metabolic stability of the test item was analysed using pooled liver S9 fractions from male Sprague Dawley (SD) rats. Based on the knowledge gained during method development, in the present metabolic stability study of BDGMA the following test conditions were used: 20 μM of BDGMA were incubated in glass vials with 0.5 mg/ml S9 fractions and after 0, 2, 5,10,15 and 30 minutes samples were collected for analytical detection via LC-MS.

Two types of negative control (NC; n=3) i.e. heat-inactivated S9 fraction and pure assay buffer w/o S9 mix, respectively, were run in parallel to the experimental incubations to verify that any apparent loss of test article in the assay incubation was due to metabolism.

As positive control 1 μM verapamil was incubated in parallel to the test item (n=3), and the depletion of the compound was monitored to demonstrate the enzymatic activity of the S9 fractions. Positive control samples were taken after 0 and 30 minutes.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-butoxyethoxy)ethyl methacrylate

EC Number:

230-813-8

EC Name:

2-(2-butoxyethoxy)ethyl methacrylate

Cas Number:

7328-22-5

Molecular formula:

C12H22O4

IUPAC Name:

2-(2-butoxyethoxy)ethyl 2-methylprop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

TEST MATERIAL:
- Name of test material: BDGMA

Radiolabelling:

no

Test animals

Species:

other: Rat liver S9 fractions

Strain:

Sprague-Dawley

Sex:

male

Administration / exposure

Vehicle:

DMSO

Duration and frequency of treatment / exposure:

sampling after 0, 2, 5,10,15 and 30 minutes

Doses / concentrations

Dose / conc.:

20 other: µM

Remarks:

The final test concentration was determined during experimental development.

No. of animals per sex per dose / concentration:

not applicable; in vitro test

Control animals:

other: not applicable, in vitro test

Positive control reference chemical:

As positive control 1 μM verapamil was incubated in parallel to the test item (n=3), and the depletion of the compound was monitored to demonstrate the enzymatic activity of the S9 fractions. Positive control samples were taken after 0 and 30 minutes.

Details on study design:

- Dose selection rationale: based on experimantal method development (analytical detection)

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Time and frequency of sampling: after 0, 2, 5, 10, 15 and 30 minutes


METABOLITE CHARACTERISATION STUDIES
- Method type(s) for identification: Liquid chromatography – mass spectrometry (LC-MS)
- Limits of detection and quantification:
- Other: after incubation time samples were samples were processed for ACN precipitation and quantitative bioanalysis (addition of two volumes (i.e. 400 μl) stop solution (ACN containing the ISTD).

Statistics:

Descriptive statistics were used, i.e., mean ± standard deviation. All calculations in the database were conducted using Microsoft Excel.

Results and discussion

Main ADME results

Type:

metabolism

Results:

The ester was rapidly converted into MAA and the respective alcohol. (approx. 1.3 min)

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Methacrylic acid (MAA) and Butyl diglycol

Any other information on results incl. tables

The metabolic stability of the test item butyldiglycol methacrylate (BDGMA) was analysed using rat liver S9 fractions fortified with Phase I metabolism cofactor NADPH. The time-dependent decrease of the initial test concentration of 20 µM BDGMA was investigated after incubation with liver S9 mix (0.5 mg microsomal protein/ml) for several incubation time points (i.e. 0, 2, 5, 10, 15 and 30 minutes). Moreover, the formation of the primary metabolites, i.e. methacrylic acid and butyldiglycol was detected as an additional proof of the enzymatic metabolism.

Negative controls served as a monitoring system for non-enzymatic degradation or non-specific binding effects of the test item. One negative control (NC) with heat-inactivated S9 fractions and another one with buffer only (i.e. negative control without S9 mix and NADPH) were integrated in the assay.

Remaining BDGMA concentrations and percentages of test item remaining after 30 minutes of incubation with rat liver S9 fractions as well as the formation of the two metabolites methacrylic acid and butyldiglycol are shown in Table 1 - Table 3 . Depletion of parent compound and formation of the two metabolites were in good accordance reflecting well the rapid and complete hydrolysis of BDGMA. However, found BDGMA concentrations were only 35% of the initial test concentration (i.e. 7038.8 nM, see Table 1), indicating a very fast degradation immediately after the reaction start.

In the negative control group with heat-inactivated S9 mix, 97.9% remaining BDGMA was measured after 30 min of incubation. In the negative control group using only buffer without S9 mix, BNMA was also stable. 108% of the test item could be detected after 30 minutes of incubation. These findings confirm that non-CYP mediated metabolic degradation processes as well as unspecific binding of the test item to the assay system can be excluded.

Table 2: Remaining BDGMA (nominal initial concentration: 20 µM): measured concentration and calculated percentage of remaining test item after incubation with rat liver S9 fractions for different time points, (n=3)

 

Remaining BDGMA concentration				% remaining BNMA of initial concentration
Time [min] (±0.25 min)	Mean (nM)	SD (nM)	%CV[1]	Time [min] (±0.25 min)	Mean (%)
0	7038.8	1032.5	14.7	0	100.0
2	2603.2	222.5	8.5	2	37.0
5	484.4	25.4	5.2	5	6.9
10	483.7	110.9	22.9	10	6.9
15	178.8	16.8	9.4	15	2.5
30	0.0	0.0	n.a.	30	0.0

 

Table 3: Measured concentration and calculated percentage of formed metabolite benzyl alcohol after incubation of BDGMA (nominal initial concentration: 20 µM) with rat liver S9 fractions for different time points, (n=3)

Formed butyldiglycol concentration				% formed butyldiglycol of initial concentration in BDGMA
Time [min] (±0.25 min)	Mean (nM)	SD (nM)	%CV	Time [min] (±0.25 min)	Mean (%)
0	16032.1	3793.5	23.7	0	80.2
2	18597.8	642.6	3.5	2	93.0
5	21735.7	885.1	4.1	5	108.7
10	17063.4	5768.2	33.8	10	85.3
15	18961.3	2316.1	12.2	15	94.8
30	20902.4	1419.3	6.8	30	104.5

 

Table 3: Measured concentration and calculated percentage of formed metabolite methacrylic acid after incubation of BDGMA (nominal initial concentration: 20 µM) with rat liver S9 fractions for different time points, (n=3)

Formed methacrylic acid concentration				% formed methacrylic acid of initial concentration in BDGMA
Time [min] (±0.25 min)	Mean (nM)	SD (nM)	%CV	Time [min] (±0.25 min)	Mean (%)
0	15998.0	1286.3	8.0	0	80.0
2	18639.3	485.5	2.6	2	93.2
5	19576.3	1584.3	8.1	5	97.9
10	15923.2	5189.9	32.6	10	79.6
15	18533.2	360.7	1.9	15	92.7
30	22321.8	1424.4	6.4	30	111.6

 

 

 

Applicant's summary and conclusion

Conclusions:

The aims of the present study were to characterise the test item butyldiglycol methacrylate (BDGMA) with respect to its metabolic stability in rat liver S9 fractions and the quantitative determination of its two primary metabolites methacrylic acid and butyldiglycol.
Metabolic stability of the test item was analysed using pooled liver S9 fractions from male Sprague Dawley (SD) rats. In the present metabolic stability study of BDGMA the following test conditions were used: 20 µM of BDGMA were incubated in glass vials with 0.5 mg/ml S9 fractions and after 0, 2, 5, 10, 15 and 30 minutes samples were collected for analytical detection via LC-MS. Test item BDGMA was rapidly metabolised resulting in a half-life of 1.3 minutes and a Clint value of 1074.5 µl/min/mg protein.
In the negative control groups using only buffer without S9 mix or heat-inactivated S9 mix, BDGMA was stable over the investigated incubation period: 108.0% and 97.9% remaining compound could be detected after 30 minutes of incubation, respectively.
Additionally, quantitative determination of the two BDGMA metabolites methacrylic acid and butyldiglycol was performed reflecting well the rapid hydrolysis of the test item.

Executive summary:

The aims of the present study were to characterise the test item butyldiglycol methacrylate (BDGMA) with respect to its metabolic stability in rat liver S9 fractions and the quantitative determination of its two primary metabolites methacrylic acid and butyldiglycol.

Metabolic stability of the test item was analysed using pooled liver S9 fractions from male Sprague Dawley (SD) rats. In the present metabolic stability study of BDGMA the following test conditions were used: 20 µM of BDGMA were incubated in glass vials with 0.5 mg/ml S9 fractions and after 0, 2, 5, 10, 15 and 30 minutes samples were collected for analytical detection via LC-MS. Test item BDGMA was rapidly metabolised resulting in a half-life of 1.3 minutes and a Cl_int value of 1074.5 µl/min/mg protein.  

In the negative control groups using only buffer without S9 mix or heat-inactivated S9 mix, BDGMA was stable over the investigated incubation period: 108.0% and 97.9% remaining compound could be detected after 30 minutes of incubation, respectively.

Additionally, quantitative determination of the two BDGMA metabolites methacrylic acid and butyldiglycol was performed reflecting well the rapid hydrolysis of the test item.

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