Benzyl methacrylate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

dermal absorption

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Specific details on test material used for the study:

- Name of test material (as cited in study report): Benzyl methacrylate

Details on test animals or test system 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 4.563 μg/cm²/h; the relative dermal absorption is low

Based on a molecular weight of 176.21 g/mol and ag Kow of 3.1, the predicted flux of BNMA is 4.563 μg/cm²/h; the relative dermal absorption is low.

Conclusions:

The dermal absorption of BNMA is predicted to be low; the predicted flux is 4.563 μg/cm²/h.

Executive summary:

The dermal absorption (steady-state flux) of BNMA has been estimated by calculation using the principles defined in the Potts and Guy prediction model.

Based on a molecular weight of 176.21 g/mol and ag Kow of 3.1, the predicted flux of BNMA is 4.563 μg/cm²/h; the relative dermal absorption is low.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

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 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 BNMA the following test conditions were used: 20 μM of BNMA 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

Specific details on test material used for the study:

Name of test material: Benzyl methacrylate

Radiolabelling:

no

Species:

other: Rat liver S9 fractions

Strain:

Sprague-Dawley

Sex:

male

Vehicle:

DMSO

Duration and frequency of treatment / exposure:

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

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.

Type:

metabolism

Results:

The ester was rapidly converted into MAA and the respective alcohol. (<0.25 min)

Metabolites identified:

yes

Details on metabolites:

Methacrylic acid (MAA) and benzyl alcohol

As shown in the Table 2, already at the first time point 0 min (+0.25 min), only 0.3 μM BNMA was detectable which corresponds to 1.5% of initial BNMA concentration. At the same time, the analytical measurements revealed the formation of the two metabolites Benzyl alcohol (Table 3) and Methacrylic acid (only qualitative detection of MAA). These findings clearly indicate that BNMA was rapidly metabolised within the first minute.

Table 2: Remaining BNMA (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 BNMA concentration				% remaining BNMA of initial concentration
Time [min] (±0.25 min)	Mean (nM)[1]	SD (nM)	%CV[2]	Time [min] (±0.25 min)	Mean (%)
0	301.4	120.0	39.8	0	1.5
2	0.0	0.0	n.a.	2	0.0
5	0.0	0.0	n.a.	5	0.0
10	0.0	0.0	n.a.	10	0.0
15	0.0	0.0	n.a.	15	0.0
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 BNMA (nominal initial concentration: 20 µM) with rat liver S9 fractions for different time points, (n=3)

Formed Benzyl alcohol concentration				% formed Benzyl alcohol of initial concentration in BNMA
Time [min] (±0.25 min)	Mean (nM)	SD (nM)	%CV	Time [min] (±0.25 min)	Mean (%)
0	22385.6	2407.1	10.8	0	111.93
2	21714.8	2761.8	12.7	2	108.57
5	22218.3	1915.1	8.6	5	111.09
10	19567.2	423.3	2.2	10	97.84
15	20073.7	3283.2	16.4	15	100.37
30	23577.3	5884.2	25.0	30	117.89

[1]values were at or below quantification limit (LOQ 400 nM)

[2]Coefficient of variation expressed as percentage of SD divided by mean value

Taken all findings together it can be concluded that 20 μM BNMA was completely metabolized within very short time (< 0.25 min; at time point 0 min) in presence of 0.5 mg/ml rat liver S9 fractions (i.e. in presence of phase I and II enzymes). At time point 0 min already no BNMA was detectable and the formation of the primary metabolites could be shown as a proof of the very rapid enzymatic hydrolysis of BNMA. Due to the immediate cleavage of BNMA in presence of rat liver S9 fractions, half-life and intrinsic clearance could not be calculated.

Conclusions:

Taken all findings together it can be concluded that 20 μM BNMA was completely metabolized within very short time (< 0.25 min; at time point 0 min) in presence of 0.5 mg/ml rat liver S9 fractions (i.e. in presence of phase I and II enzymes). At time point 0 min already no BNMA was detectable and the formation of the primary metabolites could be shown as a proof of the very rapid enzymatic hydrolysis of BNMA. Due to the immediate cleavage of BNMA in presence of rat liver S9 fractions, half-life and intrinsic clearance could not be calculated.

Executive summary:

Taken all findings together it can be concluded that 20 μM BNMA was completely metabolized within very short time (< 0.25 min; at time point 0 min) in presence of 0.5 mg/ml rat liver S9 fractions (i.e. in presence of phase I and II enzymes). At time point 0 min already no BNMA was detectable and the formation of the primary metabolites could be shown as a proof of the very rapid enzymatic hydrolysis of BNMA. Due to the immediate cleavage of BNMA in presence of rat liver S9 fractions, half-life and intrinsic clearance could not be calculated.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Description of key information

BNMA is likely to be readily absorbed by oral and dermal route. Due to the low vapour pressure, inhalation is unlikely.

The ester is rapidly hydrolysed by carboxylesterases to methacrylic acid (MAA) and Benzyl alcohol. The rapid hydrolysis of BNMA has been shown in a metabolism study. Due to the small size and the solubility of the breakdown products in aqueous media, it is likely that they will be widely distributed. The available data do not show accumulation in any organ or tissue. For BNMA, no specific target has been identified.

The primary metabolite, MAA, is subsequently cleared rapidly from blood by standard physiological pathways, with the majority of the administered dose being exhaled as CO2. Benzyl alcohol is oxidised to Benzoic acid, which is subsequently conjugated with Glycine and renally excreted.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100

Additional information

Absorption

a) Oral

Based on the some physico-chemical properties of BNMA (molecular weight, physical state, water solubility, lipophilicity) the absorption from the gastrointestinal tract is the main route of absorption.

For chemical safety assessment a dermal absorption rate of 100% is assumed as a worst-case default value according to R8 guidance (ECHA, 2012).

b) Dermal

Based on the physico-chemical properties of BNMA, the dermal availability of the substance is very limited. The water solubility of the substance can be classified as moderate and favours the partitioning from the stratum corneum into the epidermis. Besides, the logPow favours the penetration into the stratum corneum and hence the absorption across the skin. In a well conducted QSAR model, the predicted steady-state flux of BNMA is 4.563 μg/cm²/h (Heylings, 2013) and is classified by the author as low in comparison to other methacrylates. The low absorption rate of BNMA after dermal application is supported by data of an acute dermal toxicity study showing no effects and consequently the LD50-value is above 2000 mg/kg bw/d. It is well-known that the ester bonds of BNMA can be hydrolysed by carboxylesterases in the skin, although to a much lesser extent than in the gastrointestinal tract due to the lower level of enzymes in the skin.

The breakdown products (MAA and 1,3-BDDMA) may then be absorbed and enter the bloodstream.

Based on LLNA data, the substance is classified as skin sensitizer. This indicates that the substance can be taken up after dermal application and induce a positive reaction in the skin.

For chemical safety assessment a dermal absorption rate of 100% is assumed as a worst-case default value according to R8 guidance (ECHA, 2012).

c) Inhalation

The vapour pressure of BNMA was determined to be 0.03 hPa (0.003 kPa) at 20°C. This is clearly below the general cut-off value of 0.5 kPa indicating a low volatility and hence poor availability for inhalation as vapour (ECHA, 2017). Solid particles, however, may be available for absorption after inhalation of an aerosolized substance, although this does not seem likely considering the size of the molecule. There are no studies regarding absorption of BNMA from the respiratory tract.

Nevertheless, for chemical safety assessment an inhalation absorption rate of 100% is assumed as a worst-case default value according to R8 guidance (ECHA, 2012).

Distribution

Since BNMA undergoes enzymatic hydrolysis especially in the gastrointestinal tract, the breakdown products (MAA and BA) are likely to be widely distributed due to their small size and solubility in aqueous media. The parent compound has a high permeability across lipid membranes (log P_OW 3.10), but the degradation products do not contain any lipophilic groups. The available data do not show accumulation in any organ or tissue, either. No target organs have been identified for BNMA.

 

Metabolism 

Ester hydrolysis induced by carboxylesterases has been established as the primary step in the metabolism of methacrylate esters. Hence, the first step in metabolism of aromatic methacrylate esters, e.g. BNMA is the cleavage of the ester bond to produce thus the carboxylic acid (i.e. methacrylic acid) and the corresponding alcohol, i.e. benzyl alcohol.

These primary metabolites are further metabolised in vivo which highlights the hydrolysis as key event in the detoxification process of methacrylates. Consequently, the hydrolysis rate provides information about the toxicity potential and indicates whether the hazard assessment can be based on the metabolites MAA and Benzyl alcohol.  

The methacrylic metabolite, MAA is cleared rapidly from blood by standard physiological pathways, with the majority of the administered dose being exhaled as CO₂.

The alcohol metabolite, Benzyl alcohol, is oxidised rapidly via benzyl aldehyde to benzoic acid. Most of the ingested benzyl alcohol, benzaldehyde and benzoic acid is excreted renally after transformation to hippuric acid.

 

In a metabolism study, the above-mentioned rapid ester hydrolysis of BNMA catalysed by unspecific carboxylesterases present in rat liver S9 fractions has been shown. In this in vitro assay, it was observed that 20 µM BNMA was completely metabolized within very short time (< 0.25 min; at time point 0 min) in presence of 0.5 mg/ml rat liver S9 fractions (i.e. in presence of phase I and II enzymes). At time point 0 min already no BNMA was detectable and the formation of the primary metabolites could be shown as a proof of the very rapid enzymatic hydrolysis of BNMA.

 

Excretion

The parent compound BNMA is not likely to be excreted as such due to the rapid hydrolysis of the ester bond. The metabolites of the substance will be cleared from blood circulation by physiological pathways. The methacrylic moiety will mainly be exhaled as CO₂. The alcohol moiety will mainly be excreted via urine.