Isobutyl acrylate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 Jan 2001 to 30 Jan 2001

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

Isobutyl acrylate was tested for relative rates of hydrolysis by mammalian esterase.

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Isobutyl acrylate
- Analytical purity: 99.88 %
- Lot/batch No.: S0308-01-GA

Radiolabelling:

no

Dose / conc.:

0.2 other: mM

Dose / conc.:

0.5 other: mM

Dose / conc.:

2 other: mM

Key result

Toxicokinetic parameters:

other: Isobutyl acrylate was rapidly hydrolyzed at rates ranging between 45 - 47 µmoles/min/mg protein (3 - 27 %) after 2 minutes and 38 - 49 µmoles/min/mg protein (7 - 57 %) after 5 minutes of incubation at 37°C.

Metabolites identified:

no

The % conversion results of isobutyl acrylate (an average of 3 determinations)

Concentration (mM)	% Conversion / Rate*
	At 2 min.	At 5 min.
2.00	2.83 / 47.17	7.28 / 48.53
0.50	14.88 / 62.00	34.17 / 56.95
0.20	27.07 / 45.12	57.44 / 38.29

* in μmoles / min / mg protein

Isobutyl acrylate was rapidly hydrolyzed at rates ranging between 45 - 47 µmoles/min/mg protein (3 - 27 %) after 2 minutes and 38 - 49 µmoles/min/mg protein (7 - 57 %) after 5 minutes of incubation at 37°C.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

n-Butyl acrylate (BA) (2,3-14C-labelled) was administered by iv injection to male Fischer 344 rats. Radioactivity was determined in the urine, feces and different tissue samples. Binding of BA derived radioactivity to components of blood was investigated and identification of metabolites in urine, bile and kidney extracts was performed.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): n-Butyl acrylate
- Analytical purity: >99%
- Impurities (identity and concentrations): 10-55 ppm hydroquinone monomethyl ether
- Radiochemical purity (if radiolabelling): >98%
- Specific activity (if radiolabelling): 218 μCi/mmol
- Locations of the label (if radiolabelling): n-Butyl [2,3-14C]acrylate

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Raleigh, NC)
- Age at study initiation: 8-10 week
- Weight at study initiation: 180-220 g
- Individual metabolism cages: yes
- Diet: NIH31 rat chow, ad libitum
- Water: ad libitum

Route of administration:

intravenous

Vehicle:

other: 1:1:8, v/v, solution of ethanol, Emulphor EL-620 (GAF, New York, NY), and water

Duration and frequency of treatment / exposure:

single

Dose / conc.:

40 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

18

Control animals:

no

Details on study design:

Radiolabeled BA was diluted as needed with unlabeled BA to administer 20 µCi/kg bw for iv dose. At sacrifice, animals were immediately dissected and tissues were removed, and weighed. Tissues were stored at -20°C and analysed later for radioactivity. Excretion of BA derived radioactivity was determined by complete collection of urine and feces from each rat. Biliary excretion was determined by collection of serial bile samples for 4 h after injection of BA. The volume of each sample was measured and the radioactivity was determined using liquid scintillation counter.

Rats maintained 6 hr or longer were housed in individual metabolism cages for separate collection of urine and feces. Animals were killed (3 per time point) by cervical dislocation at time points from 15 min to 3 days after BA administration. Radioactivity was determined in the urine, feces and different tissue samples. Binding of BA derived radioactivity to components of blood was investigated and identification of metabolites in urine, bile and kidney extracts was performed.

Binding ot BA-derived radioactivity to components of blood was investigated by collection of blood 24 h after administration of BA. The red blood cells were separated and hemolyzed by osmotic pressure. Centifugation resulted in formation of pellet which was washed with ethanol/ether (3:1, v/v) to extract lipid-bound radioactivity. The radioactivity was determined in a liquid scintillation counter and the amount of protein was estimated in the pellet using method of Lowry et al (J. Biol. Chem. 193:265-275, 1951).
Collection of CO2 and volatiles was done by placing rats in glass metabolism cages after administration of BA. The percentage of the total dose of 14C-BA eliminated as 14CO2 and volatiles was determined by counting triplicate 1 ml aliquots of each trapping solution.

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, bile, 0-15-min kidney extract
- Method type(s) for identification: HPLC, NMR, FAB-MS

Type:

absorption

Results:

Butyl acrylate is rapidly absorbed after i.v. administration.

Type:

distribution

Results:

The greatest quantitity of BA-derived radioactivity was found in the adipose tissue.

Type:

metabolism

Results:

The two major metabolites in urine were identified as N-acetyl-S-(2-carboxyethyl)cysteine and N-acetyl-S-(2-carboxyethyl)cystein-S-oxide. S-(2-carboxyethyl) glutathione butyl ester was isolated in bile.

Type:

excretion

Results:

The acrylate moiety is oxidised to CO2 which is eliminated via the exhaled air (up to 45 % of the administered radiolabel).

Details on absorption:

After iv administration the labelled butyl acrylate was rapidly absorbed and metabolized. The acrylate moiety was metabolized primarily to CO2, accounting for elimination of up to 45 % of the administered radiolabel. After i.v. administration the inital clearance of radioactivity from the tissues was very rapid and the elimination decreased to a negligible rate after 2 hours.

Details on distribution in tissues:

Total radioactivity in the major tissues (adipose, muscle, liver, skin) was relatively constant from 2 to 24 hours. The blood plasma contained 21% of the radioactivity with the remaining 70% in the red blood cells. The cells were hemolyzed and fractionated further to reveal 5% of the total in the cellular contents with the remaining radioactivity in the membranous portion. Solvent extraction of the membranes removed another 12% of the total in the blood as lipid-bound radioactivity. Less than 3% of the total was found in soluble nucleic acids, with over 55% of the total in blood being covalently bound to the protein fraction of the red blood cell membranes. The amount of bound BA-derived radioactivity was determined to be 1.17 nmol/mg of protein.

Details on excretion:

After iv administration, elimination as CO2 was up to 45 % of the administered radiolabel. Elimination in urine and feces accounted for approximately 16 and 1 % of the dose, respectively.

Metabolites identified:

yes

Details on metabolites:

The two major metabolites in urine were identified as N-acetyl-S-(2-carboxyethyl)cysteine and N-acetyl-S-(2-carboxyethyl)cystein-S-oxide. In addition, S-(2-carboxyethyl) glutathione butyl ester was isolated in bile.

Distribution and excretion of BA-derived radioactivity 24 h administration

Tissue	% of total dose*
Blood	3.9
Liver	2.0
Kidney	0.3
Skin	2.7
Adipose	12.2
Muscle	5.2
Urine	15.6
Feces	1.2
CO2	45.3
Volatiles	1.3

*Mean from three animals

After i.v. administration it seemed that there was a slight shift of metabolism towards GSH conjugation. The authors discussed that the conjugation of this minor pathway may occur between GSH and intact n-butyl acrylate, the isolation of S-(2-carboxyethyl) glutathione butyl ester in bile provides further evidence that this conjugation occurs before hydrolysis of the ester. Results of this study indicated that the major portion of butyl acrylate dose was hydrolyzed to acrylic acid, which was further metabolized to compounds available for oxidative metabolism.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

n-Butyl acrylate (BA) (2,3-14C-labelled) was administered by gavage to male Fischer 344 rats. Radioactivity was determined in the urine, feces and different tissue samples. Identification of metabolites in urine, bile and kidney extracts was performed.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): n-Butyl acrylate
- Analytical purity: >99%
- Impurities (identity and concentrations): 10-55 ppm hydroquinone monomethyl ether
- Radiochemical purity (if radiolabelling): >98%
- Specific activity (if radiolabelling): 218 μCi/mmol
- Locations of the label (if radiolabelling): n-Butyl [2,3-14C]acrylate

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Raleigh, NC)
- Age at study initiation: 8-10 week
- Weight at study initiation: 180-220 g
- Individual metabolism cages: yes
- Diet: NIH31 rat chow, ad libitum
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

other: corn oil or 1:1:8, v/v, solution of ethanol, Emulphor EL-620 (GAF, New York, NY), and water

Duration and frequency of treatment / exposure:

single

Dose / conc.:

4 mg/kg bw (total dose)

Dose / conc.:

40 mg/kg bw (total dose)

Dose / conc.:

400 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

18

Control animals:

no

Details on study design:

Radiolabeled BA was diluted as needed with unlabeled BA to administer 20 µCi/kg bw for 40 and 400-mg/kg bw doses. Undiluted radiolabeled BA was adminsitered at 7µCi/kg bw for 4-mg/kg bw dose. At sacrifice, animals were immediately dissected and tissues were removed, and weighed. Tissues were stored at -20°C and analysed later for radioactivity. Excretion of BA derived radioactivity was determined by complete collection of urine and feces from each rat.

Collection of CO2 and volatiles was done by placing rats in glass metabolism cages after administration of BA. The percentage of the total dose of 14C-BA eliminated as 14CO2 and volatiles was determined by counting triplicate 1 ml aliquots of each trapping solution.

To eliminate the possibility that metabolism was effected by the vehicel, 3 rats were administed with 40 mg/kb bw BA in the ethanol/emulphor/water solution and comapared with the animals dosed in corn oil.

Rats maintained 6 hr or longer were housed in individual metabolism cages for separate collection of urine and feces. Animals were killed (3 per time point) by cervical dislocation at time points from 15 min to 3 days after BA administration. Radioactivity was determined in the urine, feces and different tissue samples. Identification of metabolites in urine, bile and kidney extracts was performed.

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, bile, 0-15-min kidney extract
- Method type(s) for identification: HPLC, NMR, FAB-MS

Type:

absorption

Results:

Butyl acrylate was rapidly absorbed after oral administration.

Type:

distribution

Results:

The greatest quantitity of BA-derived radioactivity was found in the adipose tissue.

Type:

metabolism

Results:

The two major metabolites in urine were identified as N-acetyl-S-(2-carboxyethyl)cysteine and N-acetyl-S-(2-carboxyethyl)cystein-S-oxide.

Type:

excretion

Results:

The acrylate moiety is oxidised to CO2 which is eliminated via the exhaled air (up to 75 % of the administered radiolabel).

Details on absorption:

After oral administration the labelled butyl acrylate was rapidly absorbed and metabolized.

Details on distribution in tissues:

The distribution of BA-derived radioactivity in major tissues (except adipose tissue) was not significantly affected by the dose in the range studied. The greatest quantitity of BA-derived radioactivity was found in the adipose tissue. There was no significant difference in the cumulative 24-h excretion data of the oral dose in the ethanol/emulphor/water solution when compared with the oral dose in corn oil.

Details on excretion:

After oral administration, elimination as CO2 was up to 75 % of the administered radiolabel. Elimination in urine and feces accounted for approximately 10 and 2 % of the dose, respectively.

Metabolites identified:

yes

Details on metabolites:

The acrylate moiety was metabolized primarily to CO2. N-acetyl-S-(2-carboxyethyl)cysteine and N-acetyl-S-(2-carboxyethyl)cystein-S-oxide are the two major metabolites in urine.

Distribution and excretion of BA-derived radioactivity 24 h administration

Tissue	% of total dose*
	4 mg/kg	40 mg/kg	400 mg/kg
Blood	1.9	2.0	2.0
Liver	1.9	2.6	2.3
Kidney	0.3	0.3	0.4
Skin	3.4	2.9	3.2
Adipose	1.6	8.6	5.7
Muscle	5.9	5.4	5.7
Urine	12.6	7.7	7.6
Feces	2.2	2.1	2.1
CO2	74.2	65.5	78.0
Volatiles	1.6	0.8	1.7

*Mean from three animals

Comparison of tissue distribution (with the exception of adipose tissue) and excretion data indicated that gastrointestinal absorption and metabolism were not affected by dose in the range studied. The appearance of minor metabolites in the initial urine (0-4-hr) of rats dosed with 400 mg/kg BA indicated that some minor saturation of metabolic pathways occurred at this high dose. BA dose in male Fischer rats is hydrolyzed by carboxylesterases to acrylic acid and butanol, with a smaller portion being conjugated with endogenous GSH to be subsequently excreted as mercapturic acids in the urine.

Reference 4

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012 - 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study which meets basic scientific principles.

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

Enzymatic hydrolysis of butyl acrylate after incubation with different physiological media (whole blood, blood plasma, rat liver S9 fraction) in vitro.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Remarks:

Doses / Concentrations:
butyl acrylate: 1 mM, methyl methacrylate: 0.5 mM

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: S9 fraction: 0.74 min.

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: blood plasma: 8.15 min.

Metabolites identified:

yes

Details on metabolites:

Acrylic acid

Results:

S9 -fraction:

Time (min.)	0	0.5	1.0	2.5	5.0
iso-butyl acrylate (mM)	1.248	0.752	0.423	0.119	0
acrylic acid (mM)	0	0.299	0.460	0.861	0.756

Blood plasma:

Time (min.)	0	5	10	30
iso-butyl acrylate (mM)	1.251	0.679	0.421	0.090
acrylic acid (mM)	0	0.258	0.506	0.636

Summary:

After incubation of iso-butyl acrylate with S9 -fraction of rat liver, the test substance was rapidly and completely (100%) hydrolyzed. The half-life with regards to elimination of iso-butyl acrylate was 0.74 min.

Halfe-life	S9 -fraction	blood plasma
iso-butyl acrylate T1/2 (min.)	0.74	8.15

Reference 5

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012 - 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study which meets basic scientific principles.

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

Enzymatic hydrolysis of butyl acrylate after incubation with different physiological media (whole blood, blood plasma, rat liver S9 fraction) in vitro.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Remarks:

Doses / Concentrations:
butyl acrylate: 1 mM, methyl methacrylate: 0.5 mM

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: S9 fraction: 0.84 min.

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: whole blood: 2.61 min.

Test no.:

#3

Toxicokinetic parameters:

half-life 1st: blood plasma: 8.45 min.

Metabolites identified:

yes

Details on metabolites:

Acrylic acid

Results:

S9 -fraction:

Time (min.)	0	0.5	1.0	2.5	5.0
butyl acrylate (mM)	1.148	0.760	0.560	0.146	0
acrylic acid (mM)	0	0.273	0.392	0.879	0.963

Whole blood:

Time (min.)	0	5	10	30	60
butyl acrylate (mM)	1.053	0.240	0.074	0	0
acrylic acid (mM)	0	0.069	0.081	0.093	0.101

Blood plasma:

Time (min.)	0	5	10	30
butyl acrylate (mM)	1.008	0.589	0.420	0.082
acrylic acid (mM)	0	0.102	0.189	0.273

Summary:

After incubation of butyl acrylate with S9 -fraction of rat liver, the test substance was rapidly and completely (100%) hydrolyzed. The half-life with regards to elimination of butyl acrylate was 0.84 min. associated with a concurrent increase of acrylic acid (T1/2: 1.21 min.).

Halfe-life	S9 -fraction	whole blood	blood plasma
butyl acrylate T1/2 (min.)	0.84	2.61	8.45
acrylic acid T1/2 (min.)	1.21	64.20	20.72

Reference 6

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

other: Hydrolysis and glutathione conjugaison

Principles of method if other than guideline:

ln Vitro Hydrolysis in rat liver microsomes and whole rat blood and glutathione conjugation rates in the presence of glutathione transferases (GST).

GLP compliance:

no

Specific details on test material used for the study:

Other test substances:
- Glacial Acrylic Acid (AA)
Chemical Name: 2-Propenoic acid
Synonyms Acrylic acid
Lot/Reference/Batch Number: A829I25023
Purity: 99.69 wt%

- Methyl Acrylate (MA)
Chemical Name: 2-Propenoic acid methyl ester
Lot/Reference/Batch Number: SHBH6895
Purity: 99.7 wt%

- Ethyl Acrylate (EA)
Chemical Name: 2-Propenoic acid ethyl ester
Lot/Reference/Batch Number: A829H7K000
Purity: 99.9 wt%

- n-Butyl acrylate (BA)
Chemical Name: 2-Propenoic acid butyl ester
Synonyms: Butyl acrylate, BA - 15 PPM MEHQ (4-Methoxyphenol)
Lot/Reference/Batch Number: A829H7D000
Purity: 99.70 wt%

- iso-Butyl Acrylate (iso-BA)
Chemical Name: 2-Propenoic acid, 2-methylpropyl ester
Lot/Reference/Batch Number: 08520136W0
Purity: 99.67 wt%

- tert-Butyl Acrylate (tert-BA)
Chemical Name: 2-Propenoic acid 1,1-dimethylethyl ester
Lot/Reference/Batch Number: SHBJ2365
Purity: 99.9 wt%

- Hydroxyethyl Acrylate (HEA)
Chemical Name: 2-Propenoic acid 2-hydroxyethyl ester
Lot/Reference/Batch Number: A022HC4006
Purity: 97.52 wt%

- Hydroxypropyl Acrylate (HPA)
Chemical Name: 2-Propenoic acid monoester with 1,2-propanediol
Lot/Reference/Batch Number: A022H6F007
Purity: 97.87 wt%

- 2-Propylheptyl Acrylate (2-PHA)
Chemical Name: 2-Propenoic acid 2-propylheptyl ester
Lot/Reference/Batch Number: 0017077376
Purity/Characterization: The non-GLP certificate of analysis lists the water content as 0.02%

Radiolabelling:

yes

Remarks:

Deuterium labeled acrylates were obtained for each acrylate and were used as internal standards.

Species:

rat

Strain:

Fischer 344

Route of administration:

other: in vitro

Positive control reference chemical:

Hydroxyethyl Acrylate (HEA) was used as the positive control for microsomal and blood incubations to verify the enzyme hydrolysis activity in both microsomes and blood. This material was utilized during each run of incubations.

Details on study design:

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.

Stage I – Acrylate Hydrolysis in Liver Microsomes and Blood from F344 Rats
Stage IA – Incubation with Rat Liver Microsomes or Whole Blood
The rat liver microsomes (20 mg/mL) were removed from -80°C storage and thawed on wet ice. Blood was removed from cold storage (4°C) and also placed on wet ice. Microsomal incubations and blood incubations were conducted according to Text Table 1. In consideration of the extreme volatility of some of these acrylates, one incubation per time point was conducted in separate 4-mL (1 dram) glass vials with Teflon-seal caps to avoid exposing a volatile sample to air at multiple time points. For liver microsomal incubations, an aliquot (10 µL) of each diluted acrylate dose solution (50 mM) was added to incubation buffer (0.1 M phosphate, pH 7.4) containing 1 mg/mL of rat liver microsomes and NADPH (1.3 mM) for a final concentration of 500 µM of acrylate with 1% or less of organic solvent. For blood incubations, an aliquot (10 µL) of the diluted acrylate dose solution (50 mM) was added to 990 µL of rat blood for a final concentration of 500 µM of acrylate with 1% or less of organic solvent. For both sets of incubations, after the addition of the test material the whole system with the Teflon-seal cap was incubated at 37°C in a vortex incubator. After incubation, each incubation sample was removed from the incubator and put into an ice-bath, and 50 µL of trifluoroacetic acid (TFA) was added to the incubation. After quickly vortexing, 1 mL (for microsomal incubation) or 2 mL (for blood incubation) of ethyl acetate containing the corresponding deuterated acrylate (i.e., internal standard; IS), was added to the incubation sample. After centrifugation for 10 minutes at 2000 rpm, the resulting supernatant (top organic layer) was transferred to a clean GC glass vial and quantitatively analyzed for remaining parent compound and the hydrolysis metabolite (AA) by gas chromatography and tandem mass spectrometry (GC/MS-MS) according to the method described below.

Stage IB – Determination of Enzymatic Kinetic Constants (Km and Vmax) for Ester Hydrolysis of Acrylates in Rat Liver Microsomes
Based on the Phase IA results, five concentrations for each acrylate were selected (31.25, 62.5, 125, 250, 500 µM) and incubated with rat liver microsomes
(0.5 mg/mL or 0.1 mg/mL) and NADPH (1.3 mM) for 10 minutes in 0.1 M phospahate buffer (pH 7.4).
The rat liver microsomes were removed from -80°C and thawed on wet ice. The microsomes were diluted 2-fold or 10-fold with cold 0.1 M phosphate buffer (pH 7.4) to afford a microsome concentration of 10 mg/mL or 2 mg/mL. The diluted microsome solution was kept cold on wet ice. The incubations were conducted according to Text Table 2 and the conditions described in Stage IA. The test material was dissolved in ACN and introduced into the rat liver microsomes in a volume of 1% of the total incubation volume (10 µL). For each acrylate, 2 replicates per concentration were incubated in the presence of microsomes and cofactor (NADPH) under physiological conditions (37°C). After incubation, each sample was removed from the incubator and put into an ice-bath, and 50 µL of TFA was added to the incubation. After quickly vortexing, 1 mL of the appropriate IS (internal standard) was added to the incubation samples. After centrifugation for 10 minutes at 2000 rpm, the resulting supernatant (top organic layer) was transferred to a clean GC glass vial and quantitatively analyzed for the hydrolysis metabolite AA by GC/MS-MS according to the method described below.

Stage II – Glutathione Reactivity
All acrylates were incubated individually with appropriate amounts of tritiated glutathione (10 µCi, 47.8 Ci/mmol) and non-tritiated glutathione (1000 µM) with the same substrate molar concentration (100 µM) in the presence of glutathione transferase (GST) (1 mg/mL) or absence of GST under physiological conditions in 0.1 M phosphate buffer (pH 7.4). After an incubation period of 60 minutes, 50 µL of TFA was added to the incubation buffer to stabilize any potential GSH conjugates. The whole solution was transferred to a 2-mL microcentrifuge tube and centrifuged for 10 minutes at 15000 rcf. The resulting supernatant was transferred to a clean glass autosampler vial for analysis. Qualitatively these samples were analyzed for glutathione conjugates via high performance liquid chromatography with time-of-flight mass spectrometry (HPLC/Q- TOF/MS-MS) and quantitatively via HPLC with eluent fractions collected. Based on the radioactive peaks observed in the reconstructed radiochromatograms from the corresponding incubations, the relative rate of GSH-conjugate formation was measured.

Details on dosing and sampling:

Test Material Dose Stock Solutions
Dose Solution
A stock solution (500 mM) of each test material was prepared on each day of use and diluted 10-fold in acetonitrile (ACN) to a concentration of 50 mM based on the water solubility of each individual test material. Final incubations were administered as a substrate concentration equivalent to 500 µM of the applicable acrylate and had a total organic solvent content of 1% or less.

Solubility
Based on the experimental water solubility, the maximum substrate concentration (500 µM) was used for incubations for all acrylates.
Stability
The stability of the test materials was not performed as test materials were prepared within 24 hours of use.

Positive Control Dose Stock Solution
Dose Solution
Due to non-volatility and similar reactivity, HEA was used as the positive control. A stock solution (500 mM in acetonitrile) of the positive control was prepared on each day of use with a 10-fold dilution in ACN to form 50 mM based on water solubility. Final incubations were administered as a substrate concentration equivalent to 500 µM and had a total organic solvent content of 1% or less.

Stability
Stability was not performed for dose solutions as the dose solutions were used within 24 hours of preparation.

Sample Analysis
Method A: GC/MS and GC/MS-MS Analysis of Acrylates and Acrylic acid
All GC/MS and GC–MS/MS analyses were performed using an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled to a triple quadrupole mass spectrometer Agilent 7000B MS (Agilent Technologies) operated in electron ionization (EI) mode.
The GC system was equipped with an Agilent 7693A autosampler (Agilent Technologies). For the separation, a Phenonemex 7HM-G009-22; ZB-FFAP column (15 m_0.18 mm i.d._0.07 mm film thickness; Phenomenex) was used. The triple quadrupole was operated either in Q1 full scan mode or in multiple reaction monitoring (MRM) mode for detecting the transitions for all analytes (acrylates and AA). The GC/MS and GC/MS-MS methods were used during the study and the optimized conditions of GC set-up and MRM transitions for each analyte and for each GC/MS-MS method are summarized in Text Table 3.

Method B: HPLC/RAM (Fraction Collection) Analysis of Acrylate GSH conjugates
The HPLC system with eluent fractions comprised of an Agilent HP 1200 HPLC system (Agilent, Palo Alto, California) and LCJet Pro (AIM Research Company, Hockessin, Delaware, USA) fraction collector. The HPLC system was equipped with a pump, an autosampler, a column compartment, a degasser, and an UV detector. The analytes were separated on Phenomenex Luna Omega 1.6 µm, Polar C18, 2.1 x 100 mm column (Phenomenex, California, USA). The components were eluted with a gradient mode at a flow rate of 0.4 mL/minute. Mobile phases consisted of ultrapure water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The gradient elution run lasted 20 minutes (0-5 minutes 0.00% B; 5-16 minutes 90% B; 16-18 minutes, 90% B; 18-18.2 minutes, 0.00% B;
18.2-20 minutes, 0.00% B). The LC eluent was introduced into a 96-well plate fraction collector (LCJet Pro) at 10 second intervals. After collection, the well plates were dried at room temperature and counted for radioactivity for each fraction with a PerkinElmer MicroBeta2 (6-dectector) top-count system (Waltham, Massachusetts, USA). The individual fraction radioactivities of each fraction were combined to reconstruct an HPLC/RAM chromatogram.

Method C: HPLC-QTOF/MS-MS Analysis of Acrylate GSH conjugates
A HPLC-QTOF/MS-MS system comprised of a Sciex API 5500 QTOF mass spectrometer (Concord, Ontario, Canada) coupled to an Agilent HP 1200 HPLC system (Agilent, Palo Alto, California) was used. The HPLC system was equipped with a pump, an autosampler, a column compartment, a degasser, and an UV detector. The analytes were separated on Phenomenex Luna@ Omega 1.6 µm, Polar C18, 2.1 x 100 mm column (Phenomenex, CA, USA). The components were eluted with a gradient mode at a flow rate of 0.4 mL/minute. Mobile phases consisted of ultrapure water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The gradient elution run lasted 20 minutes (0-5 minutes 0.00% B; 5-16 minutes 90% B; 16-18 minutes, 90% B; 18-18.2 minutes, 0.00% B; 18.2-
20 minutes, 0.00% B). The eluent from the HPLC was diverted to waste for the first 2 minutes of the run using a Valco valve from Valco Instrument Co. (Houston, Texas). The LC eluent was introduced into an electrospray ion source. Mass spectrometry was performed on the Triple TOF5600 (AB SCIEX, Foster City, CA, USA) a hybrid triple quadrupole time–of–flight mass spectrometer equipped with an electrospray (ESI) source. The system was operated with Analyst TF 1.6 software (AB SCIEX). The conditions of the MS/MS detector were as follows: first ion source gas 40 psi, second ion source gas 40 psi, curtain gas 25 psi, temperature 500ºC, ion spray voltage floating 4500 V, collision energy 35 V, collision energy spread 15 V, declustering potential 80 V. The TOF–MS range was set at m/z 50- 1500 and product ions mass range was set at m/z 50–1250. Both positive and negative ion modes were used for compound ionization. Nitrogen was used as nebulizer and auxiliary gas.

Statistics and Calculations
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 values of the acrylates and the rates of hydrolysis to AA for the nine acrylates 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 the parent esters 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

Type:

other: Rat Liver Microsomal Incubations

Results:

Ke from parent (min): 0.306; Half-Life from parent (min): 2.26; Ke from AA formation (min): 0.244; Half-Life from AA formation (min): 2.83

Type:

other: Rat Blood Incubations

Results:

Ke from parent (min): 0.180; Half-Life from parent (min): 3.85; Ke from AA formation (min): 0.179; Half-Life from AA formation (min): 3.87

Type:

other: GSH incubations in the presence of GST

Results:

Rate formation (nmol/mg protein.min): 1.73

Stage I – Acrylate Hydrolysis in Rat Liver Microsomes and Blood from F344 Rats

Stage IA–Incubation with Rat Liver Microsomes or Rat Blood (Tables 1, 2; figure 1)

As shown Table 1 and Figure 1, the similarity in the graph lines (measured vs calculated) indicate that all acrylates except tert-BA were metabolized by rat liver microsomes in the presence or absence of NADPH to form the hydrolysis metabolite, AA. Without microsomes, all acrylates were relatively stable under the incubation conditions, indicating the hydrolysis of acrylates was mainly catalyzed by the enzymes contained in rat liver microsomes. At the same time points of 0, 30, or 60 min, the concentrations of the remaining acrylates and the acrylates calculated from the formation of AA are very comparable between the microsomal incubations with NADPH and the microsmal incubations without NADPH, implying that the hydrolysis of acrylate in rat liver microsomes is mainly due to the esterases which do not require NADPH for the enzymatic hydrolysis of acrylates.

In some cases, as shown in Table 4, the half-life values calculated from the concentration of the remaining parent acrylate are comparable to the half-life values calculated from the concentration of parent acrylate based on AA formed in the microsomal incubations (i.e., EA, BA, iso-BA). According to the half-life values, acrylates (MA, EA, BA, iso-BA, and 2-EHA) have a half-life of less than

8.5 minutes (0.77-8.2 min). Under the same incubation conditions, HEA, HPA, and 2-PHA have half-lives of longer than 15 minutes, but less than 62 minutes. Possibly due to the steric hindrance, tert-BA is relatively stable under the same microsomal incubation conditions.

TABLE 4. Elimination Rates, Intrinsic Clearance and Half-life in Rat Liver Microsomes and Whole Rat Blood for Acrylates at 500µM Substrate Concentration

 

	Rat Liver Microsomal Incubation						Rat Blood Incubation
Compound Name	Clint from parent (µl/min/mg)	Ke from parent (min)	Half-Life from parent (min)	Clint from AA formation (µl/min/mg)	Ke from AA formation (min)	Half-Life from AA formation (min)	Clint from parent (µl/min)	Ke from parent (min)	Half Life from parent compound (min)	Clint from AA formation (µl/min/mg)	Ke from AA fromation (min)	Half Life from AA formation (min)
MA (Methyl Acrylate)	84.5	0.0845	8.20	121	0.121	5.73	465	0.465	1.49	ND	ND	ND
EA (Ethyl Acrylate)	372	0.372	1.87	385	0.385	1.80	303	0.303	2.29	ND	ND	ND
BA (n-Butyl Acrylate)	771	0.771	0.770	711	0.711	0.710	297	0.297	2.33	259	0.259	2.68
iso-BA (i-Butyl Acrylate)	866	0.866	0.798	778	0.778	0.888	281	0.281	2.47	250	0.250	2.77
tert-BA (tert-Butyl Acrylate)	ND	ND	ND	ND	ND	ND	94.1	0.0941	7.37	ND	ND	ND
HEA (2-Hydroxyethyl Acrylate)	11.3	0.0113	61.1	8.14	0.00814	85.2	271	0.271	2.56	ND	ND	ND
HPA (Hydroxypropyl Acrylate)	29.5	0.0295	23.5	23.1	0.0231	30.0	704	0.704	0.985	ND	ND	ND
2-EHA (2-Ethylhexyl Acrylate)	306	0.306	2.26	244	0.244	2.83	180	0.180	3.85	179	0.179	3.87
2-PHA (2-Propylheptyl Acrylate)	33.6	0.0336	20.6	45.6	0.0456	15.2	61.7	0.0617	11.2	58.5	0.0585	11.8
ND: not determined due to no AA formation or much less AA formation Ke: elimination rate; Clint in microsomal incubation: Intrinsic clearance (Ke x volume of incubation/mg/mL microsomal protein) Clint in blood incubation: Intrinsic clearance (Ke x volume of incubation)

Stage IA–Incubation with Pooled Rat Blood (Table 3, Figure2)

As shown Table 3 and Figure 2, all acrylates are metabolized in rat blood. However, unlike rat liver microsomal incubations, where the concentrations of the acrylate calculated from the formed AA correlated well with the remaining corresponding acrylates, the acrylate concentration calculated from AA formation in rat blood incubations are significantly different from the concentrations of the remaining corresponding acrylates (Figure 2). In fact, AA was not quantifiable from the blood incubations of MA, tert-BA, and HPA except the background level of AA (Table 3). To investigate whether the non-quantifiable AA in some of the blood incubations of acrylates was due to the stability of AA in rat blood, a rat blood time-course of AA was conducted and the result indicated that AA was relatively stable under rat blood incubation conditions (Figure 2,AA blood time course). As shown in Figure 2, AA is relatively stable in rat blood over a 60-minute period. This stability study result clearly showed that the AA was either not formed or formed at considerably lower levels than the corresponding rat liver microsomal incubations for the acrylates (MA, EA, BA, iso-BA, tert-BA, HEA, and HPA) during the rat blood incubations.

As shown in Table 4, the half-life values for all acrylates are less than 12 minutes (0.99-11.2 min), implying that all acrylate esters are quickly metabolized in rat blood. Whether measured by loss of parent or calculated from AA formation when possible (ie. BA, iso-BA, 2-EHA and 2-PHA), half-life values were similar in rat blood.

Stage IB–Determination of Enzymatic Kinetics (Kmand Vmax) of Ester Hydrolysis of Acrylates in Rat Liver Microsomes (Table 5, figure 3)

The final concentrations of the formed metabolite AA in the corresponding microsomal incubations at the microsomal protein level of 0.5 mg/ml, are summarized in Table 5. Based on the concentrations of AA and the substrate concentrations, the initial Michaelis-Menten kinetic plots were depicted in Figure 3. As shown in Figure 3, due the stability of tert-BA in the rat liver microsomal incubation conditions, tert- BA was hardly hydrolyzed and therfore, Km and Vmax values could not be calculated.

At the same microsomal protein level of 0.5 mg/mL, MA, HPA, HEA, and 2-PHA were partially hydrolyzed and their corresponding Km and Vmax values were able to be calculated and summarized in Table 7 except HEA which did not show Michaelis-Menten kinetics.

EA, BA, iso-BA, and 2-EHA were almost 100% hydrolyzed at each substrate concentration (Table 5), indicating that microsmal enzymes were not saturated by the substrate concentration at the current levels, therfore, no Kmand Vmaxvalues could be calculated for EA, BA, iso-BA, and 2-EHA at the microsomal protein concentration level of 0.5 mg/mL. In order to saturate the microsomal enzymes at the same range of substrate concentrations, the microsomal protein level was reduced to 0.1 mg/mL for EA, BA, iso-BA, and 2-EHA. At this new microsomal protein level, all incubations and analyses were repeated for EA, BA, iso-BA, 2- EHA, and MA. While Kmand Vmaxvalues were determined for MA at the higher microsomal protein concentration, this compound was included in the second analysis to verify similar performance across the assays. The final results and Michaelis-Menten kinetic plots were shown in Table 6 and Figure 4, respectively. As shown in Figure 4, at this lower level of microsomal protein, EA, BA, iso-BA, 2-EHA, and MA exhibited Michaelis-Menten kinetics; therefore their corresponding Kmand Vmaxvalues were able to be calculated and summarized in Table 7. The Kmand Vmaxvalues for MA were comparable (see study files); therefore, averaged values are shown in Table 7.

TABLE 7. Hydrolysis Rates of Acrylates in Rat Liver Microsomes

Test Material	Vmax (µM)a	Vmax (nmol/min/mg)	Km (µM)a
MAb	512	216	2002
EA	410	410	1157
BA	788	788	731
iso-BA	1188	1188	1293
HEA	ND	ND	ND
HPAc	847	169	829
2-EHA	602	602	503
2-PHAc	538	108	979
a: V max and Km  were calculated by GraphPad Prism based on the Michaelis-Menten kinetic model b: Averaged values from both microsomal protein concentrations (0.1 and 0.5 mg/mL) c: V max and Km were obtained from rat liver microsmal incubation at 0.5 mg/mL ND: Km and Vmax cannot be determined based on Michaelis-Menten kinetic model

Stage II–Glutathione Reactivity

According to the experimental procedure described above, each acrylate was incubated with GSH and tritiated GSH. After centrifugation, the resulting supernatants were analyzed by the HPLC with fractions collection (Method B) and HPLC/Q-TOF/MS analysis (Method C) for acrylate GSH (Acrylate-SG) conjugate formation (detailed analytical procedures are included in the study files). The final reconstructed radiochromatograms for all acrylate esters are depicted in Figure 5. As shown in Figure 5, all acrylate esters can react with GSH in the presence of GST to form one major peak (one adduct) in addition to the GSH peak. Under the same conditions, AA does not form GSH adduct. Further HPLC/Q-TOF/MS analysis of the new peak showed it was an Acrylate-SG adduct (see study files) (Table 8). Using the peak percentage of each GSH conjugate, the ratio of cold GSH to tritiated GSH, and the specific activity of tritiated GSH, the total GSH conjugate and rate formation of the GSH conjugate are summarized in Table 8. Overall, the rates of formation of the GSH conjugates in the presence of GST for all the acrylates ranged from 1.20 to 3.94 nmol/mg protein/min. Without GST, the rate of GSH conjugation of 2-EHA was slower than the GSH conjugation rate in the presence of GST.

TABLE 8. Formation of Acrylate GSH conjugates from incubation of acrylate with GSH in the presence of GST

 

Substrate name	Substrate concentration (µM)	Cold GSH concentration (µM)	Cold GSH concentration (µmmol)	Tritiated GSH (hot GSH)(µCi)a	Formed GSH Conjugate	Retention time in HPLC/RAM	Detected m/z of [M+H]+ in high resolution MS	Calculated m/z of [M+H]+ based on the conjugate structure	Peak Area (percetage)	Total triated GSH conjugate (µmol)	Total cold GSH conjugate (µmol)b	Total GSH conjugate (µmol)	Rate formation (nmol/mg protein.min)
AA	100	1000	1	10	AA-SG	ND	ND	378.1342	NDc	ND	ND	ND	ND
MA					MA-SG	7.50	394.1277	394.1278	8.31	0.0174	0.0831	0.100	1.67
EA					EA-SG	8.20	408.1430	408.1435	10.9	0.0228	0.109	0.132	2.19
BA					BA-SG	9.30	436.1738	436.1748	19.6	0.0409	0.196	0.237	3.94
iso-BA					Iso-BA-SG	9.30	436.1746	436.1748	11.3	0.0236	0.113	0.137	2.28
tert-BA					tert-BA-SG	9.20	436.174	436.1748	9.06	0.0190	0.091	0.110	1.83
HEA					HEA-SG	8.10	424.1376	424.1284	9.29	0.0194	0.0929	0.112	1.87
HPA					HPA-SG	8.30	438.1525	438.154	11.7	0.0244	0.116	0.141	2.35
2-EHA					2-EHA-SG	11.2	492.2362	492.2374	8.59	0.0180	0.0859	0.104	1.73
2-EHA (no GST)					2-EHA-SG	11.2	492.2362	492.2374	5.15	0.0108	0.0515	0.0623	1.04
2-PHA					2-PHA-SG	12.0	520.2692	520.2687	5.96	0.0125	0.0596	0.0721	1.20
a:Specific activity of hot GSH: 48.7 Ci/mmol; b: based on the ratio (4.78) of cold GSH versus hot GSH ; c: Not detected in HPLC/RAM, but detedted in HPLC/Q-TOF/MS; ND: Not detected based on the Low limit quantitation (LLQ) of 50 CPM

Overall, the half-life values and elmination rates in both rat microsomal and blood incubation conditions and GSH conjugation rates in the presence of GST for all acrylates, are summarized in Table 9.

TABLE 9. Summary of Half-life Values, Elimination Rates, and Rates of GSH Conjugation

 

Incubation Types	Rat Liver Microsomal Incubations				Rat Blood Incubations				GSH incubations in the presence of GST
Compound Name	Ke from parent (min)	Half-Life from parent (min)	Ke from AA formation (min)	Half-Life from AA formation (min)	Ke from parent (min)	Half Life from parent compound (min)	Ke from AA fromation (min)	Half Life from AA formation (min)	Rate formation (nmol/mg protein.min)
MA (Methyl Acrylate)	0.0845	8.20	0.121	5.73	0.465	1.49	ND	ND	1.67
EA (Ethyl Acrylate)	0.372	1.87	0.385	1.80	0.303	2.29	ND	ND	2.19
BA (n-Butyl Acrylate)	0.771	0.770	0.711	0.710	0.297	2.33	0.259	2.68	3.94
iso-BA (i-Butyl Acrylate)	0.866	0.798	0.778	0.888	0.281	2.47	0.250	2.77	2.28
tert-BA (tert-Butyl Acrylate)	ND	ND	ND	ND	0.0941	7.37	ND	ND	1.83
HEA (2-Hydroxyethyl Acrylate)	0.0089	61.1	0.0111	62.4	0.271	2.56	ND	ND	1.87
HPA (Hydroxypropyl Acrylate)	0.0295	23.5	0.0231	30.0	0.704	0.985	ND	ND	2.35
2-EHA (2-Ethylhexyl Acrylate)	0.306	2.26	0.244	2.83	0.180	3.85	0.179	3.87	1.73
2-PHA (2-Propylheptyl Acrylate)	0.0336	20.6	0.0456	15.2	0.0617	11.2	0.0585	11.8	1.20
Ke: Elimination rate.

Conclusions:

Nine acrylates, including methyl acrylate (MA), ethyl acrylate (EA), butyl acrylate (BA), iso-butyl acrylate (iso-BA), tert-butyl acrylate (tert-BA), 2-hydroxyethyl acrylate (HEA), 2- hydroxypropyl acrylate (HPA), 2-ethylhexyl acrylate (2-EHA), and 2-propylheptyl acrylate (2-PHA), were 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) and 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 evaluate the ability of each acrylate to conjugate with glutathione in the presence of glutathione transferases (GST).
In rat liver microsomes, eight acrylate esters (excluding tert-BA) were hydrolyzed to form the metabolite acrylic acid (AA). For some acrylates (i.e., EA, BA, iso-BA), the half-life values calculated from the concentration of the remaining parent acrylate were comparable to the half-life values calculated from the parent acrylate concentration calculated from AA formation. The small acrylates (MA, EA, BA, iso-BA) and the large acrylate (2-EHA) have a half-life of less than 8.5 minutes (0.77-8.2 min); HEA, HPA, and 2-PHA have half-life values of ranging from 20 minutes to 61 minutes. Under the rat liver microsomal conditions, tert-BA is relatively stable.
In whole rat blood, all nine acrylate esters are rapidly 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, tert-BA, HEA, and HPA. Thus, despite significant loss in parent acrylates, there is not a corresponding formation of AA in blood. Based on the measured concentrations of the remaning parent acrylates, the half-life values for all acrylates were less than 12 minutes in rat blood (0.99-11min).
In the serial substrate concentrations (31.25, 62.5, 125, 250, and 500 µM) of acrylate esters, further experiments were conducted to determine Km and Vmax values for acrylate ester hydrolysis to AA in rat liver microsomes. Km and Vmax values were determined for all acrylate esters except HEA and tert-BA.
All nine acrylates formed a single GSH conjugate in the presence of GST. The rate of formation of these GSH conjugates ranged from 1.2 to 3.94 nmol/mg protein/min.
Overall, these in vitro metabolism results imply that all nine acrylate esters can be quickly metabolized through hydrolysis to AA and/or glutathione conjugation in vivo.

Executive summary:

The hydrolysis and glutathione conjugation rates of methyl acrylate (MA), ethyl acrylate (EA), butyl acrylate (BA), iso-butyl acrylate (iso-BA), tert-butyl acrylate (tert-BA), 2-hydroxyethyl acrylate (HEA), 2-hydroxypropyl acrylate (HPA), 2-ethylhexyl acrylate (2-EHA), and 2-propylheptyl acrylate (2-PHA), were evaluated in vitro. Initial experimental determination of metabolism rates was performed with 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) and 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 evaluate the ability of each acrylate to conjugate with glutathione in the presence of glutathione transferases (GST). In rat liver microsomes, eight acrylate esters (excluding tert-BA) were hydrolyzed to form the metabolite acrylic acid (AA).  In whole rat blood, all nine acrylate esters are rapidly metabolized. Km and Vmax values were determined for seven of nine acrylate esters excluding HEA and tert-BA. All nine acrylates formed a single GSH conjugate in the presence of GST. The half-life values and elmination rates in both rat microsomal and blood incubation conditions and GSH conjugation rates in the presence of GST for all acrylates, are summarized in the following Table.

 

Incubation Types	Rat Liver Microsomal Incubations				Rat Blood Incubations				GSH incubations in the presence of GST
Compound Name	Ke from parent (min)	Half-Life from parent (min)	Ke from AA formation (min)	Half-Life from AA formation (min)	Ke from parent (min)	Half Life from parent compound (min)	Ke from AA fromation (min)	Half Life from AA formation (min)	Rate formation (nmol/mg protein.min)
MA (Methyl Acrylate)	0.0845	8.20	0.121	5.73	0.465	1.49	ND	ND	1.67
EA (Ethyl Acrylate)	0.372	1.87	0.385	1.80	0.303	2.29	ND	ND	2.19
BA (n-Butyl Acrylate)	0.771	0.770	0.711	0.710	0.297	2.33	0.259	2.68	3.94
iso-BA (i-Butyl Acrylate)	0.866	0.798	0.778	0.888	0.281	2.47	0.250	2.77	2.28
tert-BA (tert-Butyl Acrylate)	ND	ND	ND	ND	0.0941	7.37	ND	ND	1.83
HEA (2-Hydroxyethyl Acrylate)	0.0089	61.1	0.0111	62.4	0.271	2.56	ND	ND	1.87
HPA (Hydroxypropyl Acrylate)	0.0295	23.5	0.0231	30.0	0.704	0.985	ND	ND	2.35
2-EHA (2-Ethylhexyl Acrylate)	0.306	2.26	0.244	2.83	0.180	3.85	0.179	3.87	1.73
2-PHA (2-Propylheptyl Acrylate)	0.0336	20.6	0.0456	15.2	0.0617	11.2	0.0585	11.8	1.20
Ke: Elimination rate.

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

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Based on the results from an in vitro study with isobutyl acrylate (iBA) and in vivo data from the structural analogue n-butyl acrylate (n-BA), it can be presumed that isobutyl acrylate will be rapidly absorbed and metabolized after oral exposure in rats. The major portion of iBA will be hydrolysed by carboxyesterase to acrylic acid and iso-butanol. The subsequent metabolism follows that for acrylic acid, and involves metabolism to carbon dioxide via the propionate degradation pathway (acrylic acid --> 3-hydroxypropionic acid --> malonyl semialdehyde --> acetyl S CoA --> --> tricarboxylic acid cycle --> --> CO2). Metabolism of iso-butanol proceeds via the alcohol and aldehyde dehydrogenase pathway.

 

In vitro study:

Isobutyl acrylate was tested for relative rates of hydrolysis by a representative mammalian esterase (Porcine hepatic esterase). At concentrations of 0.2, 0.5 and 2.0 mM, conversion rates ranged between 45 - 47 μmol/min/mg protein (3 - 27 %) after 2 minutes, and 38 – 49 μmol/min/mg protein (7 - 57 %) after 5 minutes of incubation at 37°C. Thus, IBA was hydrolysed by hepatic esterase activity under in vitro conditions. The results of this study indicate that isobutyl acrylate and n-butyl acrylate are hydrolyzed at comparable rates by a representative mammalian enzyme (BASF AG, 2001).

The ester hydrolysis was examinedin vitroin rat liver S9 and rat plasma for the lower acrylate esters, showing a fast hydrolysis especially for the linear alkyl acrylate, but to a lesser extent to the tertiary structure (BASF SE, 2017b; Roos, 2015).

For the whole lower acrylate category in vitro the ester hydrolysis and glutathion conjugation was determined. (ARTF (Zhang et.al), 2018 ).The in vitro evidence consistently suggests a rapid metabolism of acrylate esters, catalysed by hepatic enzymes for the linear alkyl acrylate but to a lesser extent to the tertiary structure, with both ways forming AA as the common primary metabolite responsible for the systemic effects of these acrylate esters. In rat liver, the enzymatic hydrolysis of acrylate esters did not appear to involve NADPH. The acrylate esters were metabolised by rat liver microsomes in the presence or absence of NADPH. The exception was tBA where the metabolism was slow due to the presence of steric hindrance. In addition in vitro assay, a group of acrylate esters (MA, EA, BA, iBA, tBA and 2EHA) was individually incubated with tritiated- and non-tritiated glutathione in the presence or absence of GST at pH 7.4 for 60 minutes (Zang et al, 2018). All acrylate esters can react with GSH in the presence of GST to form one major peak (Acrylate-SG adduct) in addition to the GSH peak. Under the same conditions, AA did not form GSH adduct. Overall, the rates of formation of the GSH conjugates in the presence of GST for all the acrylate esters were similar (ranging from 1.20 to 3.94 nmol/mg protein/min), suggesting the involvement of GST to conjugate GSH with the acrylate esters in the category.

 

In vivo studies:

There are no in vivo studies for isobutyl acrylate available, but sufficient information on the structurally-related n-Butyl acrylate.

After oral administration (gavage), n-Butyl [2,3-14C]-acrylate was rapidly absorbed and metabolized in male Fischer 344 rats (75 % was eliminated as CO2, approximately 10 % via urine and 2 % via faeces). The major portion of n-butyl acrylate was hydrolysed by carboxyesterase to acrylic acid and n-butanol and eliminated as CO2. A smaller portion was conjugated with endogenous GSH to be subsequently excreted as mercapturic acids in the urine (Sanders, 1988).

After i.v. administration, the labelled n-butyl acrylate was rapidly absorbed and metabolized. The acrylate moiety was metabolized primarily to CO2, accounting for elimination of up to 45 % of the administered radiolabel. The second major route of elimination was in urine, with only trace amounts in faeces and as volatiles (Sanders, 1988).