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Basic toxicokinetics

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basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sep. 2012 - March 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
State of the art metabolism/toxicokinetcs study, GLP

Data source

Reference Type:
study report

Materials and methods

Objective of study:
Principles of method if other than guideline:
In vitro metabolism study (rat liver microsomes, rat whole blood) in combination with toxicokinetic modelling (ADME model)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene dimethacrylate
EC Number:
EC Name:
Ethylene dimethacrylate
Cas Number:
Molecular formula:
ethane-1,2-diyl bis(2-methylacrylate)
Test material form:

Test animals

Details on test animals or test system and environmental conditions:
in vitro study

Results and discussion

Metabolite characterisation studies

Details on metabolites:
By the action of the non-specific carboxylesterases in the microsomes and in whole blood the esters were hydrolysed quantitatively to methacrylic acid and the corresponding alcohols/diols.

Applicant's summary and conclusion

Interpretation of results (migrated information): no bioaccumulation potential based on study results
Seven methacrylate esters (including MMA, which served as a reference chemical) were initially chosen for experimental determination of metabolism rates in whole rat blood and rat liver enzymes at a single substrate concentration (Phase I). All seven methacrylates were quickly hydrolyzed to methacrylic acid (MAA) in, both, whole rat blood and rat liver microsomes. Hydrolysis half-lives of the esters in rat liver microsomes ranged 0.06 minutes to 4.95 minutes. Hydrolysis half-lives of the esters in whole rat blood ranged 1.56 minutes to 99 minutes.
Five methacrylate esters (including MMA, which served as a reference chemical) were chosen for further experiments to determine Km and Vmax values for these in rat liver microsomes. These values, along with QSAR-estimated partition coefficients were used for PBPK modeling to simulate in vivo blood concentrations of each molecule and its MAA hydrolysis product. Resulting blood concentrations were very similar between the five molecules. Differences in parent molecule blood concentrations (mg/L) varied by less than 2-fold and differences in MAA blood concentrations (mg/L) varied by less than 4-fold.
It is important to note that the PBPK model used for this effort was designed for methacrylate esters that have a single ester group, such that for each mol of parent ester hydrolyzed, one mol of methacrylic acid is formed. This is not the case for EGDMA and 1,4-BDDMA, which both contain two ester groups that can be hydrolyzed, resulting in two mol of methacrylic acid for every mol of parent ester. In the Phase II hydrolysis experiments, two mol of methacrylic acid were produced for every mol of methacrylate ester substrate introduced into the incubations. For these molecules, the PBPK model simulates only hydrolysis of the first ester group, resulting in one mol methacrylic acid per mol of parent ester.
Overall, these metabolism data and modeling results show that all five methacrylate esters studied in the definitive experiment are expected to be rapidly hydrolyzed in the rat, with greater than 86-99% cleavage by the oral route. Additionally, these simulated blood levels represent conservative estimates for those that would be expected to occur in the real world. In this study, only hydrolysis in the liver and blood has been considered. In reality, metabolism in other tissues would also be expected to occur (Brebner and Kalow, 1970; Fukami and Yokoi, 2012; Prusakiewicz et al., 2006; Satoh and Hosokawa, 1998; Zhu et al., 2000). Generally, exposures would be expected to occur via dermal, inhalation or oral ingestion. For any of these routes, pre-systemic hydrolysis would be expected to occur, significantly reducing the total amount of material reaching the systemic circulation. This has been illustrated by modeling of an oral dose route. However, even these simulations are expected to be highly conservative in terms of levels of parent ester and MAA metabolite present in the blood as the model assumes an oral bioavailability of 100 percent to the liver with no GI metabolism and no first-pass metabolism of MAA. In reality, esterase enzymes in the gut, as well as the lungs and skin in cases of dermal or inhalation exposure, would be expected to reduce the amount of methacrylate ester available to be absorbed (Brebner and Kalow, 1970; Fukami and Yokoi, 2012; Imai et al., 2003; Inoue et al., 1979; Li et al., 2007; Prusakiewicz et al., 2006). Additionally, further downstream metabolism of MAA, likely with significant first-pass metabolism, would be expected to reduce blood levels of this metabolite. Thus, real in vivo exposures are expected to result in lower blood levels of the methacrylate esters and their metabolic products than those simulated in this study.
Brebner, J., and Kalow, W. (1970). Soluble esterases of human lung. Canadian Journal of Biochemistry 48(9), 970-978.
Fukami, T. and Yokoi, T. (2012). The emerging role of human esterases. Drug Metabolism and Pharmacokinetics 27(5), 466-477.
Imai, T., Yoshigae, Y., Hosokawa, M., Chiba, K., and Otagiri, M. (2003). Evidence for the involvement of a pulmonary first-pass effect via carboxylesterase in the disposition of a propranolol ester derivative after intravenous administration. The Journal of Pharmacology and Experimental Therapeutics 307(3), 1234-1242.
Inoue, M., Morikawa, M., Tsuboi, M., and Sugiura, M. (1979). Species difference and characterization of intestinal esterase on the hydrolizing activity of ester-type drugs. The Japanese Journal of Pharmacology 29, 9-16.
Li, P., Callery, P. S., Gan, L-S., and Balani, S. K. (2007). Esterase inhibition attribute of grapefruit juice leading to a new drug interaction. Drug Metabolism and Disposition 35(7), 1023-1031.
Prusakiewicz, J. J., Ackermann, C., and Voorman, R. (2006). Comparison of skin esterase activities from different species. Pharmaceutical Research 23(7), 1517-1524.
Satoh, T. and Hosokawa, M. (1998). The mammalian carboxylesterases: from molecules to functions. Annual Review of Pharmacology and Toxicology 38, 257-288.
Zhu, W., Song, L., Zhang, H., Matoney, L., LeCluyse, E., and Yan, B. (2000). Dexamethasone differentially regulates expression of carboxylesterase genes in humans and rats. Drug Metabolism and Disposition 28(2), 186-191.
Executive summary:

All seven methacrylate esters were rapidly converted to MAA in whole rat blood and rat liver microsomes. Hydrolysis half-lives ranged from 1.56 to 99 minutes, and from 0.06 to 4.95 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. Table 6 shows elimination rates (ke), intrinsic clearance (Clint) and half-life values for each molecule in whole rat blood and rat liver microsomes at 0.25 mM starting concentrations. 

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 remaining six molecules exhibited rat liver microsome hydrolysis rates approximately 10 fold lower than MMA. However, all seven molecules were completely, or nearly completely, hydrolyzed to MAA within 15 minutes incubation.



Jones, R. D .O. (2002). Using physiologically based pharmacokinetic modeling to

predict the pharmacokinetics and toxicity of methacrylate esters. Thesis

submitted to theoffor the degree of Doctor of Philosophy

in the Faculty of Medicine, Dentistry, Nursing and Pharmacy.


Mainwaring, G., Foster, J. R.,, V., and Green, T. (2001). Methyl methacrylate

toxicity in rat nasal epithelium: studies of the mechanism of action and

comparisons between species.Toxicology158,109 -118.