Registration Dossier

Toxicological information

Basic toxicokinetics

Currently viewing:

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-26-2017 to 09-06-2017
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:
2017
Report Date:
2017

Materials and methods

Objective of study:
metabolism
Principles of method if other than guideline:
The objective of this study was to investigate the metabolic stability of tert.-Butylacrylate in subcellular fraction of liver of rats and humans. The test substance was incubated in duplicates at a nominal concentration of 500 µM at 37°C for 0 (t=0), 10, 30, 60, 120, 180 and 240 min. with continuous agitation. In addition to the active in vitro system, buffer controls (BC) and heat deactivated controls (HDC) were included in the assay. The buffer control (test substance in the incubation buffer) was used for calculation of recoveries in the HDC and t=0 controls. The incubations were stopped by addition of three times one volume of ethylacetate to denature the proteins and to extract remaining test substance from the incubate. For analysis, the organic phase(s) were separated from the aqueous phase and analysed for tert.-Butylacrylate. In the aqueous phase, the potential hydrolysis product acrylic acid was quantified. Analyses were performed by HPLC/UV.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
tert.-Butylacrylate;
Batch Identification: B60218042017
Purity: 99.72 %
Radiolabelling:
no

Test animals

Species:
other: liver S9 fraction of male Han-Wistar rats (Charles River, Sulzfeld, Germany) and humans (TebuBio; manufacturer: Xenotech, H0620.S9, LotNo. 1610015)

Administration / exposure

Route of administration:
other: in vitro
Details on exposure:
Tert.-Butylacrylate was incubated with liver S9-fraction of each species at a nominal concentration of 500 µM. The incubation was performed at 37°C for 0 (t=0), 10, 30, 60, 120, 180 and 240 min. with continuous agitation in a cofactor containing buffer system. The cofactor was continuously reproduced by a NADPH generating system, consisting of Glucose-6-phosphate and Glucose-6-phosphate-dehydrogenase.

In addition to the active incubations, buffer controls and heat deactivated controls were included in the assay. Total incubation volume was 500 µL. The incubations were stopped by addition of three times 500 µL ethylacetate to denature the proteins and to extract remaining test substance: after defined incubation times one vol. ethylacetate was added to each respective incubate and mixed well. After centrifugation the organic phase (on the top) was carfully collected in a new tube. This “extraction-step” was repeated two more times. The aqueous phase was remained in the original tube. Samples (organic extract and aqueous phase) were performed in two replicates. All samples derived from the incubations were stored in a freezer (about -20°C) until analyses.

Testosterone (positive control) was incubated with liver S9-fraction of each species at a nominal concentration of 200 µM. The incubation was performed at 37°C for 4 hours with continuous agitation in a cofactor containing buffer system. In addition to the active incubations, a heat deactivated control was included in the assay. Total incubation volume was 500 µL. To stop the reaction, the protein of incubates was precipitated by the addition of 1 vol. acetone. Supernatants were used for analyses and stored in a freezer (about -20°C) until analyses.
Duration and frequency of treatment / exposure:
incubation: 0, 10, 30, 60, 120, 180 and 240 min.
Doses / concentrations
Remarks:
nominal concentration of 500 µM
Positive control:
Testosterone
Details on study design:
Tert.-Butylacrylate was incubated with liver S9-fraction of each species at a nominal concentration of 500 µM. The incubation was performed at 37°C for 0 (t=0), 10, 30, 60, 120, 180 and 240 min. with continuous agitation in a cofactor containing buffer system. The cofactor was continuously reproduced by a NADPH generating system, consisting of Glucose-6-phosphate and Glucose-6-phosphate-dehydrogenase.

In addition to the active incubations, buffer controls and heat deactivated controls were included in the assay. Total incubation volume was 500 µL. The incubations were stopped by addition of three times 500 µL ethylacetate to denature the proteins and to extract remaining test substance: after defined incubation times one vol. ethylacetate was added to each respective incubate and mixed well. After centrifugation the organic phase (on the top) was carfully collected in a new tube. This “extraction-step” was repeated two more times. The aqueous phase was remained in the original tube. Samples (organic extract and aqueous phase) were performed in two replicates. All samples derived from the incubations were stored in a freezer (about -20°C) until analyses.

Testosterone (positive control) was incubated with liver S9-fraction of each species at a nominal concentration of 200 µM. The incubation was performed at 37°C for 4 hours with continuous agitation in a cofactor containing buffer system. In addition to the active incubations, a heat deactivated control was included in the assay. Total incubation volume was 500 µL. To stop the reaction, the protein of incubates was precipitated by the addition of 1 vol. acetone. Supernatants were used for analyses and stored in a freezer (about -20°C) until analyses.

Results and discussion

Any other information on results incl. tables

The current experiment was started in liver S9-fractions of rats and humans. Tert.-Butylacrylate was incubated in these in vitro systems at nominal concentrations of 500 µM to investigate the metabolic behaviour of this test substance in these in vitro systems.

 

In S9 fraction from rat liver, mean recoveries of tert.-Butylacrylate (quantities recovered in heat deactivated controls(HDC) and t=0 controls versus buffer controls (BC)) were 50.1% and 106.3%. It is assumed that the low recovery in HDC depends on abiotic reactivity of the test substance with proteins in the sample. In the active incubate, amounts of recovered tert.-Butylacrylate were 550.7, 323.6, 191.4, 153.5, 101.5, 80.3 and 72.4 µM, reflecting 100.0, 58.7, 34.7, 27.8, 18.4, 14.5 and 13.1 % of the initial concentration of the incubate. These data demonstrate that tert.-Butylacrylate was metabolized in liver S9 fraction of rats under the chosen incubation conditions. Corresponding amounts of quantified acrylic acid were below the limit of quantification at the start of incubation and at early measurement time points (until 10 min) and up to 27.3 µM at the latest measurement time point. Since these amounts of the hydrolysis product of tert.-Butylacrylate are stochiometrically underproportional to the degredated tert.-Butylacrylate, it can be concluded that tert.-Butylacrylate is either partially metabolized to acrylic acid in the liver and partially via an alternative pathway or that acrylic acid is futher metabolized to secondary metabolites.

Based on the degradation data, an inital half life of 13 min. was calulated.

 

A comparable situation was observed in S9 fraction of human liver: Mean recoveries of tert.-Butylacrylate(quantities recovered in HDC and t=0 controls versus BC) were 78.3% and 127.5%. In the active incubate, amounts of recoveredtert.-Butylacrylatewere 548.9, 397.3, 338.3, 250.4, 174.2, 105.0 and 71.4 µM, reflecting 100.0, 72.4, 61.6, 45.6, 31.7, 19.1 and 12.9 % of the initial concentration of the incubate. These data demonstrate that tert.-Butylacrylatewas metabolized in liver S9 fraction of humans under the chosen incubation conditions. Corresponding amounts of quantified acrylic acid were below the limit of quantification at the start of incubation and at early measurement time points (until 10 min) and up to 37.5 µM at the latest measurement time point. Since these amounts of the hydrolysis product of tert.-Butylacrylate are stochiometrically underproportional to the degredated tert.-Butylacrylate, it can be concluded that tert.-Butylacrylate is either partially metabolized to acrylic acid in the liver and partially via an alternative pathway or that acrylic acid is futher metabolized to secondary metabolites.

Based on the degradation data, an inital half life of 21 min. was calulated.

Applicant's summary and conclusion

Conclusions:
In conclusion, the current in-vitro data demonstrate that tert.-Butylacrylate is metabolized in liver S9 fraction of rats and humans. Detected amounts of acrylic acid in the incubates are able to proof hydrolysis of the tested ester in principle, although quantified amounts of this hydrolysis product do not correlate stochiometrically to the degraded amounts of tert.-Butylacrylate. Based on these findings, it can be concluded that in liver, tert.-Butylacrylate is either partially metabolized to acrylic acid and partially via an alternative pathway or that acrylic acid is futher metabolized to form secondary metabolites that were not addressed within the current investigtions.