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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
February till August 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study was performed for investigative purposes, to investigate the metabolism of TMPTA in liver S9 fractions or whole blood of rats.

500 µM of TMPTA and positive control substance Methyl Acrylate was used based on laboratory experience.

In S9 fractions, TMPTA was incubated ate a nominal concentration of 500 µM at 37°C for 1, 2.5, 5 and 10 min (first experiment) and 10, 30 and 60 min (second experiment). The reaction was stopped by three times the addition of a defined volume of Ethyl Acetate.
In whole blood, TMPTA was incubated at a nominal concentration of 500 µM at 37°C for 10, 30 and 60 min. The reaction was stopped by the addition of a defined volume of Hydrochloric Acid followed by two times the addition of a defined volume of Butyl Acetate. The organic phases were analyzed for TMPTA. In the aqueous phase of S9 incubates, the potential hydrolysis product Acrylic Acid was quantified by HPLC/UV.

The incubation of positive control substance Methyl Methacrylate was analogously performed in rat S9 fractions for 1, 2.5, 5, 10 and 30 min (first experiment) and 0.5 and 10 min (second experiment) as well as in whole blood for 10, 30 and 60 min.
In addition to the active in vitro incubation, a zero-point determination (t=0), a buffer control (BC) and a heat-deactivated control (HDC) were included in the assays with TMPTA and positive control substance. The BC (TMPTA in incubation buffer) was used for calculation of recoveries in the HDC and t=0.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate
EC Number:
239-701-3
EC Name:
2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate
Cas Number:
15625-89-5
Molecular formula:
C15H20O6
IUPAC Name:
2,2-bis[(acryloyloxy)methyl]butyl acrylate (non-preferred name)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Trimethylolpropantriacrylate (TMPTA), CAS No.: 15625-89-5.
- Batch number: Tankprobe (Tank 1) from 12.02.2013
- Test substance No: 04/0187-2
- Expiration date of the lot/batch: No information
- Purity: >98 %
- Purity test date: NA

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (RT), protect against heat and light
- Stability under test conditions and in solvent/vehicle: nromal stability


TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing: test substance formulated in DMSO, and stable over the investigated storage periods as confirmed by analyses

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Test substance is colorless, clear liquid
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The in vitro metabolsim of TMPTA was investigated in liver S9 fraction and whole blood of male Han-Wistar rats (Charles River, Sulzfeld, Germany).
Sex:
male
Details on test animals or test system and environmental conditions:
Rats were kept at in airconditioned laboratories at 20-24 degrees celcius and 30-70% relativ humidity under conventional hydienic conditions as described in the international standard procdues of the test facility and revceived feed and water ad libitum. The in vitro studies was performed in an AAALAC-approved laboratory in accordance wih the German Animal Welfare ACt and the effective uropean Council Directive.

Administration / exposure

Route of administration:
other: In vitro study, test substance formulated in DMSO was tested in rat liver S9 fractions and whole blood prepared from Han-Wistar rats
Vehicle:
DMSO
Doses / concentrations
Dose / conc.:
500 other: µM
No. of animals per sex per dose / concentration:
NA
Control animals:
no
Positive control reference chemical:
Methyl Acrylate
Details on study design:
Rat liver S9 fractions and whole blood sampoles were prepared to standard operating procedures of the test facility. For preparation of the S9 fraction, the liver from sacrificed rats (under isoflurane anesthesia) was perfused with cold NaCl and weighed. Liver samples was cut into small pieces and mixed with buffer. Standard procedures for preparation fo S9 fractions was used (see further information in attached study report).

Experimenmtal porocedures for incubation in liver S9 fractions and whole blood was performed in accordance to standard protocols used in the testing lab (see attached study report).

The analytical investigation s of the stock solutions of TMPTA and the positive control substance as well as the incubates was carried out at the analytical lab at BASF SE, Germany. TMPTA nd Methyl Methacrylate in the organic phases of the liver S9 fraction and whole blood incubates wre determined by GC-FID. Acrylic Acid and Methacrylic Acid in the aqueous phaseds of the S) fraction and whole blood were analysed by HPLC-UV. Details of the analytical methods used can be found in the attached study report.
Statistics:
No information

Results and discussion

Main ADME results
Type:
metabolism
Results:
TMPTA is hydrolysed fast in rat liver S9 fractions with a half-life of 1.1 min. Stoichiometric amounts of hydrolysis product acrylic acid was detected. TMPTA was degraded completely after 10 min in rat whole blood samples.

Toxicokinetic / pharmacokinetic studies

Toxicokinetic parametersopen allclose all
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: 1.1 min.
Remarks:
S9 fraction
Test no.:
#2
Toxicokinetic parameters:
half-life 1st: 10 min
Remarks:
Whole blood

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Acrylic Acid

Any other information on results incl. tables

Details on study results can be found in the attached study report.

In S9 fractions, TMPTA was incubated at a nominal concentration of 500 µM at 37°C for 1, 2.5, 5 and 10 min (first experiment). The analytical results demonstatte that TMPTA was comletely hydrolysed after 10 min and simultaneously, an equivalent anount of Acrylic Acid was formed. The calculated half-life of TMPTA was 1.1 min. The recovery of the zero-point determination (t=0) was 92%, the heat-deactivated control (HDC) was 73% and referred to the nominal concentration 105%.

In the second experiment, TMPTA was incubated in S9 liver fractions at a nominal concentration of 500 µM at 37°C for 10, 30 and 60 min in order to confirm the complete turn-over and formation of Acrylic Acid after 10 min. The results indicate that after 10 min., TMPTA was completely hydrolysed and the maximal concentrationj of Acrylic Acid was reached (see attached study report).

The positive control substance Methyl Methacrylate was incubated in S) liver fractions at a nominal coincentration of 0.5 mM for 1,2.5, 5, 10 and 30 min. The results demonstrate that the substance was completely hydrolysed after 2.5 min and in the same time, the cleavage product Methacrylic Acid was formed stoichiometrically. The recovery of the zero-point determination (t=0) is 102%, of the heat-deactivated (HDC) is 82% and referred to the nominal concentration of 103%. The second experiment, in which Methyl Methacrylat was incubated in S9 liver fractions at a nominal concentration of 0.5 mM for 0.5 and 10 min, generally confirmed these results (see attached study report).

The hydrolysis of Methyl Methacrylate to Mehtacrylis Acid afeter a time span of 2.5 min was seen in previous experiments in the laboratorium. The recoveries of the incubations lie in the analytical and preparative range of acceptance. These data prove the validity of the applied in vitro systems and the chosen incubation conditions.

In whole blood, TMPTA was incubated at a nominal concentration of 500 µM at 37°C for 10, 30 and 60 min. The results revealed that TMPTA was completely degraded after 10 min. However, it should be noted that TMPTA was also completely degraded in the heat-deactivated control (HDC) indicating that the degradation pathway in blood may be different from to the degradation pathway in the liver leading to other metabolites of TMPTA, such as Glutathion conjugates and not the the hydrolysis products or Acrylic Acid reacts with blood components and consequently, this hydrolysis product cannot be detected in the respective active incubates of whole blood samples. The recovery rate of the zero-point determination (t=0) is 113%, that of the heat-deactivated control (HDC) and of the nominal concentration could not be determined because TMPTA was not detectable in the HDC.

The positive control substance Methyl Methacrylate was incubated in whole blood at a nominal concentration of 500 µM at 37°C for 10, 30 and 60 min. The results displayed a continuous turn-over of the positive control substance with the lowest amount at the latest observation time point of 60 min. The recovery rate of the zero-point determination (t=0) is 128% and that of heat-deactivated control (HDC) is 46%.

A comparable turn-over of Methyl Methacrylate was seen in previous experiments in the laboratorium. The recoveries of the controls lie in the analytical and preparative range. The data prove the validity of the aplied in vitro systsmes and the chosen incubation conditions.

Applicant's summary and conclusion

Conclusions:
The current in vitro data demonstrate that TMPTA is hydrolysed fast in rat liver S9 fractions with a half-life of 1.1 min. Detected stoichiometric amounts of acrylic acid in the incubates proved that hydrolysis of TMPTA is taking place. TMPTA was also degraded in rat whole blood samples, degradation was complete after 10 min. The metabolism was though different in blood compared to the liver as indicated by a complete degradation of TMPTA in heat-deactivated control samples. The validity of the in vitro systems as well as the incubation conditions was seen using methyl methacrylate as positive control substance.
Executive summary:

The objective of this study was to investigate the hydrolysis of TMPTA in rat liver S9 fraction and whole blood samples. In S9 fractions, TMPTA was incubated at a nominal concentration of 500µM at 37°C for 1, 2.5, 5 and 10 min (first experiment) and 10, 30 and 60 min (second experiment). The reaction was stopped by three times the addition of a defined volume of Ethyl Acetate.

In whole blood, TMPTA was incubated at a nominal concentration of 500 µM at 37°C for 10, 30 and 60 min. The reaction was stopped by the addition of a defined volume of Hydrochloric Acid followed by two times the addition of a defined volume of Butyl Acetate. The organic phases were analyzed for TMPTA. In the aqueous phase of S9 incubates, the potential hydrolysis product Acrylic Acid was quantified by HPLC/UV.

The incubation of positive control substance Methyl Methacrylate was analogously performed in rat S9 fractions for 1, 2.5, 5, 10 and 30 min (first experiment) and 0.5 and 10 min (second experiment) as well as in whole blood for 10, 30 and 60 min.

In addition to the active in vitro incubation, a zero-point determination (t=0), a buffer control (BC) and a heat-deactivated control (HDC) were included in the assays with TMPTA and positive control substance. The BC (TMPTA in incubation buffer) was used for calculation of recoveries in the HDC and t=0.

The current in vitro data demonstrate that TMPTA is hydrolysed fast in rat liver S9 fractions with a half-life of 1.1 min. Detected stoichiometric amounts of acrylic acid in the incubates proved that hydrolysis of TMPTA is taking place. TMPTA was also degraded in rat whole blood samples, degradation was complete after 10 min. The metabolism was though different in blood compared to the liver as indicated by a complete degradation of TMPTA in heat-deactivated control samples. The validity of the in vitro systems as well as the incubation conditions was seen using methyl methacrylate as positive control substance.

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