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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Dec 2011 - 28 Jun 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Objective of study:
metabolism
Principles of method if other than guideline:
The objective of this study was to investigate the hydrolytic stability of Etocrilene in the liver and gastrointestinal tract. For this purpose, appropriate in vitro systems were applied that are established to investigate the stability of test substances in the liver and in the GI tract.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Etocrilene
EC Number:
226-029-0
EC Name:
Etocrilene
Cas Number:
5232-99-5
Molecular formula:
C18H15NO2
IUPAC Name:
etocrilene
Test material form:
solid
Details on test material:
- Physical state: solid/white
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
Radiolabelling:
no

Test animals

Details on test animals or test system and environmental conditions:
Test system: liver S9 fraction, intestinal-fluid simulant and gastric juice simulant.

Administration / exposure

Vehicle:
DMSO
Duration and frequency of treatment / exposure:
2 hours exposure at 37°C
Doses / concentrations
Remarks:
Doses / Concentrations:
100 and 250 μM
No. of animals per sex per dose / concentration:
All test item incubations were performed in duplicates.
Control animals:
other: heat deactivated control incubations as well as controls that were directly stopped by the addition of one volume of acetone after the start of the enzymatic reaction were performed.
Positive control reference chemical:
Testosterone and Benzyl benzoate
Details on study design:
In vitro metabolism of the test article was performed in liver S9 fraction, intestinal-fluid simulant and gastric juice simulant.

Results and discussion

Any other information on results incl. tables

Positive Controls

Testosterone, incubated at 200 μM under the given incubation conditions (phosphate buffer, 37 °C for 2 h) was metabolized in rat liver S9 fraction to an extend of 99.8 %. Benzyl benzoate was hydrolyzed to 99.3 % mean value of two incubations (in intestine fluid simulant (phosphate buffer, 37 °C for 2 h). In gastric juice simulant low peak areas as compared to BC (about 2 %) were found for Benzyl benzoate in t=0 control and in AI (active incubate). It is assumed (although these data are inconsistent in respect of expected recovery) that degradation of Benzyl benzoate was fast and occurred in both samples (t=0 control and in AI) tested. Based on the results, the validity of liver S9 fraction, intestine fluid simulant and the chosen incubation conditions was clearly demonstrated. Since no enzymatic activity is present in gastric juice (it consists of hydrochloric acid), the validity of this in vitro system is given, although the data for the positive control are inconsistent in respect of the amounts of Benzyl benzoate in t=0 control and AI.

Hydrolytic Stability of the test article

For experiments in liver S9 fraction and intestine fluid simulant, HDC and t=0 controls showed generally test material with sufficient recovery and stability. In gastric juice simulant peak areas of the test item were comparable in t=0 controls and in AIs. Therefore it can be concluded that the test substance is stable under the incubation conditions chosen and that peaks in the metabolic profiles of active incubates different from the test item (as e.g. in AIs of liver S9 fraction) are attributable to degradation products of the test article. Analytical investigations were performed that demonstrated the stability of Etocrilene in this stock solution over 24h.

Liver S9 fraction

In S9 fraction from rat liver, recoveries of the test item (HDC versus BC and t=0 control versus BC) were between 80.5% and 99.5% for a concentration of 100 μM. At 250 μM, recoveries were between 66.7% and 97.0%. In the active incubate, 100 and 250 μM test material were metabolized to 97.9 and 99.3 % (mean values of two incubations), respectively. One peak could be detected in the active incubations and was attributed to a formed metabolite, since this peak was not present in any of the controls analyzed.

Intestine fluid simulant

Incubations of the test material (100 and 250 μM) in intestine fluid simulant resulted in recoveries between 88.7 and 105.7 % (HDC and t=0 control versus buffer control). Comparing the amounts of Etocrilene in AI with the amounts in HDC and t=0 controls, no degradation of the test item could be observed. Therewith, it can be concluded that the test item is stable in intestine fluid simulant under the incubation conditions chosen.

Gastric juice simulant

In gastric juice simulant, peak areas of the test item in the t=0 control and the AI were comparable. The values were low and accounted for about 2 % of the value detected for BC. Based on these data, it is assumed that degradation of the test item was fast under the chosen incubation conditions and degradation occurred faster than the t=0 control was stopped. Although no metabolite peak was detected in this matrix, degradation of the test item is assumed based on the low peak areas in all samples investigated.

Summary of Results

  Metabolic turn over [%]
liver S9 fraction intestinal fluid simulant gastric fluid simulant
Test substance 250
µM
99.3 0.0 hydrolysis assumed based on low peak area in AI and t=0 control
Test substance 100 µM 97.9 0.0 hydrolysis assumed based on low peak area in AI and t=0 control
benzyl benzoate - 99.3 hydrolysis assumed based on low peak area in AI and t=0 control
testosterone 99.8 - -

Applicant's summary and conclusion

Conclusions:
The current in-vitro data demonstrate that the test article is degradated almost completely in liver S9 fraction but is stable in intestinal-fluid simulant. In gastric juice simulant, spontaneous degradation of the test item is assumed based on low amounts of test substance analyzed in the performed incubates.
Executive summary:

The objective of this study was to investigate the hydrolytic stability of the test article in liver and gastrointestinal tract. To determine hydrolysis in either compartment, the test substance was incubated in replicates at nominal concentrations of 100 and 250 μM for 2 h at 37°C with S9 fraction from rat liver, intestinal-fluid simulant including pancreas lipase and gastric juice simulant. After incubation, the amount of remaining substrate was analysed in the appropriate incubate by HPLC/UV. Heat deactivated controls (HDC) and controls, directly stopped after addition of test substance (t=0 control) served as control samples. A buffer control (BC, test substance in the incubation buffer) was used for calculation of recoveries in the HDC and t=0 contols. Positive controls were performed with Testosterone at a concentration of 200 μM for rat liver S9 fraction and Benzyl benzoate with a concentration of 250 μM for intestinal-fluid and gastric juice simulant. It could be demonstrated that Testosterone was metabolized extensively in liver S9 fraction. Benzyl benzoate was hydrolyzed almost completely in intestine fluid simulant. In gastric juice simulant low peak areas as compared to BC were found for Benzyl benzoate in t=0 control and in the active incubate (AI) and it is assumed that degradation of Benzyl benzoate was fast and occurred in both samples tested. In S9 fraction from rat liver, recoveries of the test substnce (peak areas in HDC and t=0 control versus BC) were between 81% and 100% and 67% and 97% for incubations at concentrations of 100 μM and 250 μM, respectively. In the active incubate, the test article was almost completely metabolized under the applied incubation conditions. In addition, one metabolite could be detected that was present in AI but not in the performed control incubations. The incubations with intestinal-fluid simulant resulted in recoveries of 89% and 105% and 99% and 106% for the incubations at concentrations of 100 μM 250 μM, respectively. The test article was not metabolized in any of the active incubates. The peak areas of the t=0 control and the AI in gastric juice simulant showed comparable peak areas that were low compared to the BC. Based on these data, it is assumed that degradation of the test item was fast under the chosen incubation conditions and occurred in both samples tested. Taken together, the current in-vitro data demonstrate that the test article is degradated almost completely in liver S9 fraction but is stable in intestinal-fluid simulant. In gastric juice simulant, spontaneous degradation of the test item is assumed based on low amounts of test substance analyzed in the performed incubates.