Desmedipham

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014-06-10 to 2015-02-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Remarks:

This study is new. No OECD test guideline is available for this study. The study is acceptable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The comparative metabolism of [Phenoxy-UL-14C] Desmedipham (14C-Desmedipham) was investigated in animal in-vitro systems by incubating the test item with liver microsomes from male Wistar rats (RLM) and humans (HLM) in the presence of NADPH cofactor. The test item concentration was 10 µM and the protein concentration 1 mg/mL. The 10 µM test item concentration was chosen in order to have enough sample material for possible identification of metabolites by chromatographic or spectroscopic methods. The sampling times were 0, 0.5 and 1 hour after test start. The test durations of 0.5 and 1 hour for the test item were considered as reasonable because positive results were obtained from the enzymatic reaction of Testosterone to Hydroxy-Testosterone already after 5 minutes. Samples were analyzed following protein precipitation by reversed phase HPLC with radiochemical detection (HPLC-RAD).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Desmedipham

EC Number:

237-198-5

EC Name:

Desmedipham

Cas Number:

13684-56-5

Molecular formula:

C16 H16 N2 O4

IUPAC Name:

ethyl 3´-phenylcarbamoyloxycarbanilate

Test animals

Species:

other: Rats and Humans

Strain:

other: Wister Rats and Humans

Sex:

male

Details on test animals or test system and environmental conditions:

Pooled liver microsomes from male Wistar rats (pool of 200 individuals) and humans (pool of 50 donors from both genders)

Administration / exposure

Route of administration:

application in vitro

Vehicle:

other: Acetonitrile (AcN)

Details on exposure:

IN VITRO APPLICATION

- Concentration of test material and reference chemical: Final concentrations of the incubates were: 5 mM MgCl2; 1 mg/mL microsome protein; 10 µM 14C-Desmedipham (0.133 µCi/incubate); 1 mM reduced NADPH.

The final concentrations were 5 mM MgCl2, 0.15 mg/mL microsome protein, 150 µM testosterone and 1 mM reduced NADPH.

- Method of preparation of stock solution(s) of test material and reference chemical:
Working solution of 14C-Desmedipham (499.5 µM; 13.30 µCi/mL): 30 µL of 14C-Desmedipham stock solution were diluted with 1098 µL of AcN in a 10-mL polypropylene tube and the solution was stirred gently. This solution was freshly prepared and kept at room temperature until use.

1 mM non-Radiolabelled Desmedipham Solution was prepared by weighing 8.28 mg of non-radiolabelled Desmedipham in a 25-mL volumetric flask. The level mark was filled up with AcN, and the solution was stirred until complete dissolution of the solid. This solution was freshly prepared and kept at room temperature until use.

- Cell culture medium characteristics (temperature, pH): Room temperature until the end of the
experiment and were further stored at -80°C±10°C until analysis, pH 7.4

- Incubation temperature: 37 ± 1°C.

- Number of replicates: Triplicate samples at T=0 (R-0-1, R-0-2, R-0-3, H-0-1, H-0-2 and H-0-3; not incubated) were prepared by adding the same components as test samples but in different order (i.e., AcN was added prior to NADPH and 14C-Desmedipham).

- Time points: These samples were incubated for 0.5 and 1h, respectively.

Duration and frequency of treatment / exposure:

These samples were incubated for 0.5 and 1h, respectively.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Buffer control

Doses / concentrations 2

Dose / conc.:

10 ppm (nominal)

Positive control reference chemical:

Metabolite Standard Used in Positive Metabolism Controls of the In-vitro System:
6ß-hydroxytestosterone.
Internal Standard Use in Positive Metabolism controls of the In-vitro Systems:
Dexamethasone Vetranal

Details on study design:

- Dose selection rationale: The 10 µM test item concentration was chosen in order to have enough sample material for possible identification of metabolites by chromatographic or spectroscopic methods.

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- 14C-Desmedipham was incubated separately with RLM and HLM (n=3) at 37±1°C in a final volume of 500 µL. Incubations were performed in a thermomixer device (Eppendorf) with shaking at 1000 rpm. The microsomal incubates were centrifuged at 16000 x g for 15 minutes at 20°C. After centrifugation, 100 µL of each supernatant were diluted with 400 µL of HPLC mobile phase A. The samples were directly analysed by HPLC-RAD without any further extraction procedure.

ANALYTICAL METHOD
- Samples were analysed by HPLC-RAD for the unchanged test item and metabolites using the analytical method listed in the table that is attached in "Overall remarks, attachments".
The chromatograms were recorded electronically and quantitatively evaluated using the MassLynx® Chromatography software (V4.0). The 14C-trace of a chromato-gram, which should be integrated, was divided into regions of interest, corresponding to the separated radioactive peaks. The area counts from all regions of interest were used for the percentage calculation of the individual compounds.

Expression of the Results:
Relative Percentages of Unchanged 14C-Desmedipham and Metabolites: The relative percentages were calculated from the radio chromatographic profiles at the different incubation times.

Testosterone 6ß-hydroxylase Activity in Metabolism Positive Controls: The results from the positive metabolism control incubations will be expressed as testosterone 6ß-hydroxylase enzyme activity (CYP3A).

Results and discussion

Main ADME results

Type:

metabolism

Results:

The study did not identify any unique human metabolites

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Metabolite Profile of 14C-Desmedipham:

14C-Desmedipham showed high instability in the incubation buffer at pH 7.4, in the samples incubated for 0.5 and 1 hours at 37°C. A 14C-labelled product (namely Dm-5) was found after 0.5- and 1-hour incubation. The radioactive peak corresponding to Dm-5 accounted for 53.6% and 76.6% of the total peak areas in the chromatogram. Dm-5 degradation product was also detected in RLM and HLM incubated for 0 hours and accounted for 6.3% and 6.0% of the relative percentage of peak area, respectively, indicating that 14C-Desmedipham was instable under the microsomal test conditions. 31.1% and 13.4% of 14C-Desmedipham remained unchanged when incubated with RLM for 0.5 h and 1 h, respectively. In these incubations, a total of eight 14C-labelled potential metabolites could be detected in addition to compound Dm-5 (namely Dm-1, Dm-2, Dm-3, Dm-4, Dm-6, Dm-7, Dm-8 and Dm-9).

At both incubation time periods, three of the above mentioned 14C-labeled metabolites were detected below the LLOQ and three of them accounted for mean 5% or more of the relative percentage at 0.5 h incubation: Dm-1 (6.8%), Dm-6 (5.4%) and Dm-7 (16.8%) and only two at 1 h incubation: Dm-1 (13.4%) and Dm-7 (14.7%). The remaining metabolites (Dm-3, Dm-8 and Dm-9) showed mean relative percentages ranging from 0% to 2.8%. The relative percentages of the degradation product Dm-5 were 38.8% and 55.7% after 0.5- and 1-hour incubations, respectively, in RLM. These percentages were remarkably lower as compared with the ones in the samples incubated in buffer only, meaning that Dm-5 is subsequently metabolized in the RLM system. Figure 2 shows the representative HPLC-RAD profiles of 14C-Desmedipham in RLM.

44.0% and 23.5% of 14C-Desmedipham remained unchanged when incubated with HLM for 0.5 and 1 hours, respectively.

In these incubations, a total of 6 14C-labelled potential metabolites could be detected in addition to compound Dm-5 (namely Dm-1, Dm-3, Dm-4, Dm-6, Dm-7, and Dm-9). The rat metabolites Dm-2 and Dm-8 were not found at detectable levels in HLM. Two of the above mentioned 14C-labeled metabolites accounted for mean 5% or more of the relative percentage: Dm-6 (6.2% at 0.5 h and 8.0% at 1 h) and Dm-7 (11.6% at 0.5 h and 12.2% at 1 h). The remaining metabolites showed mean relative percentages ranging from 0% to 4.5%. The relative percentages of the degradation product Dm-5 were 37.6% and 49.8% after 0.5- and 1-hour incubation, respectively, in HLM. These percentages were lower as compared with the ones in the samples incubated in buffer only, meaning that Dm-5 is also subsequently metabolized in the HLM system. No human-specific metabolites of 14C-Desmedipham were found in the liver microsomes system in the present study.

The results of the present in vitro study demonstrated a slightly different metabolic pattern of 14C-Desmedipham when comparing rat and human liver microsomes that were incubated with the test item for 0.5 h and 1 h. This observation belongs however only to the lower number of metabolites detected in HLM. All metabolites, of which the two largest (Dm-6 and Dm-7) must be specifically mentioned, were also detected in the tests with rat liver microsomes.

Any other information on results incl. tables

Peak areas were recorded for each chromatogram. The LLOQ value was set at the 500 dpm level for radioactivity detection (cv <20%). These results indicate that after analysis of test samples, compounds showing radioactive peak areas below the mean peak area value obtained for the LLOQ (peak area of 1683.7) were not considered for quantification.

 

 

 

Positive Metabolism Controls

 

Formation of 6β-hydroxytestosterone from testosterone demonstrated sufficient metabolic capability of the microsome batches used in the study. However, due to the short incubation time (5 minutes) it was affected by the lack of the usual 2-minutes preincubation at 37°C mostly in the RLM incubations. Testosterone 6 β-hydroxylase activities was found to be 749.6 pmol/mg/minute in male rat liver microsomes and, 2415.5 pmol/mg/minute in pooled human liver.

 

 

 

Recovery of Radioactivity

 

The mean recovery of radioactivity after microsome incubations and sample preparation (i.e., protein precipitation with AcN and centrifugation) at T=0 hours was found to be 102.1% and 94.4% in RLM and HLM, respectively, after 0.5-hour incubation the recoveries were 97.6% in RLM and 98.2% in HLM, after 1 hour incubation the recoveries were 99.3% in RLM and 96.9% in HLM.

Applicant's summary and conclusion

Conclusions:

14C-Desmedipham was highly instable in incubation buffer at 37°C and at pH 7.4. A single degradation product (Dm-5) was formed in incubations with buffer alone accounting for 53.6% of the radioactivity after 0.5 h incubation and 76.6% after 1 h incubation.

In rat and liver microsomes, Dm-5 amounted to 37.6-38.8% after 0.5 h incubation and 49.8-55.7% after 1 h incubation. These percentages were lower as compared with the ones in the samples incubated in buffer only, meaning that Dm-5 is also subsequently metabolized in the rat liver and human liver microsome systems.

The in-vitro metabolite profile of 14C-Desmedipham when incubated with liver microsomes was slightly different between rats and humans.

In incubations with rat liver microsomes, 31.1% and 13.4% of the initial 14C-Desmedipham remained unchanged at 0.5 h and 1 h incubation, respectively. 14C-Desmedipham was metabolized towards a high number of metabolites. Under the experimental conditions used in the present study, a total of 8 metabolites were detected in addition to the degradation product Dm-5, three (Dm-1, Dm-6 and Dm-7) of them were above 5% of the relative percentage at 0.5 h incubation and two (Dm-1 and Dm-7) at 1 h incubation.

In human liver microsomes, 14C-Desmedipham was metabolized to lower number of metabolites (six in addition to compound Dm-5). The percentage of 14C-Desmedipham remaining after 0.5 h and 1 h incubation was slightly higher (44.0% and 23.5%, respectively), indicating a slightly slower metabolism rate of 14C-Desmedipham in human liver microsomes.

From the six detectable metabolites formed by human liver microsomes two (Dm-6 and Dm-7) were the most important compounds because of their higher relative percentage values (from 6.2% to 12.2%). These metabolites were also detected as major metabolites in incubations with rat liver microsomes.

In summary, it can be assumed that in incubations with human liver microsomes not specifically different 14C-Desmedipham metabolites are formed in comparison with rat liver microsomes.

Executive summary:

The comparative metabolism of [Phenoxy-UL-¹⁴C] Desmedipham (¹⁴C-Desmedipham) was investigated in vitro by incubating the test item with liver microsomes from male Wistar rats (RLM) and humans (HLM) in the presence of NADPH cofactor. The test item concentration was 10 μM and the protein concentration 1 mg/mL. The 10 μM concentration was chosen in order to have enough sample material for possible identification of metabolites by chromatographic or spectroscopic methods. The sampling times were 0, 0.5 and 1 hours after test start. The test durations of 0.5 and 1 hours were considered as reasonable because positive results were obtained from the enzymatic reaction of Testosterone to Hydroxy-Testosterone already after 5 minutes. Samples were analyzed following protein precipitation by reversed phase HPLC with radiochemical detection (HPLC-RAD).  The recovery of radioactivity was measured in both microsome incubations and amounted to ≥96.9% for the 0.5- and 1-hour samples.  The metabolic activity of the microsomes was clearly demonstrated by determining 6β-hydroxytestosterone that was formed from testosterone by testosterone 6β-hydroxylase. This biochemical reaction is a well-known marker for the CYP3A microsomal enzyme.  ¹⁴C-Desmedipham was highly instable after incubation with buffer at 37^°C and pH 7.4. A single radiolabeled compound (Dm-5) was produced and accounted for >53.6% of the radioactivity. Dm-5 was also detected in the liver microsome incubations from both species but in lower amounts, meaning that Dm-5 was subsequently metabolized by the liver microsome preparations.  The results of the tests indicated that the in vitro metabolism of ¹⁴C-Desmedipham when incubated with liver microsomes was slightly different between rats and humans.  In rat liver microsomes, 31.1% and 13.4% of the initial ¹⁴C-Desmedipham remained unchanged after 0.5 h and 1 hour incubation, respectively. ¹⁴C-Desmedipham was metabolized towards a high number of metabolites. A total of 8 metabolites were detected in addition to the degradation product Dm-5, three of them were above 5% of the relative percentage.  In human liver microsomes, a total of 6 metabolites were found in addition to Dm-5. The percentage of ¹⁴C-Desmedipham remaining after 0.5 h and 1 h incubation was slightly higher as compared to the rat liver microsomes system indicating a marginally slower metabolism rate of ¹⁴C-Desmedipham in human liver microsomes. From the five detectable metabolites formed by human liver microsomes, two (Dm-6 and Dm-7) were the most important compounds because of their high relative percentage values (from 6.2% to 12.2%). Metabolites Dm-6 and Dm-7 were also detected as major metabolites in incubations with rat liver microsomes.  The slightly different metabolic pattern of ¹⁴C-Desmedipham when comparing rat and human liver microsomes belongs however only to the lower number of metabolites detected in human liver microsomes. All metabolites, of which the two largest (Dm-6 and Dm-7) must be specifically mentioned, were also detected in the tests with rat liver microsomes.  In summary, it can be assumed that in incubations with human liver microsomes not specifically different ¹⁴C-Desmedipham metabolites are formed in comparison with rat liver microsomes. No human-specific metabolites of ¹⁴C-Desmedipham were found in the liver microsomes system.