1,4-Benzenediamine, 2-(methoxymethyl)-

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

other: the study was performed with the sulfate salt of 1,4-diamino-2-methoxymethylbenzene

Adequacy of study:

key study

Study period:

From April 08, 2010 to Mar. 02, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed scientific standards/principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Study was performed to determine the in-vitro metabolism of test substance using human hepatocytes. Hepatocytes were incubated with test substance and the supernatants were analyzed for determination of metabolites by using HPLC and Mass Spectrometry.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

337906-37-3

Cas Number:

337906-37-3

IUPAC Name:

337906-37-3

Constituent 2

Reference substance name:

-

EC Number:

474-270-7

EC Name:

-

IUPAC Name:

474-270-7

Constituent 3

Reference substance name:

1,4-diamino-2-methoxymethyl benzene sulfate

IUPAC Name:

1,4-diamino-2-methoxymethyl benzene sulfate

Details on test material:

RADIOALBELLED TEST SUBSTANCE
- Name of test material: 14C 1,4-diamino-2-methoxymethyl benzene sulfate
- TSIN: RC09039.00
- Molecular formula: Not reported
- Molecular weight: 252 (at specific gravity)
- Smiles notation: Not reported
- InChl: Not reported
- Substance type: Pure active substance
- Physical state: Not reported
- Specific activity: 2.04 GBq/ mmol (55 mCi/mmol) (measured by mass spectroscopy) and 6.99 MBq/ mmol (189 µCi/mg) (measured by gravimetric analysis)
- Locations of the label: [ring-U-14C]MEOME PPD sulphate
- Stability under test conditions: Not reported
- Storage condition of test material: Not reported

Radiolabelling:

yes

Remarks:

14C

Administration / exposure

Route of administration:

other: In vitro

Vehicle:

other: Supplemented Hepatocyte Maintenance Media (HMM) and hepatocyte growth media

Duration and frequency of treatment / exposure:

- Suspended method: 3 h
- Plated method: 24 h

Doses / concentrations

Remarks:

Doses / Concentrations:
- Suspended human hepatocyte method: 1 and 10 µg/mL
- Plated hepatocyte method: 1.08, 10.8 and 107.8 µg/mL (7, 70 and 700 µM, respectively)

Positive control reference chemical:

- Suspended human hepatocyte method: 7-ethoxycoumarin was tested at 500 µM for 0 and 3 h
- Plated human hepatocyte method: 7-Ethoxycoumarin was tested at 1000 µM for 24 h

Details on study design:

TEST SYSTEM: Human hepatocytes were used as test system to determine the possible metabolism pathways of test substance.

PREPARATION OF DOSING SOLUTION
A) Suspended human hepatocyte method: Dose solutions were prepared at 2X the desired test concentration in supplemented HMM, from a stock solution of 14C-labelled test substance at 0.208 mg/mL. The 20 µg/mL dose solution (test concentration of 10 µg/mL) was prepared directly from the stock solution, while the 2 µg/mL dose solution (test concentration of 1 µg/mL) was prepared by diluting the 20 µg/mL dose solution further with supplemented HMM. Three 10 µL aliquots of each dose solution were counted on a liquid scintillation counter to determine the radiolabeled content. The accuracy of dose concentration at 1 and 10 µg/mL was 94 and 101%, respectively.
B) Plated human hepatocyte method: Dose solutions were prepared at 2X the desired test concentration in hepatocyte growth media, from a stock solution of 0.464 mg/mL 14C-labelled test substance. The dose solution prepared at 215.6 µg/mL (test concentration of 107.8 µg/mL) was prepared directly from the stock solution. The lower dose solutions were prepared through serial dilution from the highest dose solution. Three 10 µL aliquots of each dose solution were measured on a liquid scintillation counter to calculate the actual concentration in each dose solution. The accuracy of dose concentration at 1.08, 10.8 and 107.8 µg/mL was 93.5, 97 and 100.2%, respectively.

Details on dosing and sampling:

SUSPENDED HUMAN HEPATOCYTE METHOD:
- Preparation of hepatocytes: The cryopreserved human hepatocytes were thawed and reconstituted in CHRM (Cryopreserved Hepatocyte Recovery Media). The cell suspension was centrifuged at 800 rpm for 10 minutes. The supernatant was gently aspirated off the cell pellet, the cell pellet was tapped to loosen, and the cells were diluted into approximately 5 mL supplemented Hepatocyte Maintenance Media (HMM). A cell density and viability measurement was taken using a hemacytometer. The cell suspension was then diluted to a concentration of 1,000,000 cells/mL (2X the test concentration) with additional HMM. The cell suspension was pipetted (150 µL) to the appropriate wells in a 96 well plate. The plate was placed in a 37°C incubator for an acclimation period of approximately 15 to 30 minutes.
- Procedure for treatment: Prior to dosing, all 0 minute wells were quenched with 300 µL acetonitrile to stop enzyme activity. Each well was dosed with 150 µL of the appropriate dose solution. The prequenched wells were moved to a plate on wet ice for the duration of the method. The test plates were placed in the 37°C incubator. At the designated time point, the wells were quenched with 300 µL acetonitrile to stop the enzyme activity. The plates were centrifuged at 4000 rpm for 10 minutes to sediment any debris or protein precipitation. A 200 µL aliquot was removed from the supernatant of each well and combined with the other two replicates into an HPLC vial. The samples were frozen at -80°C until shipment to the Trace Analysis Core group at MBC.
- Each test concentration was tested in triplicates.

PLATED HUMAN HEPATOCYTE METHOD:
- Preparation of hepatocytes: The plateable cryopreserved human hepatocytes were thawed and reconstituted in CHRM (Cryopreserved Hepatocyte Recovery Media). The cell suspension was centrifuged at 800 rpm for 10 minutes. The supernatant was gently aspirated off the cell pellet, the cell pellet was tapped to loosen, and the cells were diluted into approximately 5 mL hepatocyte plating media. A cell density and viability measurement was taken using a hemacytometer. The density of the cell suspension was adjusted to 700,000 cells/mL with warm plating media, and the plates were seeded with 350,000 cells/well (0.5 mL/well). The plates were placed in a 37°C incubator for 4 to 6 hours for attachment. After the attachment period, the plating media was aspirated off the cell layer and replaced with ice cold growth media containing Geltrex. The plates were returned to the 37°C incubator for overnight. Upon warming, the Geltrex forms a gel overlay over the cells. The following morning the growth media was gently aspirated off the cell layer and replaced with 250 µL warm growth media. The wells were then dosed with 250 µL of the appropriate 2X dose solution.
- Procedure for treatment: After dosing the wells with 250 µL of the appropriate dose solution, the plates were placed in a 37°C incubator on a rotating shaker, set at 80 rpm. After the 24 hour incubation period, the plates were removed from the incubator and the supernatant drawn off the cell layer. The supernatant was centrifuged at 10,000 rpm for 10 minutes to sediment any cells or debris, and the resulting supernatant was drawn off the cell pellet and frozen at -80°C until shipment to TAC for analysis. After sampling from each well, the wells were washed with 1 mL methanol. The methanol wash was collected from each well and frozen at -80°C for future analysis if needed.
- Each test concentration was tested in triplicates.

ANALYSIS
- Incubations of radiolabelled test substance with human hepatocytes, dose and stability samples were analyzed with HPLC-UV system. Radio analytical detector (RAD)/Q-ToF-mass spectroscopy was used to determine the metabolites. Accurate mass identification was made with mass spectroscopy while quantitation was performed with radio analytical detection. The sample were split and half the volume was analyzed as is, with the other half derivatised with Propionic anhydride in order to retain the compounds of interest and obtain better MS signal. Incubates were quantitated against a calibration curve prepared from the dose solution standards. The details are provided in the study report.

Results and discussion

Main ADME results

Type:

metabolism

Results:

Two metabolism pathways, N-mono-acetylation and cysteine conjugation of test substance were observed.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Suspended human hepatocyte method:
- In the suspended hepatocyte incubations only the N-mono-acetylated MBS metabolite was observed.
- The amount of N-acetylated MBS did increase with the higher dose level of MBS, but did not increase proportionally (0.16 µg/mL at the low dose and 0.49 µg/mL at the high dose).

Plated human hepatocyte method:
- In the plated hepatocyte method, two metabolites were observed, N-mono-acetylated MBS and Cysteine MBS.
- The N-acetylated MBS increased in the MBS dose range of 1.08 to 10.8 µg/mL, but decreased in the MBS dose range of 10.8 to 107.8 µg/mL, suggesting a possible saturation of the enzyme (1.01 µg/mL in the low dose, 6.94 µg/mL in the mid dose and 3.89 µg/mL in the high dose).
- The Cysteine MBS was only found in the highest MBS test concentration (7.56 µg/mL in the high dose).
- Two non-metabolic modifications of 2-Methoxy-methyl-pphenylenediamine sulfate occurred and these compounds were detected in dose solutions, stability samples, and incubated samples. One of these involved conjugation of 2- Methoxy-methyl-p-phenylenediamine sulfate with glucose. The other modification was hypothesized to be a cyclised form of 2-Methoxy-methyl-pphenylenediamine sulfate but this structure could not be confirmed by MS analysis.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: Two metabolism pathways, N-mono-acetylation and cysteine conjugation of test substance were observed.
N-Acetylation of 2-Methoxy-methyl-p-phenylenediamine sulfate was found to occur in both suspended and plated hepatocytes at all dose levels. At the highest substrate concentration in the plated hepatocytes, cysteine conjugation occurred in addition to N-mono-acetylation.

Executive summary:

The purpose of this in-vitro study was to determine the possible metabolism pathways of 1,4-diamino-2-methoxymethyl benzene sulfate (MBS) after incubation with human hepatocyte cells.

Prior to use, cryopreserved human hepatocytes were thawed and reconstituted in CHRM (Cryopreserved Hepatocyte Recovery Media). The test was performed by two methods, suspended and plated method.

In the suspended human hepatocyte method, 150 µL (1,000,000 cells/mL) of hepatocytes were pipetted (150 µL) into wells in a 96 well plate and incubated for an acclimation period of approximately 15 to 30 minutes. After incubation, hepatocytes were treated with final test concentrations of 1 and 10 µg/mL for period of 3 h. Each test concentration was tested in triplicates.

In the plated human hepatocyte method, 350,000 cells/well (0.5 mL/well) were seeded in the 96 well plate. After processing of cells, hepatocytes were incubated with final test concentration of 1.08, 10.8 and 107.8 µg/mL (7, 70 and 700 µM, respectively) for 24 h. Each test concentration was tested in triplicates.

In both the suspended and plated methods, 7-ethoxycoumarin served as positive control. The concentration of 7-ethoxycoumarin in suspended and plated method was 500 µM (for 3 h) and 1000 µM (for 24 hours), respectively.

After treatment, the resulting supernatant was drawn off the cell pellet and frozen at -80°C until shipment for analysis. Incubations of radiolabelled test substance with human hepatocytes, dose and stability samples were analyzed with an HPLC-UV system. Radio analytical detector (RAD)/Q-ToF-mass spectroscopy was used to determine the metabolites.

In the suspended hepatocyte incubations only the N-mono-acetylated MBS metabolite was observed. The amount of N-acetylated MBS did increase with the higher dose level of MBS, but did not increase proportionally (0.16 µg/mL at the low dose and 0.49 µg/mL at the high dose).

In the plated hepatocyte method, two metabolites were observed, N-mono-acetylated MBS and Cysteine MBS. The N-acetylated MBS increased in the MBS dose range of 1.08 to 10.8 µg/mL, but decreased in the MBS dose range of 10.8 to 107.8 µg/mL, suggesting a possible saturation of the enzyme (1.01 µg/mL in the low dose, 6.94 µg/mL in the mid dose and 3.89 µg/mL in the high dose). The Cysteine MBS was only found in the highest MBS test concentration (7.56 µg/mL in the high dose). Two non-metabolic modifications of 2-Methoxy-methyl-pphenylenediamine sulfate occurred and these compounds were detected in dose solutions, stability samples, and incubated samples. One of these involved conjugation of 2- Methoxy-methyl-p-phenylenediamine sulfate with glucose. The other modification was hypothesized to be a cyclised form of 2-Methoxy-methyl-pphenylenediamine sulfate but this structure could not be confirmed by MS analysis.

Based on above, N-Acetylation of 2-Methoxy-methyl-p-phenylenediamine sulfate was found to occur in both suspended and plated hepatocytes at all dose levels. At the highest substrate concentration in the plated hepatocytes, cysteine conjugation occurred in addition to N-acetylation.