1-Hexyl 4,5-diamino pyrazole hemisulfate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given, followed scientific standards/principles.

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:

An in-vitro study was conducted to assess the metabolism of test substance following incubation with human keratinocytes (HaCaT cells) for 3 and 24 hours. Samples were collected at 3 and 24 hours after incubation. The metabolic loss and formation of potential metabolites of 1-hexyl-1H-pyrazole-4,5 diamine sulfate were analyzed using HPLC-UV/RAD/Q-ToF-mass spectrometer for quantification and structure elucidation.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-hexylpyrazole-3,4-diamine;sulfuric acid

EC Number:

696-231-5

Cas Number:

1361000-03-4

Molecular formula:

C9H18N4 x 0.5 H2SO4

IUPAC Name:

2-hexylpyrazole-3,4-diamine;sulfuric acid

Constituent 2

Reference substance name:

C6 Pyrazole hemisulfate

IUPAC Name:

C6 Pyrazole hemisulfate

Constituent 3

Reference substance name:

4,5-diamino-1-hexyl-1H-pyrazole hemisulfate

IUPAC Name:

4,5-diamino-1-hexyl-1H-pyrazole hemisulfate

Test material form:

solid: crystalline

Details on test material:

- Name of test material: 1-Hexyl-1H-pyrazole-4,5-diamine hemisulfate- Substance type: Pure active substance- Physical state: White Crystalline Solid- Stability under test conditions: Bench top stability was good, ≥ 74% at 3 hours and ≥ 59% at 24 hours- Storage condition of test material: Not reported

Radiolabelling:

yes

Remarks:

( 14 C)

Administration / exposure

Duration and frequency of treatment / exposure:

3 hours and 24 hours

Doses / concentrations

Remarks:

Doses / Concentrations:7, 70 and 200 µM in treatment medium (1.276, 12.76 and 36.45 µg/mL respectively)

Positive control reference chemical:

PABA (p-aminobenzoic acid) was used as a positive control for the N-Acetyltransferase-1 (NAT-1) enzyme.

Details on study design:

TEST SYSTEM: - Type of test system: HaCaT cells, an immortalized human keratinocyte cell line- Justification for selection of test system: Keratinocytes account for approximately 95% of the cells in the epidermis, and HaCaT cells are described as a relevant model to analyze the dermal metabolism of aromatic amines- Medium and buffers: Following medium and buffer were used: Growth medium: DMEM - Dulbecco's Modified Eagles Medium (Gibco #118855) supplemented with 10% Fetal Bovine Serum and 1% Pen-StrepTreatment medium: DMEM with 5% Fetal Bovine Serum and 1% Pen-Strep- Preparation of test system: Each test well was seeded with approximately 5,00,000 cells and allowed to grow for 24 hours in a 37°C incubator prior to dosing with the test substance.POSITIVE CONTROL: p-Aminobenzoic acid (PABA) was used as a positive control, to measure metabolic activity in the cells at 3 hours, 24 hours and the last 3 hours of the study. The 3 hour and the 24 hour wells were dosed at the same time as the other wells, while the last 3 hour wells were only given treatment media. With 3 hours remaining in the study, the last 3 hour wells were dosed with PABA to determine if the activity at the end of the study was comparable to that at the beginning of the study. The formation of the known metabolite, 4-acetamidobenzoic acid, was measured using established analytical methods, to assess the metabolic activity of the HaCaT cells.CELL DENSITY MEASUREMENT: Cell density was measured in all the test wells, including all the PABA positive control wells. Additional wells were seeded for cell density measurement at the time of dosing and after 3 and 24 hours in the treatment media for baseline results to determine the effects of test substance on the HaCaT cells. The Easy Count, an automated cell counter, was used to measure the cell density.

Details on dosing and sampling:

PREPARATION OF DOSING SOLUTIONS:- Dosing solutions at final concentration of 7, 70 and 200 µM (based on free base weight) were prepared in treatment medium.- The 7 µM dose solution was prepared using only radiolabeled test substance, while the 70 and 200 µM dose solutions were prepared using both radiolabeled and non-radiolabeled test substance.TEST PROCEDURE: - Treatment: After the growth phase, the growth media was gently aspirated off the cells and replaced with two mL of dosing solution.- No. of replicates: 3SAMPLING:- Period of sampling: After 3 and 24 hours of incubation- Procedure: After 3 hours, 1.5 mL of the supernatant was drawn off the cell layer of those designated wells. The samples were centrifuged at approximately 10,000 rpm for 10 minutes to sediment any cells or debris. An aliquot of the centrifuged supernatant was collected from each replicate and pooled with the other two replicates and frozen at -80°C. After 24 hours, the samples were processed in the similar manner and placed in the -80°C freezer for at least two hours- Storage of samples: The remainder of each sample was placed in a -80°C ultra cold freezer for storageANALYSIS OF SAMPLES: All samples were analyzed for metabolic loss and formation of potential metabolites using an HPLC-UV/RAD/Q-ToF-mass spectrometer for quantification and structure elucidation at Trace Analysis Core (TAC), at Mason Business Center.DETERMINATION OF CHEMICAL STABILITY: Evaluated by incubating each dose solution for 3 hours and 24 hours in the incubator without cells.

Results and discussion

Main ADME results

Type:

metabolism

Results:

N-Acetyl-1-Hexyl-1H-Pyrazole-4,5-Diamine was found as the single metabolic product of 1-hexyl-1H-pyrazole-4,5 diamine sulfate

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

- N-Acetyl-1-Hexyl-1H-Pyrazole-4,5-Diamine was found in the incubations with HaCaT cells at both 3 and 24 hours, and at all test concentrations.- Increasing the dose level of the test substance from 7 to 70 µM to 200 µM resulted in an increase in the amount of metabolite produced at the 3 hour time point. - At the 24 hour time point, there was an increase in the amount of metabolite produced when the test substance concentration was increased from 7 µM to 70 µM. However less amount of metabolite was produced at the 200 µM dose level than the 70 µM dose level. The increase in metabolite was not proportional to the increase in test substance dosed. - When the amount of metabolite produced within the 200 µM was compared at two time points, there was less metabolite produced at 24 hours than at 3 hours which was likely simply due to variation of the data analysis.- A small amount of a sugar conjugate (1-Hexyl-1 H-Pyrazole-4,5-Diamine hexose) was observed in the incubations as well as the stability samples (which contain no cells) indicating that this entity is not a metabolite.For details on results of test substance and its metabolites, refer to ‘table 2’ under ‘Any other information on material and method incl. results’ section.

Any other information on results incl. tables

Cell Density and Viability

- The growth or viability of the cells was not affected by either the addition of test substance or PABA during the course of the study.

For details on result of cell density and viability refer ‘table 3’ in this section.

Positive Controls Results

The PABA positive control samples showed a production of the known metabolite 4-acetamidobenzoic acid. The results indicate the cells were metabolically active for the entire duration the study. More activity was seen in the final three hours than the first three hours which was likely due to the presence of more cells in the final three hours

Table 2: Quantities of1-Hexyl-1 H-Pyrazole-4,5-Diamine (test substance) and N-Acetyl-1-Hexyl-1H-Pyrazole-4,5-Diamine (metabolite) during the study (Study# 87163)

Nominal test concentration-µg/mL	1.276 (7 µM)	12.76 (70 µM)	36.45 (200 µM)
Measured test concentration-µg/mL	1.221	12.81	36.64
LOQ-µg/mL	0.365	0.365	0.365
3 hour incubation
Tests substance-µg/mL	0.77	10.9	30.4
Metabolite-µg eq/mL	0.38	1.4	2.2
24 hour incubation
Tests substance-µg/mL	0	5.4	23.5
Metabolite-µg eq/mL	0.62	3.1	1.6

Note: LOQ - Limit of quantification

Table 3: Results on viability (Study # 87163)

Test Condition	Live cells per well	%Viability
Treatment medium (no test substance)
At time of dosing	18,85,000	99
After 3 hours	19,60,000	99.7
After 24 hours	35,70,000	99.7
3 hour incubation (with test compound)
0 µM	16,85,000	99.3
7 µM	18,00,000	99.3
70 µM	16,80,000	99.3
200 µM	20,90,000	99.3
24 hour incubation (with test compound)
0 µM	29,65,000	99.3
7 µM	30,75,000	98.3
70 µM	25,60,000	99.3
200 µM	32,65,000	99
PABA
3 hours	21,00,000	100
24 hours	33,65,000	99.7
Last 3 hours	24,25,000	99.7

Table 4: Results of metabolite production by PABA (positive control)

Test Condition	Metabolite produced
3 hours	6477
24 hours	53,426
Last 3 hours	10,430

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: N-Acetyl-1-Hexyl-1H-Pyrazole-4,5-Diamine was found as a metabolite of 1-hexyl-1H-pyrazole-4,5 diamine sulfateIncubation of 1-hexyl-1H-pyrazole-4,5 diamine sulfate with HaCaT cells at final concentrations of 7, 70 and 200 µM in treatment media for 24 hours resulted in formation of N-Acetyl-1-Hexyl-1H-Pyrazole-4,5-Diamine as the single metabolic biotransformation product.

Executive summary:

The study was performed to determine the metabolism of 1-hexyl-1H-pyrazole-4,5 diamine sulfate following incubation with HaCaT cells in an in-vitro experiment.

HaCaT cells, an immortalized human keratinocyte cell line, were used in the study to determine metabolism of the test compound. Each test well was seeded with 500,000 HaCaT cells and placed in a 37°C incubator for 24 hours. Incubations were performed in triplicate. After 24 hours growth, the wells were dosed with 2 mL of the appropriate dose solution. The following test concentrations were used: 7, 70 and 200 µM;7 µM dose solution was prepared using only radiolabeled test substance, while the 70 and 200 µM dose solutions were prepared using both radiolabeled and non-radiolabeled test substance.

After dosing, the plates were incubated for 3 and 24 hours. After the 3 and 24 hour incubation periods, the supernatant was drawn off, centrifuged at 10,000 rpm for 10 minutes and aliquots from replicates were pooled for analysis. Samples were analyzed using HPLC-UV/RAD/Q-ToF-mass spectrometer for quantification and structure elucidation.

P-Aminobenzoic acid (PABA) served as a positive control, to measure metabolic activity in the cells at 3 hours, 24 hours and incubation for last 3 hours of the study. The results indicated that the cells were active throughout the entire 24 hours.

Additional wells were seeded for cell density measurements at the time of dosing, and at 24 hours, in the treatment media for baseline results to determine the effects of test substance on the HaCaT cells. The Easy Count, an automated cell counter, was used to measure the cell density. The growth and viability of the cells did not appear to have been affected by the application of test substance at any concentration. The HaCaT cells were shown to be metabolically active during the entire incubation period.

N-Acetyl-1-Hexyl-1H-Pyrazole-4,5-Diamine was found as the only metabolite in the incubations with HaCaT cells at both 3 and 24 hours, and at all test concentrations. Increasing the dose level of the test substance from 7 to 70 µM to 200 µM resulted in an increase in the amount of metabolite produced at the 3 hour time point. At the 24 hour time point, there was an increase in the amount of metabolite produced when the test substance concentration was increased from 7 µM to 70 µM. However less amount of metabolite was produced at the 200 µM dose level than the 70 µM dose level. The increase in metabolite was not proportional to the increase in test substance dosed. A small amount of a sugar conjugate (1-Hexyl-1 H-Pyrazole-4,5-Diamine hexose) was observed in the incubations as well as the stability samples (which contained no cells) indicating that this entity is not a metabolite.

The above results conclude that theincubation of 1-hexyl-1H-pyrazole-4,5 diamine sulfate with HaCaT cells at final concentrations of 7, 70 and 200 µM in treatment media for 24 hours resulted in formation of N-Acetyl-1-Hexyl-1H-Pyrazole-4,5-Diamine as a single metabolic biotransformation product.