5-amino-o-cresol

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 19, 2003 to April 1, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed scientific principles, GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The comparative metabolic stability and metabolite profile of 4-amino-2-hydroxytoluene was investigated by incubating the test substance with cryopreserved primary human, rat, and mouse hepatocytes in suspension culture. The incubate samples were collected at different time intervals and analyzed for metabolite formation by LC-MS.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-o-cresol

EC Number:

220-618-6

EC Name:

5-amino-o-cresol

Cas Number:

2835-95-2

Molecular formula:

C7H9NO

IUPAC Name:

5-amino-2-methylphenol

Constituent 2

Reference substance name:

4-amino-2-hydroxytoluene

IUPAC Name:

4-amino-2-hydroxytoluene

Constituent 3

Reference substance name:

Haarpurpur

IUPAC Name:

Haarpurpur

Test material form:

solid: crystalline

Details on test material:

- Name of test material: 4-amino-2-hydroxytoluene
- TSIN: 23032
- Substance type: Pure active substance
- Physical state: Beige crystalline powder
- Stability under test conditions: The substance was considered to be stable for more than 5 years, if stored dry and protected from light at room temperature.
- Storage condition of test material: At room temperature for 5 years, light protected, tightly closed container.
- Solubility: The solubility of test substance was as follows:
Acetonitrile: 7.3 weight%
Water: 0.6 weight%
Acetone/water (1:1): 6 weight%
DMSO: >10 weight%
- Stability in solution: The stability of test substance (5%, w/v) in acetone/water (1:1 v/v) and DMSO, when stored at ambient temperature in the absence of light, was 99.5% and 100.3%, respectively.

Radiolabelling:

no

Administration / exposure

Route of administration:

other: In-vitro

Vehicle:

other: water (stock solution) and KHB buffer (working solution)

Duration and frequency of treatment / exposure:

Treated once and sampled at 0, 0.5, 1.5 and 4 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
10 µM

Positive control reference chemical:

- Coumarin was used as marker substrate for confirmation of general cytochrome P450 activity (represented by CYP 2A6) in human hepatocytes at a concentration of 200 µM in the positive control incubations (PCCYP).
- 7-Ethoxycoumarin was used as marker substrate for confirmation of general cytochrome P450 activity (represented by CYP 1A, 2A and 2B enzymes) in rat and mouse hepatocytes at a final concentration of 200 µM in the positive control incubations (PCCYP).
- 7-Ethoxyresorufin was used as marker substrate for confirmation of cytochrome P450 CYP1A1/2 activity in human, rat, and mouse hepatocytes at a final concentration of 2 µM in the positive control incubations (PCCYP1A2).
- Chlorzoxazone was used as marker substrate for confirmation of cytochrome P450 CYP2E1 activity in human, rat, and mouse hepatocytes at a final concentration of 50 µM in the positive control incubations (PCCYP2E1).
- P-Aminobenzoic acid was used as marker substrate for determination of the N-acetyl-transferase 1 activity in human as well as N-acetyl-transferase 2 activity in rat and mouse hepatocytes at a concentration of 1000 µM in the positive control incubations (PCNAT1).
- Sulfamethazine was used as marker substrate for determination of the N-acetyl-transferase 2 activity in human as well as N-acetyl-transferase activity in rat and mouse hepatocytes at concentrations of 10 and 100 µM in the positive control incubations (PCNAT2-10, PCNAT2-100).

Details on study design:

TEST SYSTEM: The primary hepatocytes obtained from human, rat and mouse were used. Primary hepatocytes are widely used in order to investigate phase I and II dependent metabolism. 10(6) cells /mL were used in study. Previous range finding experiments suggested that this concentration (10(6) cells / mL) yields sufficient quantities of metabolites in all test systems investigated. Details of test system are as follows:
- Cryopreserved human hepatocytes: Hepatocytes were obtained from In Vitro Technologies (Lot # KTG, IEM, CYE). Hepatocytes from three donors (male) were pooled to yield average enzyme activities and genotyped for CYP 2C9, 2C19, and 2D6 as well as for NAT2. These lots were *5/*7, wt/*5, and wt/*6 with respect to their NAT2 genotype. Therefore, in summa a rapid acetylation phenotype was expected.
- Cryopreserved rat hepatocytes: Male Sprague Dawley rat hepatocytes were used in study (from In Vitro Technologies; Lot # KTI). The rats were considered to be fast metabolizers.
- Cryopreserved mouse hepatocytes: Male ICR/CD-1 mouse hepatocytes were used in study (form In Vitro Technologies; Lot # PNM). CD-1 mice were reported to be a mixed population of rapid and slow metabolizers.

Negative control: Negative controls are as follows:
- Zero time point control.
- Incubations containing the respective solvent type but no test item (blank).

Replicates: Three each for test and negative control and one for positive control

Details on dosing and sampling:

PREPARATION OF CELLS:
- The 3 vials (human and rat hepatocytes, 13 vials of mouse hepatocytes) were prewarmed in water bath (37±3°C) and the hepatocytes were thawed. The hepatocytes were added to 43 mL (human and rat hepatocytes, 24 mL for the mouse hepatocytes) of prewarmed hepatocytes thawing media (HTM).
- The cell suspension was centrifuged at 60 x g and 22°C for 5 minutes and the supernatant was removed carefully except approximately 0.5 - 1 mL.
- 7 mL (human and rat hepatocytes, 6 mL for mouse hepatocytes) of KHB buffer were added and the pellet was carefully mixed (Human Hepatocytes: 1.75 x 10(6) cells/mL; Rat Hepatocytes: 2.13 x 10(6) cells/mL; Mouse Hepatocytes: 2.40 x 10(6) cells/mL).

PROCEDURE FOR TREATMENT:
- 250 µL of the hepatocyte suspension were pipetted in each well of a 24 well tissue culture plate (not collagen coated, since suspension cultures were used). After plating the cell number was evaluated, 10 µL of the remaining cell suspension was added to 90 µL of a trypan blue solution. The number of cells per mL was determined using a Neubauer cell count chamber.
- 250 µL of the respective test substance or marker substrate working solution (in KHB) were added to the 250 µL hepatocyte suspension in the respective wells.
- The 24 well plate was incubated at 37± 2°C, 5% (± 1%) CO2 and 95% (± 5%) relative humidity.

EVALUATION OF CELLS:
- After incubation times (0, 0.5, 1.5, 4 hours) each well was microscopically inspected in order to check for cell morphology, cytotoxicity, precipitation, contamination and unexpected observations.

PROCEDURE FOR TERMINATING REACTION
- 100 µL of cell suspension was removed and added to 100 µL of chilled stop reagent (acetonitrile on ice).
- Precipitated protein was pelleted by centrifugation for 10 min at 4°C and 3000 x g and the supernatant was analyzed. The details on reaction termination are provided in the study report.

METABOLITE CHARACTERISATION STUDIES
- Time and frequency of sampling: 0, 0.5, 1.5, 4.0 hours
- Method type(s) for identification: For the determination of the metabolite profile, a LC-MS analytical method which separates the test substance and its possible metabolite peaks was applied. A LC-MS/MS analytical method was used for the determination of the metabolic stability.
The parameters for the LC-MS/MS analytical method for the detection of test substance were optimized and validated with respect to specificity, linearity, limit of detection, limit of quantitation, and repeatability in previous "analytical method development and validation (report # 02082101-02)" experiments.
The details of LC/MS/MS system parameters are provided in the study report.
- Limits of detection and quantification: Limit of Detection (LOD) = 29 fmol on column (10 µL of 0.0029 µM); Limit of Quantitation (LOQ): 132 fmol on column (10 µI of 0.132 µM). The details on other validation parameters are provided in the study report.

Results and discussion

Main ADME results

Type:

metabolism

Results:

An extensive phase II metabolic activity, sulfatation of the phenol group of test substance, was observed in hepatocytes from all three species hepatocytes (human, rat and mouse).

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Determination of the Metabolic Stability (Time Course):
- Under the test conditions applied in this study, the test substance was extensively metabolized by primary human, rat, and mouse hepatocytes in the order human > rat > mouse.
- After incubation of an initial concentration of 10 µM with approximately 1 x 10(6) cells/mL for 1.5 hours 92.9 ± 0.6%, 74.0 ± 1.6% and 54.1 ± 4.0% of the test substance was metabolized upon incubation with human, rat, and mouse hepatocytes, respectively.
- With human and rat hepatocytes, the test substance was completely metabolized after 4 hours. The metabolism was linear with incubation time in the first 1.5 hours.
- The samples were analyzed by LC-MS/MS with respect to loss of parent compound.

DETERMINATION OF THE METABOLITE PROFILE:
- To determine the in vitro metabolite profile of the test substance, the samples of the metabolic stability experiment (time course) were analyzed by LC-MS (Scan).
- The formation of putative metabolites was analyzed after incubation of an initial concentration of 10 µM of the test substance with approximately 1 x 10(6) cells/mL for 0, 0.5, 1.5, and 4 hours (S0, S0.5, S1.5, S4) and compared to the respective blank control (no test item = TI blank) at that incubation time.
- In congruence with the decrease of the test substance (92.9 ± 0.6 %, 74.0 ± 1.6% and 54.1 ± 4.0% metabolism after incubation for 1.5 hours with human, rat, and mouse hepatocytes, respectively), there were significant amounts of different metabolites detected.

Metabolites of High Abundance / Major Metabolites:
- Under the test conditions applied in this study, sulfatation was suggested to be the major metabolic reaction of the test substance upon incubation with human, rat and mouse hepatocytes. This phase II reaction was consistent with the chemical structure of the test substance, since the hydroxy group was a prerequisite for sulfatation.

Metabolites of Low Abundance / Minor Metabolites:
- Under the test conditions applied in this study, taurine conjugation was suggested to be a minor metabolic reaction of the test substance upon incubation with rat hepatocytes.
- With mouse hepatocytes minor glucuronidation was detected. Sulfatation and glucuronidation are typically competitive.
- While sulfotransferases are generally considered high affinity / low capacity enzymes, UDP-glucuronosyl transferases are usually low affinity / high capacity enzymes. As a consequence, at higher substrate concentrations the ratio glucuronide conjugate / sulfate conjugate might increase.

Questionable Metabolites:
- Further putative metabolites of the test substance (hydroxylation by mouse hepatocytes) were questionable due to low / inconsistent signal intensities and/or simultaneous detection in the test substance blank and/or zero time point control.

Any other information on results incl. tables

CELL NUMBER AND VIABILITY/CYTOTOXICITY: The hepatocyte viability after 0, 0.5, 1.5 and 4 hour was as follows:

Human hepatocytes (Viable cells/mL: 0.88×10(6)): Ca. 90%, 90%, 90% and minor decrease ca. 10%

Rat hepatocyte (Viable cells/mL: 1.07×10(6)): Ca. 95%, 95%, 95% and minor decrease ca.10%

Mouse hepatocyte (Viable cells/mL: 1.2×10(6)): Ca. 80%, 80%, 80% and minor decrease ca. 10%

Microscopic inspection revealed no signs of test substance specific cytotoxicity up to 4 hour of incubation. The cell viability appeared to be unaffected over the incubation period with some minor decrease after 4 hour.

RESULT OF POSITIVE CONTROL: All marker reactions clearly demonstrated the functional state of the test systems and test conditions for the respective reaction. Details on the results of positive control are provided in the study report.

Table 1. Metabolite Profile of 5 -Amino-2 -methylphenol in Human, Rat, and Mouse Hepatocytes (study # 85354)

Approx. Retention Time	Ratio of Retention Time (RT metabolite/RT parent)	Metabolite	ESI	Mass Diff (mass parent - mass metabolite)	m/z found	Abundance
						HH	RH	MH
8.7	0.99	NA	+	0	124	NA
5.9	0.67	Glucuronide Conjugation	+	176	300			+
7.5	0.86	Sulfate Conjugation	+/-	80/78	204	++	++	+++
9.4	1.07	Hydroxylation	-	14	138			+?
12.4	1.42	Taurine Conjugation	-	107	229		+

Abundance: Rough estimate of metabolite abundance according to peak signal; not quantitative, since the ionization behaviour of the metabolites is unknown.

HH: Human hepatocyte

RH: Rat hepatocyte

MH: Mouse hepatocyte

NA: Not applicable

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: Test substance was extensively metabolized by sulfate conjugation.
Exposure of rodent (mice, rats) and human hepatocytes to 4-Amino-2-hydroxytoluene under identical test conditions suggests no significant differences in the metabolic rate/capacity or the metabolic profile. Under the conditions applied in this study, test substance was extensively metabolized by sulfate conjugation.
The results of this comparative in vitro metabolism study (in hepatocytes) support the validity of an extrapolation from rodent (rat) data to the human situation for phase II metabolism.

Executive summary:

The metabolic profile of 4-Amino-2-hydroxytoluene was investigated in-vitro by means of cryopreserved primary hepatocytes from male human donors, male Sprague Dawley rats and male ICR/CD-1 mice.

The metabolic capacity of the tested hepatocytes was characterized by marker substrates for phase-I (CYP 2A6, 1A, 2A and 2B, 1A1/2 and 2E1) and phase II enzymes (N-Acetyl-transferase 1/2) used routinely or considered to be relevant for the metabolism of the class of arylamines, including 4-Amino-2-hydroxytoluene.

10 μM of the test substance were incubated with approximately 1 x 10(6) cells/mL for a period of 4 hours. Samples were removed and analyzed at 0, 0.5, 1.5 and 4 hours. Final test conditions were chosen based on range finding experiments.

Incubation was performed in 24-well microtiter plates and cell conditions were microscopically evaluated at each time point.

The metabolic stability was assessed by detection of loss of parent compound by means of LCMS/ MS. The metabolic profile was also investigated by LC-MS and metabolites identified/characterized as far as possible.

Cell viability was not affected by the test substance for the incubation period. A slight decrease in viability of about 10% was noted at the end of the entire incubation period.

The marker substrates demonstrated the metabolic capacity and the validity of the test system. As expected, differences in the metabolic capacity between rat, mouse and human hepatocytes were noted for the different phase I marker reactions.

For phase II enzymes (N-acetyltransferase, NAT), liver samples from humans (pooled in order to result in a intermediate metaboliser phenotype), rats (rapid metaboliser) and mice (mixed genotype of slow and rapid metabolisers) showed similar activities for the conversion of the model substratepara-amino benzoic acid, whereas with sulfamethazine (NAT 2) a much higher activity was noted for human than for rat and mouse hepatocytes.

Test substance was rapidly metabolized in human, rat and mouse hepatocytes. A decrease of 92.9%, 74% and 54.1% of the parent compound was detected within 1.5 hour upon incubation with human, rat and mouse hepatocytes, respectively. During the first 1.5 hour, metabolism was linear with incubation time. After 4 hours, test substance was completely metabolized by both human and rat hepatocytes. In mice hepatocytes, 82% of the parent compound was metabolized.

The overall metabolism rate in rodent and human hepatocytes was comparable. The analysis of the formed metabolites revealed an extensive phase II metabolic activity with sulfatation of the phenol group of test substance predominating in hepatocytes from all three species.

Interestingly, test substance appeared not to be acetylated by human, rat, or mouse hepatocytes, though N-acetyltransferase activity of all three test systems was demonstrated by the respective marker reaction controls.

Exposure of rodent (mice, rats) and human hepatocytes to 4-Amino-2-hydroxytoluene under identical test conditions suggests no significant differences in the metabolic rate/capacity or the metabolic profile. Under the conditions applied in this study, test substance was extensively metabolized by sulfate conjugation.

Therefore, the results of this comparative in-vitro metabolism study in hepatocytes support the validity of an extrapolation from rodent (rat) data to the human situation for phase II metabolism.