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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Objective of study:
absorption
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
A single dose of 746.7 mg/kg test item was orally administered (by gavage) to 6 male Sprague Dawley rats, and the concentrations of the test item and its metabolites were determined in plasma at 0 (predose), 5, 15, 30, 45, 60, 120, 240, 360, 480, 720 and 1440 min after dosing by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
- Naringin (4',5',7-trihydroxy flavanone-7-rhamnonglucoside)
- Source: Sigma (St. Louis, MO)
- Purity: > 95%
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: College of Military Medicine Animal Research Center (Guangzhou, China).
- Weight at study initiation: 250–300 g
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
50% PEG400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the test item was dissolved into 127.9 mg/ml with 50% PEG400.
Duration and frequency of treatment / exposure:
single dose.
Doses / concentrations
Dose / conc.:
746.7 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 male rats.
Control animals:
yes
Positive control:
Not applicable.
Details on study design:
SAMPLE PREPARATION
- A 0.1 ml aliquot sample plasma was transferred to a clean glass tube. Ten microliters of methanol with 1000 ng/ml internal standard was added and vortexed. Then 400 μl ethyl acetate (LC grade) was added to the tube and vortexed again for 2 min. After the sample was centrifuged at 6000×g for 10 min, the upper organic layer was transferred into a 5 ml
glass tube. 200 μl ethyl acetate was added to the deposition again and then the deposition was extracted as those mentioned above again. The organic layer was mixed together and
dried with nitrogen at room temperature. The dried residue was dissolved in 100 μl of the mobile phase. A 20 μl aliquot of the sample was injected into the LC/MS/MS.

ANALYTICAL METHOD
- Separation method: HPLC system (SHIMADZU HPLC system, SHIMADZU, Columbia, MD), consisting of an SCL-10A system controller, an LC-10AD HPLC pump, a DGU 12A-degasser, and a manual injector.
- Conditions: The HPLC column (100 mm × 2.0 mm) was a 5 μm BetaBasic-C18 ODS column (KEYSTONE Bellefonte, PA). Column temperature was maintained at room temperature.
The mobile phase consisted of methanol (70%) and water (30%), which was filtered through a 0.2 μm nylon filter before use. The flow rate was 0.4 ml/min and the total run time was
3.0 min for each injection.
- Detection method: MS (Applied Biosystems MDS Sciex API3000 Triple Quadrapole mass spectrometer (MS/MS) equipped with a heated nebulizer interface (500ºC)). The mass spectrometer chose the positive ionization mode as the detection mode with the collision gas off. At the same time the mass spectrometer temperature was set at 370ºC. Under these conditions, the analytes yielded protonated molecules at m/z 581.3 for naringin, m/z 273.4 for naringenin and m/z 611.5 for hesperidin.
- Internal or external calibration: internal (standard: hesperidin, source: National Institute for the control of pharmaceutical and biological products, HPLC grade).
- Limits of detection and quantification: LOQ = 0.5 ng/ml.
- Extraction recovery (indicate if results are corrected or not for recoveries): The mean recovery values for the entire procedure were found to be 90.58, 91.64, 94.15% for naringin.
- Validation: The assay was validated in rat plasma. The retention time was 0.5, 0.5, and 1.07 min for naringin, hesperidin and naringenin, respectively. The absence of a signal at the
retention time of naringin, naringenin and hesperidin in the blank plasma established the specificity of the assay. For naringin, the accuracy values of the assay varied from 93.02 to 96.37%, the within-day precision values ranged from 3.9 to 5.3%, the between-day precision values ranged from 1.7 to 4.9%; the assay was linear from 5 to 1000 ng/ml, using 0.1 ml rat plasma, with a regression coefficient (r2) >0.9996.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: plasma.
- Time and frequency of sampling: Approximately 0.5 ml blood was withdrawn from each rat according to a schedule of 0 (predose), 5, 15, 30, 45, 60, 120, 240, 360, 480, 720 and 1440 min after dosing.
Statistics:
Data was collected and processed using Sciex Analyst 1.1. Plasma concentration–time profiles were analyzed by WinNonlin computer software, Version 4.0 (Pharsight Corporation, Mountain View, CA), using a noncompartmental method.

Results and discussion

Preliminary studies:
Naringenin’s conjugates in the homogenates of different tissues were confirmed to be completely hydrolyzed within 2 h in the preliminary test. Meanwhile, no obvious loss of naringin was observed during this process.
Main ADME resultsopen allclose all
Type:
absorption
Results:
the test item was quickly absorbed into the body (Tmax 45min), and declined rapidly within 2h.
Type:
excretion
Results:
After 24h, the test item was no longer detectable.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
The test item was quickly absorbed into the body and could be detected 5 min after oral administration. The concentration of naringin in plasma reached nearly 2300 ng/ml in 45 min (Tmax) and declined rapidly, within 2 h. The Cmax was 3782.50 ± 986.82 ng/ml, and the AUC(0–24) was 6026.32 ± 1562.63 ng/ml h.
Details on excretion:
The test item could not be detected after 24h.
Toxicokinetic parametersopen allclose all
Key result
Test no.:
#1
Toxicokinetic parameters:
Cmax: 3782.50 ± 986.82 ng/ml
Remarks:
in plasma.
Key result
Test no.:
#1
Toxicokinetic parameters:
Tmax: 0.75 ± 0.25 h
Remarks:
in plasma.
Key result
Test no.:
#1
Toxicokinetic parameters:
AUC: 6026.32 ± 1562.63 ng/ml h
Remarks:
in plasma (0-24).

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
The concentration of naringenin and naringenin glucuronide in plasma increased more slowly after oral administration of naringin. The Tmax of naringenin and naringenin glucuronide occurred at 9 and 7.5 h. For naringenin, the Cmax was 227.05±88.41 ng/ml, and the AUC0–24 was 1252.24±461.66 ng/ml h, whereas for naringenin glucuronide, the Cmax was 43575.00±8409.00 ng/ml, and the AUC(0–24) was 238269.00±53253.10 ng/ml h. The AUC0–24 of naringenin glucuronide was higher than naringin and naringenin, indicating that naringenin glucuronide is the main existent form in rat plasma after oral administration.

Any other information on results incl. tables

Table 1. Relevant pharmacokinetic parameters.

Parameter

Average ± S.D. (n = 6)

Naringin

Tmax (h)

0.75±0.25

Cmax (ng/ml)

3782.50±986.82

AUC [0-24] (ng/ml h)

6026.32±1562.63

MRT (h)

6.47±1.18

CL (ml/h)

0.028±0.008

HL-λz (h)

5.34±1.07

Naringenin

Tmax (h)

9.00±1.50

Cmax (ng/ml)

227.05±88.41

AUC [0-24] (ng/ml h)

1252.24±461.66

MRT (h)

8.16±1.59

CL (ml/h)

0.30±0.070

HL-λz (h)

2.98±0.65

Naringenin glucuronide

Tmax (h)

7.50±1.00

Cmax (ng/ml)

43575.00±8409.00

AUC [0-24] (ng/ml h)

238269.00±53253.10

MRT (h)

6.37±0.60

CL (ml/h)

0.0004±0.00019

HL-λz (h)

1.84±0.16

Applicant's summary and conclusion

Conclusions:
The test item was quickly absorbed into the body, and could be detected 5 min after oral administration. The concentration of naringin in plasma reached nearly 2300 ng/ml in 45 min (Tmax) and declined rapidly, within 2 h. Based on these results, the test item shows no bioaccumulation potential.
Executive summary:

A study on the absorption of the test item in rats was performed. A single dose of 746.7 mg/kg test item was orally administered (by gavage) to 6 male Sprague Dawley rats, and the concentrations of the test item and its metabolites were determined in plasma at 0 (predose), 5, 15, 30, 45, 60, 120, 240, 360, 480, 720 and 1440 min after dosing by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The test item was quickly absorbed into the body and could be detected 5 min after oral administration. The Tmax was 45 min. The plasma concentration also declined quickly, within 2 h. Its metabolites, naringenin and naringenin glucuronide, were produced slowly after oral administration and Tmax for them were 9 and 7.5 h, respectively. After 24 h, no test item could be detected. Based on these results, the test item shows no bioaccumulation potential.