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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Objective of study:
absorption
Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: “The Guidelines for the Testing of Chemicals- Health Effect” issued by the Ministry of Environmental Protection of the People’s Republic of China, second edition, September 2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Test material form:
liquid
Details on test material:
- Purity: >99%
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rats have been widely used for toxicological studies according to the standard guidelines and rats were recommended by the second edition of Guidelines for the Testing of Chemicals issued by Ministry of Environmental Protection of People’s Republic of China.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Vital River Laboratories Animal Technology Co. Ltd.
- Grade: SPF
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Male: 215.8-249.9 g, Female: 190.9-211.1 g
- Housing: Rats were group-housed in polycarbonate transparent box, three to five animals in each cage.
- Diet: ad libitum (The animals were fasted for 12 hours prior to exposure)
- Water: ad libitum
- Acclimation period: All rats were kept in the maintenance room for at least 7 days prior to the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-25°C
- Humidity: 40-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:
other: gavage and intravenous injection
Vehicle:
other: Corn oil for gavage and DMSO, cremophor el and saline for intravenous injection
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1. Toxicokinetics by intravenous injection in SD rats: An aliquot (20 mg) of the test substance was weighed and dissolved by 0.1 mL dimethyl sulfoxide, and then 0.65 mL cremophor el was added and mixed fully, diluted with normal saline to 5 mL, by which the concentration of test substance in mixture (stock solution) was 4 mg/mL. When used, the mixture was diluted with normal saline to 0.5 mg/mL.
2. Toxicokinetics by single oral gavage in SD rats: The dose formulations were prepared with corn oil, in which concentration of the suspension was 2 and 50 mg/mL, respectively.
3. Toxicokinetics by repeated oral gavage in SD rats: The dose formulations were prepared with corn oil, with the concentration of the suspension being 2 mg/mL.
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day
Remarks:
Intravenous injection
Dose / conc.:
20 mg/kg bw/day
Remarks:
Oral: gavage (Single)
Dose / conc.:
500 mg/kg bw/day
Remarks:
Oral: gavage (Single)
Dose / conc.:
20 mg/kg bw/day
Remarks:
Oral: gavage (Repeated)
No. of animals per sex per dose / concentration:
See details in study design
Control animals:
no
Details on study design:
Test 1. Toxicokinetics by intravenous injection in SD rats: Four SD animals (2 females and 2 males) were treated with test substance at a single dose level (5 mg/kg) by intravenous injection
Test 2. Toxicokinetics by single oral gavage in SD rats: Eight animals (4 males and 4 females) were treated with test substance at two dose levels (20 and 500 mg/kg body weight) by single gavage administration.
Test 3. Toxicokinetics by repeated oral gavage in SD rats: Three test groups were designed based upon single oral administration by gavage. One group was administered with single gavage of the test substance (Group I). The second group was set to measure trough concentration (Group II). The third group was administered daily by gavage with the test substance for continuous 7 days (Group III). Four animals (2 males and 2 females) were used in each group.
Details on dosing and sampling:
Test 1. Toxicokinetics by intravenous injection in SD rats: Following dosing, 0.2 mL of blood was collected via the orbital vein and was added to anticoagulant heparin solution. The samples were kept on ice until processed into plasma. Blood was collected at approximately 1, 5, 15, 30 min and 1, 2, 4, 6, 8, 12, 24 h. Pretreatment and measurement of samples were done as soon as possible after collection. Blood samples were processed and analysed as soon as possible. Pharmacokinetic parameters such as VB, AUC and t1/2, etc were calculated.
Test 2. Toxicokinetics by single oral gavage in SD rats: Following dosing, 0.2 mL of blood was collected via the orbital vein and was added to anticoagulant heparin solution. The sample was kept on ice until processed into plasma. Blood was collected at approximately 5, 15, 30 min and 1, 2, 4, 6, 8, 12, 24 h post exposure, and 48 h was additionally designed to high dose. Blood samples were processed and analysed as soon as possible. Pharmacokinetic parameters such as AUC, Cmax, F, Tmax, MRT and t1/2, etc were calculated.
Test 3. Toxicokinetics by repeated oral gavage in SD rats: Group I: Animals were treated with test substance for single oral administration by gavage. 0.2 mL blood samples were collected from the orbital venous plexus prior to exposure and at 5, 15, 30 min and 1, 2, 4, 6, 8, 12, 24 h post exposure. Heparin was used as anticoagulant.
Group II: Exposure was performed daily by gavage for 6 days (one time/day). Blood was collected daily at time prior to exposure and after 24 h for last exposure to detect trough concentration.
Group III: Exposure was performed daily by gavage for 7 days. 0.2 mL blood samples were collected from the orbital venous plexus prior to exposure, and at 5,15, 30 min and 1, 2, 4, 6, 8, 12, 24 h post exposure. Heparin was used as anticoagulant. Pretreatment and measurement of samples were done as soon as possible after collection. Pharmacokinetic parameters such as AUC, F, Tmax, MRT and t1/2, etc were calculated.
Statistics:
The chromatograms of test substance and its Rotundine internal standard were integrated by Analyst version 1.4.2. The peak area ratios of test material and its Rotundine internal standard were used as Y-axis, and the concentrations of test material were used as X-axis, and then evaluated by linear regression analysis. Weighting coefficient is 1/x2. The test material concentration in unknown sample was calculated using the following formula: x = y-b divided by m.
where:
y = the peak area ratio of test material and its Rotundine internal standard;
b = intercept of standard curve;
m =slope of the standard curve.

Results and discussion

Preliminary studies:
Three rats were administered the test substance by gavage at dose levels of 20 and 1000 mg/kg body weight, as well as at 5 mg/kg body weight by single intravenous injection, respectively. The results of the pilot test showed that parent compound was lower than detection limit in plasma at 1 minute after intravenous injection, and then through full scan mass spectrum analysis found high response certain ionic form, indicating significantly time course distribution of test substance metabolite.
Main ADME resultsopen allclose all
Type:
other: elimination half-life of test substance metabolite
Results:
0.142 ± 0.005 h after intravenous injection of a dose of 5 mg/kg body weight
Type:
other: bioavailability
Results:
11.70 ± 6.85% after single oral administration of 20 mg/kg
Type:
other: bioavailability
Results:
16.04 ± 9.48% after single oral administration of 500 mg/kg

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Toxicokinetic characteristics of the test substance by intravenous injection were in accordance with a statistic moment model. Elimination half-life value (t1/2z) of test substance metabolite was 0.142±0.005h. The 95% test substance metabolite in plasma was eliminated after 0.639 hours (approximately 4.5-fold half-life value). Clearance of the test substance (CLz) was 0.02±0.003L/h/kg, indicating relatively fast elimination of test substance in SD rats.

Toxicokinetic characteristics of the test substance after single oral dose administration (20 mg/kg or 500 mg/kg): Two absorption peaks were found for the two exposure levels. The first Tmax of the low and high dose was 0.083 ± 0 h and 6 ± 2.31 h, respectively. The second Tmax of the low and high dose was 0.083 ± 0 h and 8 ± 3.27 h, respectively. The second Tmax of the high dose had a slight time-lag compared with that of low dose. The elimination half-life values (t1/2) of the low and high dose was 5.32 ± 3.92 h and 3.7 ± 1.72 h, respectively, which was reduced with increased exposure level. The MRT0-t was 4.02 ± 1.76 h and 3.78 ± 0.57 h, without any significant change between the low and high dose. The values of the AUC0-t for the time course distribution were 122073.95 ± 71503.04 area/L*h and 4185937.52 ± 2472333.36 area/L*h, respectively. The ratio was 1.00:34.29, which was significantly higher than the ratio of the administered dose levels (1:25). According to the above findings, the absorption followed the nonlinear dynamic model by oral administration of test substance (20-500 mg/kg) in SD rats. The bioavailability (F) of the test substance when administered at dose levels of 20 and 500 mg/kg by gavage was 11.7 ± 6.85% and 16.04 ± 9.48% based on the data analysis of toxicokinetic characteristics following intravenous injection.

Repeated oral administration: Toxicokinetic characteristics of the test substance in repeated dose 7-day study: Two absorption peaks were found. The Tmax of the first administration was 0.083 ± 0 h and 6 ± 2.31 h, respectively. The Tmax of the last administration was 0.083 ± 0 h and 7 ± 4.16 h, respectively. The Tmax of the last administration had a slight time-lag compared with that of the first. The Cmax of the first administration was 187808.50 ± 222507.19 and 15172.00 ± 15463.54 area/L, respectively. The Cmax of the last administration was 70688.25 ± 31227.54 and 8901.50 ± 6903.49 area/L, respectively. The Cmax of the first administration was lower than that of the last, indicating trend of decreasing Cmax with multiple doses. The AUC0-t of the time course distribution was 122073.95 ± 71503.04 and 64367.68 ± 25851.74 area/L*h, respectively. The bioavailability (F) of the test substance was 11.70 ± 6.85 % and 6.17 ± 2.48 %, respectively. The internal exposure dose of the test substance was decreased after administration for 7 days. The elimination half-life values (t1/2) of the low and high dose were 5.32 ± 3.92 h and 4.01 ± 3.36 h, respectively. The MRT0-t was 4.02 ± 1.76 h and 3.57 ± 0.76 h, which did not change significantly over time. The trough concentrations of test substance were unchanged following oral administration for continuous seven days. According to the above results, both the bioavailability and the internal dose of test substance were slightly decreased after 7-day repeated exposure by oral administration, which accordingly did not show cumulative toxic effect.
Toxicokinetic parametersopen allclose all
Toxicokinetic parameters:
other: Clearance of the test substance (CLz) was 0.02 ± 0.003L/h/kg/Intravenous injection
Test no.:
#1
Toxicokinetic parameters:
Tmax: 20 mg/kg or 500 mg/kg: 0.083 ± 0 h and 6 ± 2.31 h, respectively
Test no.:
#2
Toxicokinetic parameters:
Tmax: 20 mg/kg or 500 mg/kg: 0.083 ± 0 h and 8±3.27 h, respectively
Toxicokinetic parameters:
other: 20 mg/kg or 500 mg/kg: 5.32 ± 3.92 h and 3.7 ± 1.72 h, respectively
Toxicokinetic parameters:
other: MRT0-t: 20 mg/kg and 500 mg/kg was 4.02 ± 1.76 h and 3.78 ± 0.57 h, respectively
Toxicokinetic parameters:
other: AUC0-t: 20 mg/kg or 500 mg/kg was 122073.95 ± 71503.04 area/L*h and 4185937.52 ± 2472333.36 area/L*h, respectively.
Toxicokinetic parameters:
other: Bioavailability (F): 20 mg/kg and 500 mg/kg was 11.7 ± 6.85% and 16.04 ± 9.48% respectively.
Toxicokinetic parameters:
Tmax: 7-day study: 0.083 ± 0 h and 6 ± 2.31 h, respectively/First administration
Toxicokinetic parameters:
Tmax: 0.083 ± 0 h and 7 ± 4.16 h, respectively/Last administration
Toxicokinetic parameters:
Cmax: first administration was 187808.50 ± 222507.19 and 15172.00 ± 15463.54 area/L, respectively.
Toxicokinetic parameters:
Cmax: last administration was 70688.25 ± 31227.54 and 8901.50 ± 6903.49 area/L, respectively.
Toxicokinetic parameters:
other: AUC0-t of the time course distribution was 122073.95 ± 71503.04 and 64367.68 ± 25851.74 area/L*h, respectively.
Toxicokinetic parameters:
other: Bioavailability (F) of the test substance was 11.70 ± 6.85 % and 6.17 ± 2.48 %, respectively.
Toxicokinetic parameters:
other: t1/2 of the low and high dose were 5.32 ± 3.92 h and 4.01 ± 3.36 h, respectively
Toxicokinetic parameters:
other: MRT0-t was 4.02 ± 1.76 h and 3.57 ± 0.76 h for low and high dose respectively.

Metabolite characterisation studies

Metabolites identified:
not specified

Applicant's summary and conclusion

Conclusions:
The toxicokinetic characteristics of the test substance in repeated dose 7-day study: Two absorption peaks were found. The Tmax of the first administration was 0.083 ± 0 h and 6 ± 2.31 h, respectively. The Tmax of the last administration was 0.083 ± 0 h and 7 ± 4.16 h, respectively. The Tmax of the last administration was slightly time-lagged compared with that of the first administration. Cmax of the first administration was 187808.50 ± 222507.19 and 15172.00 ± 15463.54 area/L, respectively. The Cmax the the last administration was 70688.25 ± 31227.54 and 8901.50 ± 6903.49 area/L, respectively. The Cmax of the first administration was a little lower than that of the last administration. The AUC0-t for the time course distribution was 122073.95 ± 71503.04 and 64367.68 ± 25851.74 area/L*h, respectively. The bioavailability (F) of the test substance was 11.70 ± 6.85% and 6.17 ± 2.48%. The internal exposure dose of the test substance was decreased after exposure for 7 days. The half-life value (t1/2) of the low and high dose was 5.32 ± 3.92 h and 4.01 ± 3.36 h, respectively. The MRT0-t was 4.02 ± 1.76 h and 3.57 ± 0.76 h, which did not change significantly over time. The trough concentrations of test substance were unchanged after oral administration for continuous seven days. According to the above results, bioavailability and internal dose of test substance were both slightly decreased after 7-day repeated exposure by oral administration, which accordingly did not have a cumulative toxic effect.
Executive summary:

The toxicokinetics study (absorption phase) of test substance in male and female Sprague Dawley (SD) rats was performed according to “The Guidelines for the Testing of Chemicals-Health Effect” issued by the Ministry of Environmental Protection of the People’s Republic of China, second edition, September 2013 (OECD TG 417, Toxicokinetics).

The pilot test showed that the test substance can be biodegraded completely and that its metabolite m/z 286.2→149.2 had good stability. The toxicokinetics characteristic of the test substance was indirectly suggested in the change process of metabolite m/z 286.2→149.2.

The quantitation of the test substance was conducted using a validated liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method. The validation results indicated that the method was reliable and highly specific. Endogenous substance in control plasma did not interfere with determination of the test substance. Liner range was between 25-12500 ng/mL. Lower limit of quantification (LLOQ) was 25 ng/mL. The intra-batch precision (RSD%) was in range of 4.49% to 9.87% for test substance in SD rat plasma measured at 75, 3750 and 9375 ng/mL. The intra-batch accuracy (RE%) was in range of -7.70% to 5.89%. The inter-batch precision (RSD%) was in range of 5.43% to 8.27%. The inter-batch accuracy (RE%) was between -5.97% and -1.97%. Average recovery of the test substance was 109.02%. Substrate inhibiting factor was 79.34%. The plasma samples of 37500 ng/ml, which was diluted 10 times, were determined for 6.40% of RSD and -3.95% of RE. Treated quality control samples remained stable in auto-sampler and test bench at room temperature for 39 hours and 16 hours, respectively. There were similar results for preconditioning quality control samples at 2-8°C for 207 hours. No residual effect was found after sample injection at the highest standard curve concentration (12500 ng/mL). The results indicated that methods mentioned above meet the requirements of toxicokinetics for the test substance.

Toxicokinetic characteristics of the test substance by intravenous injection were in accordance with a statistic moment model. Elimination half-life value (t1/2z) of test substance metabolite was 0.142 ± 0.005 h. The 95% test substance metabolite in plasma was eliminated after 0.639 hours (approximately 4.5-fold half-life value). Clearance of the test substance (CLz) was 0.02 ± 0.003L/h/kg, indicating relatively fast elimination of test substance in SD rats.

Toxicokinetic characteristics of the test substance after single oral dose administration (20 mg/kg or 500 mg/kg): Two absorption peaks were found for the two exposure levels. The first Tmax of the low and high dose was 0.083 ± 0 h and 6 ± 2.31 h, respectively. The second Tmax of the low and high dose was 0.083 ± 0 h and 8 ± 3.27 h, respectively. The second Tmax of the high dose had a slight time-lag compared with that of low dose. The elimination half-life values (t1/2) of the low and high dose was 5.32 ± 3.92 h and 3.7 ± 1.72 h, respectively, which was reduced with increased exposure level. The MRT0-t was 4.02 ± 1.76 h and 3.78 ± 0.57 h, without any significant change between the low and high dose. The values of the AUC0-t for the time course distribution were 122073.95 ± 71503.04 area/L*h and 4185937.52 ± 2472333.36 area/L*h, respectively. The ratio was 1.00:34.29, which was significantly higher than the ratio of the administered dose levels (1:25). According to the above findings, the absorption followed the nonlinear dynamic model by oral administration of test substance (20-500 mg/kg) in SD rats. The bioavailability (F) of the test substance when administered at dose levels of 20 and 500 mg/kg by gavage was 11.7 ± 6.85% and 16.04 ± 9.48% based on the data analysis of toxicokinetic characteristics following intravenous injection.

Toxicokinetic characteristics of the test substance in repeated dose 7-day study: Two absorption peaks were found. The Tmax of the first administration was 0.083 ± 0 h and 6 ± 2.31 h, respectively. The Tmax of the last administration was 0.083 ± 0 h and 7 ± 4.16 h, respectively. The Tmax of the last administration had a slight time-lag compared with that of the first. The Cmax of the first administration was 187808.50 ± 222507.19 and 15172.00 ± 15463.54 area/L, respectively. The Cmax of the last administration was 70688.25 ± 31227.54 and 8901.50 ± 6903.49 area/L, respectively. The Cmax of the first administration was lower than that of the last, indicating trend of decreasing Cmax with multiple doses. The AUC0-t of the time course distribution was 122073.95 ± 71503.04 and 64367.68 ± 25851.74 area/L*h, respectively. The bioavailability (F) of the test substance was 11.70 ± 6.85 % and 6.17 ± 2.48 %, respectively. The internal exposure dose of the test substance was decreased after administration for 7 days. The elimination half-life values (t1/2) of the low and high dose were 5.32 ± 3.92 h and 4.01 ± 3.36 h, respectively. The MRT0-t was 4.02 ± 1.76 h and 3.57 ± 0.76 h, which did not change significantly over time. The trough concentrations of test substance were unchanged following oral administration for continuous seven days.

According to the above results, both the bioavailability and the internal dose of testsubstance were slightly decreased after 7-day repeated exposure by oral administration, which accordingly did not show cumulative toxic effect.

In conclusion, the toxicokinetic characteristics of the test substance was indirectly demonstrated through detection of the time course distribution of its metabolites (m/z 286.2→149.2). The test substance can be absorbed and eliminated rapidly, and the elimination half-life value (t1/2z) was 0.142 ± 0.005 h after intravenous injection of a dose of 5 mg/kg body weight in SD rats. For single oral administration (20 and 500 mg/kg) by gavage, the bioavailability (F) of the test substance was 11.70 ± 6.85 and 16.04 ± 9.48%, respectively. Both the bioavailability and the internal dose of the test substance were slightly decreased after 7-day repeated exposure by oral administration, which accordingly did not have a cumulative toxic effect.