Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
14 May 2008 to 20 June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 94.8%
- Specific activity: 0.07 MBq/mg
- Locations of the label: not specified
- Expiration date of radiochemical substance: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, moisture and light protected under nitrogen.
- Stability under test conditions: Stability of the test item in the application solution was demonstrated by HPLC (Figure 1). The radio-purity of the test item in the formulation was found to be 99.78% after application.
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of administration, the administration solution was prepared. For administration at the target dose level of 2000 mg/kg, an aliquot of 300 mg of the radiolabeled test item, an aliquot of 2700 mg of the non-labeled test item and 12 mL corn oil were mixed to give a target concentration of 2000 mg/kg/10 mL.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Radiolabelling:
yes

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Recognized by international guidelines as an acceptable animal model for physiological, pharmacological, or toxicological studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 8 weeks
- Weight at study initiation: 31 g ± 2.1 in males and 24 g ± 1.4 in females
- Housing: During acclimatisation in groups of 7/8 in macrolon type 3 cages. During the experiment. Group of 3 animals in metabolism cages with wire floor, 1 day prior to the treatment and during the experiment.
- Diet (e.g. ad libitum): Pelleted 3433 Kliba standard diet ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 7 days to laboratory environment, including 1 day in metabolism cages

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30.0 - 70.0%
- Air changes (per hr): 10-15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light / 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of administration, the administration solution was prepared. For administration at the target dose level of 2000 mg/kg, an aliquot of 300 mg of the radiolabeled test item, an aliquot of 2700 mg of the non-labeled test item and 12 mL corn oil were mixed to give a target concentration of 2000 mg/kg/10 mL. The exact amount of radioactivity in the application solution was determined by Liquid Scinitillation Counting (LSC) resulting in 21.5 MBq, equivalent to 308 mg of undiluted 14C-Silan 449029 VP. The total amount of diluted 14C-Silan 449029 VP was 3008 mg, resulting in a new specific activity of 0.0072 MBq/mg. The concentration of Silan 449029 VP (= Silan 449029 VP & 14C-Silan 449029 VP) in the formulation was 200.5 mg/mL.

Duration and frequency of treatment / exposure:
single oral administration
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
single oral administration of 14C-Silan 449029 VP
Dose / conc.:
0.007 other: MBq/mg
Remarks:
target specific radioactivity
Dose / conc.:
10 other: mL/kg
Remarks:
target administration volume
No. of animals per sex per dose:
12 male and 12 female
Control animals:
no
Positive control:
Not used
Details on study design:
- Dose selection rationale: The current study supports a micronucleus study in bone marrow cells, where mice have been exposed through the oral route. A dose level of 2000 mg/kg was chosen, the same dose as the highest concentration in the micronucleus test.
- Rationale for animal assignment (if not random): random
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, serum, femur, stomach, large intestine, small intestine, GI tract contents, liver and kidney
- Time and frequency of sampling: Three male and three female animals each were sacrificed one and four hours after test item administration, and terminal blood, femur, stomach, combined GI tract contents, small intestine, large intestine, liver and kidney were isolated. At 24 h after test item administration the rest of the animals were sacrificed and terminal blood, femur, stomach, small intestine, large intestine, combined GI tract contents, liver, kidney as well as urine and faeces were isolated.

Results and discussion

Main ADME resultsopen allclose all
Type:
excretion
Results:
At the end of the 24 hours time period, 24.9% and 17.4% of the applied dose was detected in urine, 3.4% and 9.8% of the applied dose in cage wash of male and female mice, respectively.
Type:
absorption
Results:
At the end of the 24 hours time period, the systemic absorption (bioavailability) for Silan 449029 VP was at least 28.3% in male mice and 27.2% in female mice.
Type:
excretion
Results:
At the end of the 24 hours time period, a total of 63.8% and 64.2% of the applied dose was excreted via faeces in male and female mice, respectively.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
At the end of the 24 hours time period, the systemic absorption (bioavailability) for Silan 449029 VP was at least 28.3% in male mice and 27.2% in female mice. Overall significant mean levels of the test item were found in blood and plasma as early as 1 hour after application. This indicates that after oral administration the test item was rapidly absorbed in significant amounts.
Details on distribution in tissues:
One hour after application the mean total radioactive residue concentrations in male and female mice were found to be between 70.0 – 74.6 μgeq/g in blood, 72.7 – 78.2 μgeq/g in plasma, 42.4 – 48.9 μgeq/g in femur, 5512.9 – 9183.3. μgeq/g in stomach, 2867.6 – 3180.5 μgeq/g in small intestine, 1221.5 – 1328.9 μgeq/g in large intestine, 8480.0 – 23815.0 μgeq/g in GI tract contents, 177.0 – 179.2 μgeq/g in liver and 332.8 – 481.3 μgeq/g in kidney.
4 hours after application in stomach (6090.4 - 12986 μgeq/g), in small intestine (2211.9 – 4543.4 μgeq/g), in large intestine (1524.0 – 3170.4 μgeq/g) and in combined GI tract contents (28315.7 – 30171.4 μgeq/g). In blood (42.4 – 48.3 μgeq/g), in plasma (45.4 – 50.6 μgeq/g), in kidney (193.4 – 262.3 μgeq/g) and in liver (127.8 – 145.9 μgeq/g) a decrease in mean radioactive residue concentration was observed. In femur (32.5 – 49.2 μgeq/g) similar or slightly lower mean radioactive residue concentrations were observed.
24 hours after application only minor mean radioactive residue concentrations were left in stomach (30.9 – 47.1 μgeq/g), small intestine (16.3 – 22.4 μgeq/g), large intestine (17.1 – 21.0 μgeq/g) and combined GI tract contents (11.1 – 29.1 μgeq/g) compared to the 1 and 4 hours sampling time points. The same was true for blood (3.7 μgeq/g), plasma (4.7 – 4.9 μgeq/g) and femur (7.3 – 8.4 μgeq/g).
Toxicokinetic parametersopen allclose all
Key result
Test no.:
#1
Toxicokinetic parameters:
Cmax:
Remarks:
Males: 70.0 ± 11.2 μgeq/g (blood), 72.7 ± 12.9 μgeq/g (plasma), 49.2 ± 12.7 μgeq/g (femur), 179.2 ± 40.1 μgeq/g (liver), and 481.3 ± 340.4 μgeq/g (kidney). Females: 74.6 ± 12.3 μgeq/g, 78.2 ± 11.9 μgeq/g, 177 ± 36.3 μgeq/g and 332.8 ± 98.7 μgeq/g,
Key result
Test no.:
#1
Toxicokinetic parameters:
Cmax:
Remarks:
Males: 12986.0 ± 7940.9 (stomach), 4543.4 ± 1710.2 (small intestine), 1524.0 ± 1311.7 (large intestine) and 30171.4 ± 4649.0 μgeq/g (GI tract) Females: 9183.3 ± 3435.5, 2867.6 ± 503.0, 3170.4 ± 1792.8 and 28315.7 ± 1552.2 μgeq/g.

Metabolite characterisation studies

Metabolites identified:
no

Applicant's summary and conclusion

Conclusions:
Overall significant mean levels of the test item were found in blood and plasma as early as 1 hour after application. This indicates that after oral administration the test item was rapidly absorbed in significant amounts.