Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-21-1999 to 03-29-1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Objective of study:
distribution
excretion
Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
GLP compliance:
yes (incl. certificate)
Remarks:
NOTOX B.V., The Netherlands

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: white solid
- Radiochemical purity (if radiolabelling): 98 %
- Specific activity (if radiolabelling): 3.92 GBq/mmol (by mass spectrometry), 1.79 MBq/mg (by gravimetric analysis)
- Stability under test conditions: stable
- Storage condition of test material: room temperature in dark for Non-radiolabelled and in freezer in the dark for radiolabelled test material.
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: 7 weeks old
- Weight at study initiation: ranged between 270 and 292 g.
- Fasting period before study: 18 hours
- Housing: individually housed in Macrolon plastic cages
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): standard pelleted laboratory animal diet (Carfil Quality BVBA, Oud-Tumhout, Belgium)
- Water (e.g. ad libitum): tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25
- Humidity (%): 40-80
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: January 21, 1999 To: April 14, 1999

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The formulations were prepared immediately prior to dosing. The required amount of [14C]-test article was weighed into an empty glass container. After addition of the required amount of non-radiolabelled test material, a weighed amount of the vehicle was added to achieve the final concentration. For groups 1M and 2M (50 and 1000 mg/kg bw respectively), the nominal test material concentration was 10 and 200 mg/ml, with a nominal radioactivity concentration of 2.4 MBq/ml. The formulations were stored at ambient temperature on a magnetic stirring device for a maximum of 4 hours.

VEHICLE
- Amount of vehicle (if gavage): Dose volume of 5 mL/kg bw, the nominal radioactivity contained in each dose was approximately 3 MBq.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
Immediately after preparation and prior to dose administration, the homogeneity and radioactivity concentration of each formulation were verified by radioanalysis.
Duration and frequency of treatment / exposure:
Single dose
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
4
Control animals:
no
Details on dosing and sampling:
PHARMACOKINETIC STUDY (distribution, excretion)
- Tissues and body fluids sampled :
1) Urine samples were collected from all animals over the following time intervals: 0-24, 24-48, 48-72, and 72- 96 hours after administration of [14C]-labeled test substance. The urine was freeze-trapped to avoid atmospheric oxidation, evaporation and bacterial degradation. Faeces were collected on dry ice over the same time intervals as urine. All urine and faeces samples were stored at < -20°C until analysis. At termination the interior of the cages was rinsed with methanol/water (50/50). The cage rinse was weighed and stored at < - 20° until analysis.
2) Volatiles: For all animals, expired CO2 was collected in a 2N NaOH solution, and other volatiles in cellosolve, over the following nominal time intervals: 0-8, 8-24, 24-36, and 36-48 h after the administration of [14C]-test material. Sample weight was determined at the end of each collection period. Samples were refrigerated at approximately 4°C for a maximum of 3 days, within which period the analyses were completed. Following completion of the analysis, the remainder of the study sample was discarded.
3) All animals were anaesthetised using ether at 96 h after administration of [14C]-test article. The thorax and abdomen were opened with a midline incision. By means of aorta punction the maximum possible amount of blood was withdrawn. Care was taken to avoid contamination of other organs with blood. Sampled blood was transferred into preweighed heparinised tubes. A subsample of approximately 1 mL of the heparinised blood was removed for total C analysis. The remaining blood was centrifuged to obtain the plasma which was weighed and stored at < -20°C until analysis. Following the removal of the blood, the following tissues and organs were harvested: liver, kidneys, spleen, abdominal fat, muscle (left hind leg), G.I. tract, lymph nodes (mesenteric lymph nodes and Peyer's patches), gonads (testes, seminal vesicles, and prostate), bone (left femur), thymus, skin, brain and carcass. The weight of each tissue and blood sample was recorded at the time it was harvested. All biological samples were stored at < -20°C until analysis.

Radioactivity measurements:
All samples were subjected to radioactive measurements performed using a Packard scintillation counter (1900TR) with automatic external standard quench correction.
Statistics:
The mean and standard deviation were used to characterise the data, where appropriate (i.e., radioactivity measurement, concentration, etc.). Concentrations of radioactivity in blood, plasma and tissues were calculated as mg equivalents of [14C]-test material /kg of sample. The total amounts of radioactivity in tissue and excreta samples were calculated as a percentage of the administered radioactivity and a 14C mass balance was presented.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:
- Concentrations of radioactivity in blood, plasma and tissues: For group 1M, the highest mean tissue concentrations were recorded in the spleen and the mesenteric lymph nodes (0.99 and 1.04 mg/kg tissue, respectively). Similar mean tissue levels were recorded in the thymus, G.I tract, Peyer's patches, skin, bone, liver and kidney (0.11-0.55 mg/kg). In the remaining tissues and organs analysed (brain, blood, testes, prostate, seminal vesicles, abdominal fat and femoral muscle), mean radioactivity concentrations were about an order of magnitude lower (0.012-0.063 mg/kg). For group 2M, the highest mean tissue concentrations were recorded in the spleen and the G.I. tract (26.9 and 18.0 mg/kg, respectively). The mean tissue concentrations in thymus, Peyer's patches, skin, bone, liver and kidney of the animals of group 2M was 3.86-11.2 mg/kg. In the remaining tissues and organs analysed (brain, mesenteric lymph nodes, blood, testes, prostate, seminal vesicles, abdominal fat and femoral muscle), mean radioactivity concentrations were about an order of magnitude lower (0.11-1.96 mg/kg). The mean concentration in the plasma (0.002 mg/kg for group 1M and <0.16 mg/kg for group 2M) was a factor of at least 12 lower than in the blood (0.056 mg/kg for group 1M and 1.94 mg/kg for group 2M).
- Amounts of radioactivity (percent of dose) in blood, plasma and tissues: The mean amount of radioactivity was highest in the G.I tract (0.14-0.22%), followed by liver (0.057-0.059 %). In all other tissues and organs analysed, the mean amount of radioactivity was <0.007% of the dose. The carcass of the animals of group 1M and 2M contained a mean 1.27 and 0.84% of the dose. The total mean amount of radioactivity recovered from tissues and carcass at 96 h post dose was 1.49 and 1.14% for groups 1M and 2M, respectively.
Details on excretion:
- Volatiles: The amount of expired volatile radioactivity was minimal and the sampling was discontinued after 48 h. For group 1M and 2M, mean recoveries of radioactivity in the CO2 traps were 0.007 and 0.003%, respectively. The amount of expired organic volatiles was <0.001% in anyone sample analysed.
- Faeces: The majority of the radioactive dose was excreted in the faeces, mostly during the first 24 hours post dose (group 1M) or during the first 48 hours post dose (group 2M). Mean total recoveries from the faeces during the 96-h test period were 82 and 97% for groups IM and 2M, respectively (at a mean mass balance of 85 and 98%, respectively) (Table 1).
- Urine: Mean total recoveries from the urine during the 96-h test period were 0.60 and 0.28% for groups 1M and 2M, respectively (Table 1).

Metabolite characterisation studies

Metabolites identified:
not measured

Any other information on results incl. tables

- Each animal received a radioactive dose of [14C]-test material which ranged between 2.9 and 3.0 MBq. The animals of group 1M (nominal dose 50 mg/kg bw) received an actual dose of test material which ranged between 48 and 54 mg/kg bw corresponding with 96 and 108% of the nominal dose respectively. The animals of group 2M (nominal dose 1000 mg/kg bw) received an actual dose of test material which ranged between 1018 and 1065 mg/kg bw corresponding with 102 and 107% of the nominal dose respectively. The administered doses were considered to be sufficiently close to the target levels in order to fulfill the study's objectives.

- No mortalities occurred during the study. On day 3 of the study, animal 5 of group 2 was observed to have a scab near the tail tip. There were no other antemortem observations. During tissue harvest no abnormalities were noted.

Table: Amount (mean ± SD) of total radioactivity found in faeces and urine from male rats dosed with [14C-] test article/kg bw.

Interval (h)

% of dose in faeces

% of dose in urine

50 mg/kg bw

1000 mg/kg bw

50 mg/kg bw

1000 mg/kg bw

0-24

71.7±18.6

43.9±12.4

0.25±0.38

0.05±0.06

24-48

7.69±5.51

44.8±10.6

0.20±0.17

0.14±0.15

48-72

2.31±2.85

6.6±7.55

0.09±0.08

0.06±0.11

72-96

0.64±0.48

1.24±1.55

0.06±0.06

0.03±0.05

Total

82.3±12.0

96.6±4.46

0.60±0.67

0.28±0.35

Applicant's summary and conclusion