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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:
key study
Study period:
12.12.1995 to 17.09.1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Objective of study:
absorption
Principles of method if other than guideline:
Toxicokinetics: influence of the carrier on the intestinal absorption of D4
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS (definitive study- excluding preliminary study)
- Source: Charles River Laboratories, Raleigh, NC.

- Age at study initiation: No data

- Weight at study initiation: 149-177 g

- Fasting period before study: No data

- Housing: metabolism cages (glass or stainless steel)

- Individual metabolism cages: yes

- Diet (e.g. ad libitum): Ad libitum

- Water (e.g. ad libitum): Ad libitum

- Acclimation period: 2 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-72

- Humidity (%): 24-70

- Air changes (per hr): 10-15

- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 20.12.1995 To: 31.12.1996

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: neat, corn oil, Emulphor or Simethicone fluid.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: 14C-D4 was diluted with unlabelled D4 to achieve the target specific activity. The dosing solutions (carrier with D4) were prepared to achieve radioactivity concentration for delivery of approximately 25 µCi of radioactivity and a nominal dose of 300 mg/kg bw D4.
Dosing solutions were formulated in the following manner: corn oil, 35 cst PDMS and the Simethicone fluid, the 14C-D4 was added directly into the carrier and mixed, and EmulphorTM was prepared as a 10% solution in distilled water, and 14C-D4 was added to this solution.

Duration and frequency of treatment / exposure:
Single dose
Doses / concentrations
Dose / conc.:
300 mg/kg bw (total dose)
Remarks:
300 mg 14C-D4/kg bw
No. of animals per sex per dose:
Various, see "details on study design"
Control animals:
yes
Positive control:
None
Details on study design:
Phase I Preliminary study: jugular cannulated for oral administration.
Group 1 (8 animals): 14C-D4 in corn oil.
Group 2 (8 animals): 14C-D4 in 35 cst PDMS.

Phase II Preliminary study: 6 jugular cannulated and 2 non-cannulated.
Group 3 (8 animals): 14C-D4 in corn oil.
Group 4 (8 animals): 14C-D4 in EmulphorTM.
Group 5 (8 animals): 14C-D4 neat.

Phase III Preliminary study: 6 jugular cannulated and 2 non-cannulated.
Group 6 (8 females): 14C-D4 in Simethicone fluid.

Definitive phase I: none cannulated.
Group 7 (5 animals): 14C-D4 in corn oil (excreta group).
Group 8 (5 animals): 14C-D4 in Simethicone fluid (excreta group).
Group 9 (50 animals): 14C-D4 in corn oil (blood group).
Group 10 (50 animals): 14C-D4 in Simethicone fluid (blood group).
Group 11 (6 animals): 14C-D4 in corn oil (WBA).
Group 12 (6 animals): 14C-D4 in Simethicone fluid (WBA).

Definitive phase II: none cannulated.
Group 13 (5 animals): 14C-D4 neat (excreta group)
Group 14 (50 animals): 14C-D4 neat (blood group)
Group 15 (6 animals): 14C-D4 neat (WBA)

Analytical phase: Group 16 (9 animals): controls (extraction controls).

The study consisted of 16 groups. Groups 1 -6 were the preliminary study for blood collection only. These had 8 animals/group. Groups 7 -15 were used in the definitive study. These had 50 animals/group (5 animals/timepoint, 10 timepoints)/ carrier and 5 animals/excreta group/carrier. The excreta group served as the 168 hour timepoint for blood collection. Six animals/group/carrier were used for whole body autoradiography (1 animal/timepoint, 6 timepoints). Group 16 was used for the collection of control blood.
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption)
Phase I Preliminary study: Immediately following administration of test material, the animals were placed in stainless steel metabolism cages. Approximately 200 µl of blood was collected at each timepoint via the jugular cannula from four of the animals at 5, and 30 minutes, and at 2, 8, 24, 48, and 72 hours. The other four animals had blood collected at 15 minutes and 1, 4, 12, 24, 48, and 72 hours. This minimized the total amount of blood
collected from any one animal during the first 24 hours of collection, yet allowed for a rigorous time course.

Phases II and III Preliminary study: Following a review of the data from Phase I, the timepoints were altered to eliminate the 5 minute and 30 minute
timepoint and to add additional later timepoints, (96, 120, 144 and 168 hr.), thus eliminating the need for two groups. Immediately following administration of test material, the animals were placed in stainless steel metabolism cages. Approximately 200 µl of blood was collected via the jugular
cannula at 15, and 60 minutes, and at 6, 12, 24, 48, 72, 96, 120, 144, and 168 hours. Following the last collection the animals were euthanized via CO2, inhalation. The carcasses were stored at -80°C until the data were collected and it was determined they were no longer needed. The carcasses were then disposed of. Blood was collected from non-cannulated animals via cardiac puncture at 12 hr post administration. The animals were anesthetized with Methoxyflurane and exsanguinated by cardiac puncture. The abdominal cavity was opened by incision, the diaphragm was punctured and incised and the ribs were cut to expose the heart. The maximum volume of blood possible was collected from the heart, until the heart beat and blood flow stopped. The blood samples were placed on ice until sampled.

Definitive phase I and II: Immediately following the administration of 14C- D, by oral gavage, the excreta animals were placed into glass metabolism cages suitable for the separate collection of excreta (urine and feces) and expired air (14C02 and other volatiles). Urine, feces, and expired CO, were collected at 6, 12, 24, 48, 72, 96, 120, 144, and 168 hours. Expired volatiles were collected at 1, 2, 4, 6, 24, 48, 72, 96, 120, 144 and 168 hr post exposure. At the 168 hour timepoint, the animals were anesthetized and exsanguinated by cardiac puncture. Control blood from non-treated animals was collected in the same manner. Lung, liver, spleen, fat, adrenals, and ovaries was collected for each animal. The carcasses were immediately placed in labeled jars, weighed, and solubilized in toto. Following removal of the animals, the cages were rinsed with Dow@ Bathroom Cleaner, followed by 70%
ethanol/water. An aliquot of the rinse was analyzed for determination of radioactivity content. Following dosing, the blood group animals were placed in stainless steel metabolism cages. Blood was collected at 15 and 60 minutes, 6, 12, 24, 48, 72, 96, 120, and 144 hours. Five animals per timepoint were anesthetized and blood was collected. The blood samples were placed on ice when processing was delayed. The cages were washed to determine radioactive contamination. The carcasses were disposed of.
The metabolite profile of individual radioactive components present in the extracts was evaluated at 6, 12, 24 hr for animals administered D, in corn oil and D, in Simethicone and at 12 and 24 hr for animals administered Neat D4, using HPLC.
Selected urine samples (based on the level of radioactivity and volume of sample obtained) were directly analyzed by HPLC to determine the metabolic profile. The instrumentation and HPLC conditions for the qualitative analysis of individual radioactive urinary metaboliies of 14C- D, were the same as those for blood.
One animal at each timepoint (1, 6, 12, 24, 48, and 96 hours) was sacrificed using C02 for WBA.
Statistics:
Numerical data obtained during the conduct of the study were subjected to calculation of group mean values and standard deviations where appropriate, using Microsoft Excel@ (version 5.0 and 7.0). The concentration of radioactivity in blood and tissues was reported in µg equivalents 14C-D4/g sample. The radioactivity content in the excreta and tissues were reported in terms of percent of total radioactivity recovered relative to the amount of administered radioactivity. The concentration of parent D4 in blood was reported in µg D4/g sample. To evaluate relative absorption of parent D4 and total radioactivity for the different dosing regimens, the area under the curve (AUC) was generated for the blood data. The data were analyzed using the method of Nedelman et. al. (Applying Bailer’s Method for AUC confidence intervals in sparse sampling, Pharmaceutical Research 12:124-128, 1995).

Results and discussion

Preliminary studies:
Data for Phase I are not presented due to poor health of animals. The animals administered 14C-D4 in corn oil had the greatest concentration over time of radioactivity in blood and the animals administered 14C-D4 in Simethicone fluid had the least. Animals administered 14C-D4 in Emulphor TM had similar blood levels of radioactivity as the animals administered 14C-D4 in corn oil. Based on the results of the Preliminary Phase, two carriers, corn oil and Simethicone fluid were chosen as carriers for the Definitive Phase of this study. For comparison, D, was also delivered neat. It was determined that the timepoints as specified in Preliminary Phases II and III were appropriate for the Definitive Phases as well. It was demonstrated that there was no loss of D4 following collection via cannulae.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Absorption was ca. 52 %, 12 %, and 28 % with 14C-D4 in corn oil, Simethicone or neat respectively. In Emulphor blood levels of radioactivity were similar as in corn oil. D4 was most readily absorbed when delivered in corn oil and least available for absorption when carried in the Simethicone fluid. The major excretory pathway for the recovered radioactivity regardless of the carrier was the feces. Qualitative assessment of whole body autoradiography showed comparatively similar patterns of absorption and disposition of radioactivity but differences in transit time of radioactivity through the gastrointestinal tract following administration in the various carriers or neat.
Details on distribution in tissues:
At one hour, radioactivity appeared to be concentrated in the contents of the stomach, upper gastrointestinal (GI) tract, and a moderate amount in the fat. Twenty-fours following administration, rats receiving 14C- D4 neat or in corn oil had the highest concentration of radioactivity in the small intestines and cecum with residual amounts in the stomach contents. Moderate amounts were detected in the brown fat and urine. Levels slightly above background were detected in the liver, Harderian gland, and bone marrow. In the rat receiving the corn oil as carrier there appeared to be a moderate amount detected in the clitoral gland. In rats receiving 14C-D4 in the Simethicone fluid, radioactivity appeared to be concentrated in the cecum, lower
portion of the colon, and a detectable amount in the brown fat and clitoral gland. The clitoral gland of the rat dosed with 14C- D4 neat was not definitively identified in the section. Forty-eight hours post-dosing, rats receiving 14C-D4 neat or in corn oil showed very similar patterns of accumulation with a moderate amount appearing in the brown fat, colon, and clitoral gland. Lesser concentrations were noted in the liver, cecum, small intestine, Harderian gland, bone marrow, skin, urine, fat, and residual contents of the stomach. The rat dosed with the Simethicone carrier showed very low levels of radioactivity (slightly above background) in the brown fat, colon, and clitoral gland. At 96 hours, rats receiving the 14C-D4 neat or in corn oil showed moderately high amounts of radioactivity in brown fat and the clitoral gland. Low amounts were detected in the liver, lower GI tract, fat, skin, bone marrow, and Harder-tan gland. The rat receiving 14C-D4 in the Simethicone fluid showed only low levels of radioactivity in the brown fat and slightly detectable amounts in the fat.
Details on excretion:
Mainly faeces.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Comparison of blood radioactivity AUC with parent D4 AUC indicated that parent D4 is absorbed, however, the majority of the radioactivity can be attributed to metabolites. Metabolite profiles of pooled and concentrated blood samples demonstrated similar peaks present in the urinary profiles for animals dosed with D4 in corn oil and neat D4. Blood samples obtained from the animals dosed with 14C-D4 in Simethicone were too low in radioactivity (even when pooled and concentrated) to obtain a metabolite profile. There was one peak in the 6 hr extract of blood from the animals administered 14C-D4 in corn oil that was not present in any of the urine samples. A control sample of parent D4 analyzed under the same conditions as part of method development exhibited a similar retention time. As a result, based on retention time comparison, the peak in this sample has been identified as parent D4.
The metabolic profiles of the urine collected from animals dosed with the three different dosing carriers were compared qualitatively. The urinary metabolite profiles were similar between the three dosing carriers and to profiles obtained previously from urine collected from rats after exposure to D4. All urine samples analyzed exhibited profiles containing the five major metabolites previously identified. These five metabolites were
identified as 1) MeSi(OH)3 (methylsilanetriol); 2) Me2Si(OH)-0-Si(OH)3 (dimethyldisiloxane-1,3,3,3-tetrol); 3) Me2Si(OH)2 (monomer diol); 4) MeSi(OH)2-0-Si(OH)Me2 (trimethyldisiloxane-1,1,3-triol); and 5) Me2Si(OH)-OSi(OH)Me2 (dimer diol).

Any other information on results incl. tables

This study indicates that the oral absorption of D4 can be significantly influenced by the carrier.

Applicant's summary and conclusion

Conclusions:
In a good quality in vivo oral absorption study in rats (reliability score 1) examination of the blood radioactivity and parent D4 concentration and the mass balance of radioactivity indicated that D4 was most readily absorbed when delivered in corn oil and least available for absorption when administered in Simethicone fluid. Qualitative assessment by Whole-Body Autoradiography showed comparatively similar patterns of absorption and
disposition of radioactivity but differences in transit time of radioactivity through the gastrointestinal tract following administration of 14C-D4 in the various carriers or neat. This study indicated that the oral absorption of D4 can be significantly influenced by the carrier used to deliver D4.