Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-08-15 to 2002-08-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study and although not following any guideline, the data are still scientifically acceptable and robust
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Objective of study:
absorption
excretion
other: bioavailability
Principles of method if other than guideline:
The purpose of this study was to generate a kinetical profile of HR 02/N00002 Aq after single intravenous, oral and dermal (occlusive) administration of the test item to male Wistar rats. Additionally, the bioavailability of HR 02/N00002 Aq was calculated following oral and dermal application. Only data related to the kinetic behaviour and excretion following oral and intravenous treatment are presented in this section.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- CAS name: 1H-Benzimidazole-4,6-disulfonic acid-2,2´-(1,4-diphenylene) bis, disodium salt
- Molecular formula: C20H12N4O12S4Na2
- Molecular weight: 674g/mol
- physical state: solid

Radiolabelling:
no

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS - SPF Wistar rats
- Source: RCC Ltd, Laboratory Animal Services, CH-4414 Füllinsdorf, Switzerland
- Age at study initiation: approximately 9 weeks
- Weight and range at acclimatization/pretest: 186.5 - 267.8 grams (means 247.6 grams)
- Housing: individually in Makrolon type-3 cages with wire mesh topsand standardized softwood bedding ('Linocel' Schill AG, CH-4132 Muttenz/Switzerland, batch 02/0520701).
- Individual metabolism cages: yes, for collection of urine and feces. Animals of group 4 and group 8 were kept in metabolic cages after application.
- Diet (ad libitum): pelleted standard Provimi Kliba 3433 (batch no. 34/02) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst / Switzerland)
- Water (ad libitum): community tap-water from Itingen
- Acclimation period: 6 days under test conditions after health examination. Only animals without any visible signs of illness will be used for the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 30 - 70%
- Air changes: 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (music during the light period)

Administration / exposure

Route of administration:
other: oral (by gavage) & intravenous
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The frequency of the preparation of dose formulation was once.

Preparation of the 10% aqueous solution (stock solution): the formulation consisted of approx. 79% bi-distilled water, approx. 11% NaOH (10% in water) and 10% of the test item. For a stock-solution with a total volume of 100 mL (as an example), about 6.5 mL NaOH-solution wre solved in 79 mL bi-distilled. Using a magnetic stirrer 10 g of the test item were added. The rest of the NaOH-solution (max. 3.5 mL) was added until the solution was clear. The final pH-value lied bewteen 7.0 and 8.0. If not all of the NaOH-solution was used, bi-distilled water, was added to a total volume of 100 mL.

Preparation of dilution 1, 2 and 3: dilution 1 was prepared by diluting one part of the stock-solution with the same amount of bi-distilled water. The preparation of dilution 2 was performed by diluting one part of dilution 1 (for example 10 mL) with 3 parts (for example 30 mL) of bi-distilled water. Dilution 3 was prepared by diluting one part of dilution 1 with 9 parts of bi-distilled water.

Homogeneity of the test item in the vehicle was maintained during the administration period by using a magnetic stirrer.

Dose volumes:
- Oral: 7.51 mL/kg body weight
- Intravenous: 1.5 mL/kg body weight
The dose volumes resulted by dividing the dose levels by the factor 1.065, which was supplied by the Sponsor as the density in g/mL of the 10% aquesous solution (stock solution).

ANALYSIS OF DOSE FORMULATIONS:
Concentration of the dose formulations was determined in samples taken directly after experimental start. The samples were stored deep frozen (-78 to -83°C) on dry ice. The analyses was perfromed using a HPLC method.
Duration and frequency of treatment / exposure:
Single intravenous and oral administration
Doses / concentrations
Remarks:
Doses / Concentrations:
Group 1 (intravenous): bi-distilled water, only (vehicle)
Group 2 (oral): 7.51 mL/kg body weight HR 02/N00002 Aq (dilution 2), corresponding to 100 mg/kg body weight of active ingredient
Group 3 (oral): 7.51 mL/kg body weight HR 02/N00002 Aq (dilution 1), corresponding to 400 mg/kg body weight of active ingredient
Group 4 (oral): 7.51 mL/kg body weight HR 02/N00002 Aq (stock solution), corresponding to 800 mg/kg body weight of active ingredient
Group 8 (intravenous): 1.5 mL/kg body weight HR 02/N00002 Aq (dilution 3), corresponding to 8 mg/kg body weight of active ingredient
No. of animals per sex per dose:
Group 1: 10 males
Groups 2 and 3: 15 males each
Group 4: 20 males
Group 8: 25 males
Control animals:
yes, concurrent vehicle
Positive control:
no data
Details on study design:
- Dose selection rationale: information on dose levels were provided by the sponsor
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, excretion)
- Tissues and body fluids sampled: urine, feces, blood
- Time and frequency of sampling: blood samples (approx. 1 mL) were taken at different time points by retro-orbital bleeding into heparinized tubes under light isoflurane anesthesia.
- Control animals (group 1) were sampled at 24 hours after application. After terminal bleeding the animals were transferred to the histopathology group for necropsy. Plasma was prepared and stored at -17 to -23°C on dry ice for plasma level determination using a HPLC method.
- Urine and feces were collected from five animals of group 4 and from five animals of group 8 in metabolic cages. These animals were not used for blood sampling. Samples in group 4 were collected from 0 to 24 hours and from 24 to 48 hours. The sampling intervals for the five animals of group 8 were from 0 to 8 hours and from 8 hours to 24 hours. The urine and feces samples were stored at -17 to -23°C before determination of test item levels using an HPLC method.
- Necropsy: the animals were weighed and necropsied. The content of the stomach and the intestine was collected in all test item-treated animals. These samples were stored at -17 to -23°C on dry ice for possible further investigations.




Statistics:
A statistical analysis of data was not performed.

Results and discussion

Main ADME resultsopen allclose all
Type:
other: kinetics oral
Results:
After a single oral dose of 100, 400 and 800 mg/kg bw, maximum plasma levels of 1174, 4883 and 10476 ng/ml were reached 1-3 hours post dose. Plasma level increased linear. Elimination half-lives of 7-9 hours were found resulting in MRT of 22-26 hours.
Type:
other: kinetics intravenous
Results:
After single i.v. administration, the maximal observed average plasma concentration (Cmax) at 5 min post dose was 12965 ng/mL, and the Cmax intercept was 17208 ng/mL. The half-life was 5.4 hours and the MRT value 7.3 hours.
Type:
absorption
Results:
Comparison of AUCs (i.v. vs. oral) showed that about 1.8-2.1% of the oral doses within 100-800 mg/kg were absorbed.
Type:
excretion
Results:
After single oral administration of 800 mg/kg, exceretion of the parent test item in urine and feces amounted to 16.5% in total (48 hrs) with only about 1% found in urine.
Type:
excretion
Results:
After single i.v. administration of 8 mg/kg, excretion of the parent test item in urine and feces was 53.7% in total (24 hrs) with about 60 times less in faeces.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
1) Single oral administration (group 2, 3 and 4):
- throughout all oral groups (dose levels 100, 400 and 800 mg/kg body weight) similar Tmax values (1.25 - 3 hours), similar half lives (7.1 - 9.3 hours) and similar MRTs (mean residence times: 10.5 - 13.6 hours) were found;
- dose dependency: at about 4 and 2 times higher dose levels (from 100 to 400 to 800 mg/kg), AUC0 -t values increased similarly about 4 and 2 times; at about 4 and 2 fold higher Cmax values were also found at similar half-lives (7.1 - 9.3 hours).

2) Single intravenous administration (group 8):
- the maximal observed average plasma concentration (Cmax) at 5 min (0.08 hours) post administration (Tmax) was 12965 ng/mL;
- after PK analyses, the Cmax intercept amounted to 17208 ng/mL;
- the AUC0 -t value was 81691 ng.h/mL and the average level of HR 02/N00002 decreased to about 3.8% of the observed Cmax at 24 hours post dose.

Due to the different dose levels between the oral groups against the i.v. group, values are compared after dose normalization. As compared to the normalized AUC value after i.v. administration (100%) the percentage absorbed after oral admiistration amounted to 1.8 - 2.1%.


Details on distribution in tissues:
no data
Details on excretion:
The percentage of administered test item found in urine and feces were derived from the measured concentrations in urine and feces and related to the respecitve volumes (urine) and weights (feces).

1) Single oral administration:
- excretion via urine amounted on average to 0.9 ± 0.6% until 48 hours; an amount of 0.7 ± 0.4% was found in the urine of the first 24 hours;
- excretion via feces amounted on average to 15.6 ± 2.9%; an amount of 14.3 ± 2.5% was found in the feces of the first 24 hours;
- the total excreted amount of the test item 48 hours amounted on average to 16.5 ± 3.5%.

The about 5 - 12 times lower amounts excreted in urine as well as feces during the second 24 hour period indicate that the excretion of the non-metabolized parent item was virtually complete at 48 hours after single oral administration.

2) Single i.v. administration:
- excretion via 0-24 hours urine amounted on average to 52.8 ± 11 %.; an amount of 41.4 ± 7.6 % was found in the urine of first 8 hours;
- excretion via 0-24 hours feces amounted on average to 0.9 ± 0.4 %. (this amount was only found in the feces from 8 to 24 hours);
- the total excreted amount of HR 02/N00002 at 24 hours amounted on average to 53.7 ± 11.4 %.

The only about threefold lower amount in the urine from 8-24 hours against 0-8 hours indicates that after i.v. administration excretion of the non-metabolized parent item was not complete after 24 hours.

Based on the urine/feces ratio at complete absorption after i.v. administration, the parent test item was excreted in urine 58.7 times more than in feces. Taking the percentage excreted via urine after oral administration (0.9 %) as absorbed parent test item and assuming the same urine/feces ratio, the percentage of parent test item absorbed and excreted via the bile into feces amounts to only 0.015%.

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
no data

Any other information on results incl. tables

Viability / mortality:

All animals survived until scheduled sacrifice.

Clinical signs (daily):

No clinical signs were evident during the blood, urine and feces sampling period of up to 48 hours.

Body weights:

The body weights were determined for the automatically calculated (TecTox Release 7.0) dose volumes.

Treatment (mean weight):

Group 1: 237 g

Group 2: 255 g

Group 3: 251 g

Group 4: 247 g

Group 8: 244 g

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
After single oral administration of the test substance at dose levels of 100 to 800 mg/kg, a strict linear dose dependency was found.
Based on the normalized AUC comparison between i.v. administration of the test substance at the dose level of 8 mg/kg (100% absorbed) and oral administration of the test item at 800 mg/kg as well as via urinary excretion at the oral dose level of 800 mg/kg the percentage of absorbed test item amounted to 1-2% after oral administration.
After oral administration of the substance at the dose level of 800 mg/kg, only about one sixth of the dose was excreted in urine and faeces within 48 hours with about 17 times more in the faeces. At complete absorption after intravenous administration of the substance at the dose level of 8 mg/kg, about half of the dose was already excreted via the urine within 24 hours and about 60 times lower amounts via the faeces.
Based on the urinary excretion of about 1% after oral administration, the calculated percentage of absorbed parent item excreted into faeces via the bile becomes negligible (0.015 %).
The results indicate that absorption of the parent item is very low after oral administration. Taking into account the incomplete excretion after 48 hours the remaining part of the parent item is assumed to be still present in the intestinal tract.
The oral and i.v. results at the respective dose levels of 800 and 8 mg/kg implicate that biliary excretion of absorbed test item is negligible.