1H-Benzimidazole-5,7-disulfonic acid, 2,2'-(1,4-phenylene)bis-, sodium salt (1:2)

Dermal absorption

Administrative data

Endpoint:

dermal absorption 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 for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

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 following dermal treatment are presented in this section.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-750-0

EC Name:

-

Cas Number:

180898-37-7

Molecular formula:

C20H12N4O12S4.2Na

IUPAC Name:

disodium dihydrogen 2-[4-(5,7-disulfonato-1H-1,3-benzodiazol-2-yl)phenyl]-1H-1,3-benzodiazole-5,7-disulfonate

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 or test system 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).
- 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

Type of coverage:

occlusive

Vehicle:

water

Duration of exposure:

6 hours

Doses:

Nominal doses:
- 0 (group 1; intravenous administration),
- 100 mg/kg (group 5; dermal application), 400 mg/kg (group 6; dermal application), 800 mg/kg (group 7; dermal application)
- 8 mg/kg (group 8; intravenous administration)

Dose volumes:
- dermal: 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% aqueous solution (stock solution).

Rationale for dose selection:
- information on dose levels were provided by the sponsor

No. of animals per group:

Group 1: 10 males
Groups 5, 6 and 7: 10 males each
Group 8: 25 males

Control animals:

yes

Remarks:

concurrent vehicle

Details on study design:

DOSE PREPARATION
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.

TEST SITE / SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION / REMOVAL OF TEST SUBSTANCE
The rats were shaved on the back (approx. 3X3 cm) prior to application of the test item. After the treatment, the application site was covered with an occlusive membrane and fixed with an adhesive tape to guarantee occlusive conditions for the following 6 hours. After 6 hours, tape and the occlusive membrane were removed and the application site was cleaned with water using a sponge to avoid oral ingestion of remaining test item.

SAMPLE COLLECTION
- Collection of blood: 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.
- 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.

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.

Details on in vitro test system (if applicable):

not applicable

Results and discussion

Signs and symptoms of toxicity:

no effects

Dermal irritation:

not specified

Absorption in different matrices:

Single dermal administration:
- throughout all dermal groups (single administration at the dose levels 100, 400 and 800 mg/kg body weight) similar Tmax values (6 —8 hours), similar half-lives (13.8-16.4 hours) and a similar MRT (22.1 — 25.6 hours) were found.
- dose dependency: after single dermal administration at about 4 and 2 times higher dose levels (from 100 to 400 to 800 mg/kg), taking into account the high standard deviations and the extreme low levels at 100 mg/kg, AUC values increased similarly about 7 and 3 times; at about 8 and
2 fold higher Cmax values were found at similar half-lives (13.8-16.4 hours).

Single intravenous administration:
- 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 AUC 04 value was 81691 ng*h/mI and the average level of HR 02/N00002 decreased to about 3.8 % of the observed Cmax at 24 hrs post dose.

Due to the different dose levels between the dermal groups against the i.v. group, values are compared alter dose normalisation. As compared to the normalized AUC value after i.v. administration (100 %) the percentage absorbed after dermal administration amounted to 0.1-0.3%.

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 5: 261 g

Group 6: 240 g

Group 7: 244 g

Group 8: 244 g

Applicant's summary and conclusion

Conclusions:

After single dermal administration of the test item at dose levels of 100 to 800 mg/kg to the skin of male Wistar rats, a linear dose dependency of plasma levels was found, taking into account the large standard deviations and the low values at 100 mg/kg.
Based on AUC comparison between i.v. administration (100% absorbed) and dermal applictaion, the percentage of absorbed test item amounted to about 0.1-0.3 % after dermal administration.