Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Objective of study:
absorption
other: bioavailability
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The bioavailability of Hydroxyethyl-2-nitro-p-toluidine across the intestinal barrier was investigated in a human intestinal epithelial (TC-7) cell model in vitro. The permeability from the apical (A, pH 6.5) to the basolateral (B, pH 7.4) side was investigated at 37°C in 96-well transwell plates with shaking for a 60 min contact time. Analysis of the donor (apical) and receiver (basolateral) samples was done by means of HLPC-MS/MS and the apparent permeability coefficient (Papp) was calculated for two independent experiments. 14C-mannitol (about 4µM) was used to demonstrate the integrity of the cell monolayers. Only monolayer revealing a permeability of < 2.5x10-6 cm/sec were used. Propranolol, vinplastine and ranitidine were analysed concurrently to demonstrate the validity of the test system.
GLP compliance:
no
Remarks:
The study was not performed under GLP conditions, but a statement of the quality assurance unit of the test facility is included. However, the study was performed according to recommendations by ECVAM and is generally recognised as scientifically sound.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
red powder
Batch # : 6718 Fass 10/20

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
The total recovery for the reference substances and Hydroxyethyl-2-nitro-p-toluidine ranged from 83 to 100 %. The figures obtained for the reference substances propranolol (Papp = 29.6 x 10-6 cm/sec), a high permeability reference compound with about 90 % absorption in humans, and ranitidine (Papp= 0.4 x 10-6 cm/sec; revealing a absorption of about 50 % in humans) were well within the acceptance range of 20 –45 x 10-6 cm/sec and 0.2 – 2 x 10-6 cm/sec, respectively, and demonstrated the validity of the assay. Hydroxyethyl-2-nitro-p-toluidine revealed a Papp of 93.6 x 10-6 cm/sec and was classified to be of high permeability accordingly. The observed value indicates very good absorption from the gastro-intestinal tract.

Metabolite characterisation studies

Metabolites identified:
no

Applicant's summary and conclusion

Conclusions:
With Hydroxyethyl-2-nitro-p-toluidine a mean permeability in human intestinal epithelial (TC-7) cells of 93.6 x 10-6 cm/sec was obtained, which classifies the test item to be of high permeability. As the absorption across the intestinal epithelial is considered to be the limiting factor of the uptake through the gastro-intestinal tract, the high permeability observed in this assay indicates a good absorption Hydroxyethyl-2-nitro-p-toluidine after oral administration.
Executive summary:

The bioavailability of Hydroxyethyl-2-nitro-p-toluidine across the intestinal barrier was investigated in a human intestinal epithelial (TC-7) cell model in vitro. The permeability from the apical (A, pH 6.5) to the basolateral (B, pH 7.4) side was investigated at 37°C in 96-well transwell plates with shaking for a 60 min contact time. Analysis of the donor (apical) and receiver (basolateral) samples was done by means of HLPC-MS/MS and the apparent permeability coefficient (Papp) was calculated for two independent experiments. 14C-mannitol (about 4µM) was used to demonstrate the integrity of the cell monolayers. Only monolayer revealing a permeability of < 2.5x10-6 cm/sec were used. Propranolol, vinplastine and ranitidine were analysed concurrently to demonstrate the validity of the test system.

According to the laboratory’s classification system, a low permeability is considered for test items revealing a Papp < 2 x 10-6 cm/sec. A Papp of 2 - 20 x 10-6 cm/sec and a Papp   20 x 10-6 cm/sec classify a substance to have a moderate and a high permeability, respectively. Ranitidine, which has a  50 % absorption in humans was used as low permeability reference compound, as recommended by FDA. The total recovery for the reference substances and Hydroxyethyl-2-nitro-p-toluidine ranged from 83 to 100 %. The figures obtained for the reference substances propranolol (Papp = 29.6 x 10-6 cm/sec), a high permeability reference compound with about 90 % absorption in humans, and ranitidine (Papp= 0.4 x 10-6 cm/sec; revealing a absorption of about 50 %  in humans) were well within the acceptance range of 20 – 45 x 10-6 cm/sec  and 0.2 – 2 x 10-6 cm/sec, respectively, and demonstrated the validity of the assay. Hydroxyethyl-2-nitro-p-toluidine revealed a Papp of 93.6 x 10-6 cm/sec and was classified to be of high permeability accordingly. The observed value indicates very good absorption from the gastro-intestinal tract.