Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
14C-Trisodium Nitrilotriacetate; radiochemical purity: 99.4%
Trilon A liquid (free NaOH neutralized with Trilon AS); aqueous preparation
Aqueous dilution of Trilon A liquid (adjusted to pH 9 with Trilon AS; 1% NTA-Na3); aqueous preparation
Radiolabelling:
yes

Administration / exposure

Doses:
Dose group 1: 400 mg/kg target concentration; 381.02 mg/g nominal concentration
Dose group 2: 10 mg/kg target concentration; 10.20 mg/g nominal concentration
Dose group 1: 4000 ug/cm2 target dose; 3932 +/- 82.6 ug/cm2 actual nominal dose
Dose group 2: 100 ug/cm2 target dose; 99.96 +/- 0.72 actual nominal dose
Details on in vitro test system (if applicable):
Human skin preparations were supplied dermatomed by BIOPREDIC International. Localization: Surgically removed skin from abdomen. Only visually intact skin areas were used. Skin preparations of 4 donors were used within the study.
The radiolabeled test substance was available as a solution in water. To obtain the desired specific activity, a respective aliquot of the radiolabeled test substance solution was mixed with an adequate amount of the non-labeled test substances (concentrate and dilution). The preparation for the high dose was produced one day before application, stored over night and homogenized again just before application. The preparation for the low dose was
produced on the application day. The preparations were stirred and sonicated in order to produce homogeneity.
Before the application of the test substance, the program of the fraction collector was started for the first sampling (sampling time 0 h). The test-substance preparation was distributed uniformly on the exposed skin preparation using a displacement pipette. The exact amount applied per diffusion cell was determined by reweighing the pipette after application, the weight of which had been previously determined after filling. The application
time was documented for each cell. Application area / diffusion cell: 1 cm3 exposed skin. Target amount of test substance preparation applied: about 10 mg/cm2. Frequency of application: single topical application. Covering: The donor compartment was covered with a sheet of Fixomull Stretch (semiocclusive adhesive fleece, Beiersdorf AG).

Results and discussion

Any other information on results incl. tables

The dermal penetration of the test substance through human skin in vitro was examined after application of two different concentrations/doses to split thickness skin preparations mounted on Franz-type diffusion cells: one representing a technical concentrate and the other a representative in use dilution. For the concentrate, which has a high pH value, a 5 min exposure period was selected, as the penetration of the test substance through largely intact skin preparations should be determined. It might be taken as granted that accidental severe skin damage would greatly enhance penetration and such effects have to be avoided by suitable protection measures during handling and use of the concentrate. The study was carried out in one step, consisting of 5 cells for each dose. The target dose of about 10 mg/cm2 was achieved well for each concentration. The mean total recovery rates were 99% and 102% of the high and low dose, respectively, showing the validity of the experimental results. Within the precision of the method no penetration of test substance or its association to skin could be detected with the high dose. The mean absorbed dose was very low for the low dose (around 0.1%). These values are calculated from radioactivity measurements just above the background and have to be used with caution. In addition the mean value is mainly driven by one diffusion cell in which 0.3% of the applied dose was recovered from the receptor fluid, whereas in the other 4 cells between 0.03% and 0.08% were recovered. This might indicate that the 0.3% value is an outlier. Without the outlying value the mean absorbed dose would even decrease to about 0.06%. The amount of test substance associated to the skin membranes was in the same order of magnitude as the absorbed dose. As penetration of the test substance had stopped already 4 hours after application the test substance associated to the skin is considered to belong to the non-absorbed dose. Due to the low penetration no kinetic characterization of the diffusion process was possible for the high dose. The mean cumulative absorbed dose curve established for the low dose shows a sigmoid shape with the range of the steepest slopes up to 4 hours after application. Thereafter a sharp decrease in absorption rates was observed, already before the remaining test substance was washed quantitatively off the skin 6 hours after exposure. Indeed a plateau of the absorption curve was already reached at the 4 hour sampling time. This finding indicates that the diffusion process was interrupted already before the removal of the test substance, probably due to the drying of the test substance preparation on the skin. Drying is an expected effect in the finite dose semi-occlusive exposure regimen used, reflecting real world exposure. Even after the washing procedure, which probably put the substance back into solution no further diffusion occurred from possible residual amounts of test substance still present after the process. In addition, there is a high variability in the low absorption rates driven by one diffusion cell as indicated above (coefficient of variation is 100%), which cannot be attributed to clear differences in membrane integrity as determined by electrical resistance measurement (range of 2.1 – 9.8 kOhm for all diffusion cells, all above 1 kOhm acceptance criterion). If the calculated permeability constant derived from the steepest parts of the penetration-time curves for the low dose nonetheless is taken as valid, according to the categorization schemes suggested by Marzulli et al. 1969 and Barber et al. 1995 it shows a very slow penetration of 14C-Trisodium Nitrilotriacetate from the use dilution through human skin preparations. The presence of the lag time of absorption (about 1.6 hours) underlines the functional diffusion barrier of the skin against the tested substance preparations.

Applicant's summary and conclusion

Conclusions:
Taking into account the sensitivity of the method and the variability of results with the low dose, no appreciable diffusion of the test substance (< 0.1%) into or through skin was found under the conditions of this in vitro skin penetration study.