Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Clear colorless liquid; purity: 95 %

Test animals

Species:
other: reconstructed human skin (RHS)

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other:
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl
- Concentration (if solution): 100 %

Duration of treatment / exposure:
incubation period: 3 and 60 minutes
Details on study design:
RECONSTRUCTED TISSUES:
The experiment was carried out on a reconstructed human epidermis EST-1000 (CeIiSystems, S1. Katharinen, Germany). The tissue equivalents were shipped in 24 weil cell culture plates on Agarose supplemented with maintenance medium (Kit contents EST-1000; CeliSystems, Ca1.NO. CS-1 001). Inserts were of 0.63 cm2 size. All tests were performed in triplets for each concentration and each time point (3 min or 60 min).

ADAPTATION TO CELL CULTURE CONDITIONS:
Inserts with EST-1000 reconstructed human epidermis (0.63 cm2) were packed under sterile conditions and were shipped refrigerated on supplemented Agarose. Upon arrival, 6 well culture plates were pre-filled with 1ml of fresh and cool maintenance medium. The reconstructed tissues were placed into the prepared cell culture plates (1 insert/well) and were adapted to the recommended tissue culture conditions (5 % CO2, 37°C, max humidity) afterwards for at least 6 hours before use. In case of cultivation of the skin equivalents for more than 24 hours, a daily medium change is required by aspirating the medium and replacing it by 1ml new maintenance medium (37°C) for each well.

ENVIRONMENTAL CONDITIONS:
The environmental conditions in the incubator were standardised as folIows:
Incubator temperature: 37 ± 2° C, COz gas concentration: 5 %, Humidity: maximum.
Occasional deviations from these conditions occurred e.g. as a result of opening the incubators door, but this has no apparent effect on the course or outcome of the study. Alllncubation steps were performed in a COz atmosphere incubator (Heraeus, Osterode-Germany).

APPLICATION OF THE TEST MATERIAL AND INCUBATION:
For testing of chemical induced corrosivity the EST-1000 inserts were exposed to 50 µl of the test item for 3min (RT) and 60 min in the incubator (3 inserts per period of incubation time), respectively. 0.9 % NaCI (50µl) treated epidermal models were used as negative controls (determination in triplicates).

DETERMINATION OF CELL VIABILITY (MTT):
After the incubation period the inserts were washed carefully in PBS (3 times each insert) and MTT reduction was performed. For viability testing the inserts were placed in new 6 well plates containing 1ml of MTT solution (37°C, 1 mg/ml in MTT-assay medium, delivered by Cell Systems). The tissues were incubated for about 2 hours under cell culture conditions (5 % COz, 37°C, max humidity). The extraction of blue formazan was performed in Isopropanol (24 well plates, 300 µl inside and 300 µl outside the insert) on a vertical shaker (at least 60 min). For deterrnination of cell viability the absorption of the Isopropanol-extracts were measured in duplicates at 570 nm in an automatie reader (EL808, Bio-Tek; 96 well format, 200 µl). Data acquisition and evaluation were done with "KC4" (software by Bio-Tek). The MTT reduction assay is the most frequently used assay for the determination of cell viability. The assay depends on the intracellular capacity of living cells to chemically reduce the yellow 3-[4,5-Dimethythiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (Sigma, Deisenhofen-Germany) to blue formazan crystals. The test has shown to give accurate and reproducible results in various laboratories and has practically been modified for accurate analysis of cell viability in three dimensional skin models.

RELIABILITY CHECK:
Reliability of the test was confirmed before by interlaboratory validation.

Results and discussion

Any other information on results incl. tables

The MTT (Methylthiazoletetrazolium) method has determined the following values of cell viability after 3 min or 60 min of incubation: 92.35 % and 4.43 %, respectively. Thus, the results show that a corrosive property of Tetrabutylphosphoniumhydrogendifluorid was determined by the assay used.

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Due to the results of the 3D-Skin Test, further investigations on skin/eye irritation were omitted. The test substance is classified as corrosive (R 34).
Executive summary:

Tetrabutylphosphoniumhydrogendifluorid was characterised by a significant impact on cell viability after the 60 min period of incubation. Thus, Tetrabutylphosphoniumhydrogendifluorid should be labelled as corrosive to skin.