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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 2015-03-17; Experimental Completion Date: 2015-03-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
and OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method))
Deviations:
yes
Remarks:
one deviation from the study plan was observed: The temperatur during storage was outside the range of 20 +/- 5 °C. The temperature was 14.4 °C for one day. Due to the test item´s properties this marginal deviation was stated as uncritical for the outcome
GLP compliance:
yes (incl. certificate)
Remarks:
Statement of GLP Compliance from Rheinland Pfalz - Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear to faitful turbid viscous liquid
Details on test material:
Stabilized EMIM BF4:
Appearance: lightly yellow clear viscous liquid
Purity: 93.36% (EIM+, HPLC); 86.68% (BF4-, titration); 4.89% (Stabilizer, HPLC)

Test animals

Species:
other: EpiDerm™ tissue samples

Test system

Details on study design:
Test System:
Specification:
Commercially available EpiDermTM-Kit.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.
Origin:
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Day of delivery: 17. Mar. 2015
Batch no.: 21638

Performance of the study:
Additional tests:
Nylon mesh compatibility:
The test item was tested for possible reaction with the nylon mesh which is used to ensure sufficient contact with the tissue surface. 30 µL test item were brought onto a nylon mesh on a microscope slide.
No reaction with the mesh was visible after 1 h exposure.
Assessment of Coloured or Staining Test Items:
It was tested whether the test item develops a colour without MTT addition. 30 µL test item were given in a test tube with 0.3 mL H2O demin. and incubated at 37 ± 1°C and 5 ± 0.5% CO2 for 60 minutes.
The resulting solution was colourless, therefore no binding capacity had to be tested.
Assessment of Direct Reduction of MTT by the Test Item:
The test item was tested for the ability of direct MTT reduction. To test for this ability, 30 µL test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1°C and 5 ± 0.5% CO2 for 60 minutes. Untreated MTT solution was used as control.
The MTT solution did not change its colour within one hour, therefore, direct MTT reduction had not taken place, and no data correction was necessary.
Pre-Incubation of tissues:
Eight 6-well-plates were prepared with 0.9 mL assay medium in three of the six wells (upper row). The tissues were inspected for viability. Viable tissues were transferred (three per plate) in the wells with the medium using sterile forceps under the clean bench and placed into the incubator at 37 ± 1°C and 5 ± 0.5% CO2 for one hour.
After the pre-incubation (one hour), the other three wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1°C and 5 ± 0.5% CO2 for 18 hours.
Treatment:
The pre-incubated tissues were placed into fresh 6-well-plates containing 0.9 mL assay medium per well, using the upper row only.
One plate (three tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used as positive control; each tissue was treated with 30 µL SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used for treatment with the test item:
30 µL test item were applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
Tissues were dosed in one-minute-intervals. After dosing the last tissue, all plates are transferred into the incubator for 35 min 37 ± 1°C and 5 ± 0.5% CO2. 60 min after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in one-minute- intervals.
After rinsing, each tissue was dried with a sterile cotton tip and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). The tissue surfaces were evaluated visually under the stereo microscope, excess test item was removed, where necessary.
Then, the tissues were set in the incubator for 24 hours.
Medium renewal:
For three incubated tissues each, a new 6-well-plate with 0.9 mL assay medium in the upper row was pre-pared. The tissues were removed from the incubator and shaken for 10 min (500 rpm). Then the inserts were transferred into the new 6-well-plate and set into the incubator for 18 hours for post-incubation.
MTT Assay:
After a total incubation time of 42 hours, a 24-well-plate was prepared with 300 µL freshly prepared MTT-reagent in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours min.
After this time, the MTT reagent was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times.
At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for two hours at room temperature.
After two hours, the inserts in which formazan had been produced were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.






Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: relative absorbance values
Run / experiment:
3 tissues of the human skin model EPI'Derm
Value:
ca. 39.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Migrated information
Conclusions:
The test item is considered as skin irritant
After the treatment, the relative absorbance values were reduced to 39.8%. This value is below the threshold for skin irritation (50%)
Executive summary:

Three tissues of the human skin model EpiDermTMwere treated withstabilized EMIM BF4 for 60 minutes.

30 µL of the liquid test item (using a nylon mesh) were applied to each tissue and spread to match the tissue size (0.63 cm2; as indicated by supplier).

DPBS buffer was used as negative control, 5 % SDS solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8mean OD2.8, the OD was 1.9 . The positive control showed clear irritating effects, relative viability compared to the negative control was reduced to 2.8 %. Variation within tissues was acceptable (< 18%).

 

After the treatment with the test item, the relative absorbance values were reduced to 39.8 %. This value is below the threshold for irritation potential (50%).

 

Therefore,stabilized EMIM BF4 is considered as skin irritant in the Human Skin Model Test.