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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March 2008-6 June 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and EC guidelines and according to GLP principles.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline no. 431: In Vitro Skin Corrosion: Human Skin Model Test (adopted 13 April 2004).
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): LiPF6
- Substance type: inorganic
- Physical state: white powder
- Analytical purity: > 99.9%
- Lot/batch No.: P07102202
- Expiration date of the lot/batch: 31 December 2008
- Stability under storage conditions: stable
- Storage condition of test material: At room temperature protected from light under nitrogen

Test animals

Species:
human
Strain:
not specified
Details on test animals and environmental conditions:
Test system
EpiDerm Skin Model (EPI-200, Lot no.: 10526 kit H).
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, 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 EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
Rationale: Recommended test system in international guidelines (OECD and EC).

Tissues
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

DMEM (Dulbecco’s Modified Eagle’s Medium)
Supplemented DMEM medium, serum-free.

MTT medium
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM).

Environmental conditions
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 71 - 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.0 - 36.7°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity (with a maximum of 9%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Type of coverage:
open
Preparation of test site:
other: not applicable
Vehicle:
other: moistened with Milli-Q water
Controls:
other: negative control: Milli-Q water
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg, moistened with 25 µl Milli-Q water
The test substance was spread to match the size of the tissue.
Duration of treatment / exposure:
3 minutes and 1 hr
Observation period:
None
Number of animals:
4 tissues per test substance
Details on study design:
The skin tissues were kept in the refrigerator the day they were received. The next day, before the assay is started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The plates were incubated for 1.75 hour at 37.0 ± 1.0°C. The level of the DMEM medium is just beneath the tissue. The medium was replaced with fresh DMEM medium just before LiPF6 was applied. The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a three minutes exposure to LiPF6 at room temperature and two for a one hour exposure at 37.0 ± 1.0°C. LiPF6 (25 mg) with 25 µl Milli-Q water was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 µl Milli-Q water (negative control) and with 50 µl 8N KOH (positive control), respectively. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 µl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.
Cell viability measurement: the DMEM medium was replaced by 300 µl MTT-medium and tissues were incubated for 3 h at 37°C in air containing 5 ± 0.5% carbon dioxide. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 540 nm in triplicate with the Multiskan Spectrum (Thermo Labsystems).

Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other:
Value:
103
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 3 min. Reversibility: no data. Remarks: score is percentage of control. (migrated information)
Irritation / corrosion parameter:
other:
Value:
6
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 1 hr. Reversibility: no data. Remarks: score is percentage of control. (migrated information)

In vivo

Irritant / corrosive response data:
Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3 minutes and 1 hour treatment with LiPF6 compared to the negative control tissues was 103% and 6% respectively. Since the mean relative tissue viability for LiPF6 was below 15% after 1 hour treatment it is considered to be corrosive.

The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability of the 3 minutes exposure of the positive control was 5%. The maximum inter tissue variability in viability between two tissues treated identically was less than 21% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 12%. It was therefore concluded that the test system functioned properly.

Other effects:
None.

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
It is concluded that this test is valid and that LiPF6 is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.