Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 to 29 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch number: E010016621
Purity: Not indicated
Specific details on test material used for the study:
Batch number: E010016621
Purity: Not indicated

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Source: MatTek Corporation, Ashland MA, U.S.A.
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
Application/Treatment of the test item
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for 1 – 2.5 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test substance was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test substance and two for a 1-hour exposure. The skin was moistened with 25 μl Milli-Q water to ensure close contact of the test item to the tissue and 38.0 to 50.0 mg of the solid test item 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 item. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.
Cell viability measurement
The DMEM medium was replaced by 300 μl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 38.0 to 50.0 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
Duration of treatment / exposure:
3 minutes, 1 hour
Number of replicates:
Two tissues each group

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Value:
80
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
The exposure time is 3 minutes
Irritation / corrosion parameter:
% tissue viability
Value:
90
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
The exposure time is 1 hour
Other effects / acceptance of results:
The test substance was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test substance did not interfere with the MTT endpoint.
The maximum inter-tissue variability in viability between two tissues treated identically was less than 17% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 9% for the negative control and test item. For the positive control, the maximum inter-tissue variability in viability between two tissues treated identically was less than 31% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 18%, however since the viabilities were below 20% the acceptability criteria were met. It was therefore concluded that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Finally, it is concluded that this test is valid and that the test substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.