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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:
December 2015 - March 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
other: EU method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2008
Deviations:
no
Principles of method if other than guideline:
The quality of the environment in which the characterisation of the test item was performed was not known.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Physical appearance: white crystalline powder
Test item storage: In refrigerator (2-8°C)

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Source strain:
other: Keratinocyte strain00267
Details on animal used as source of test system:
EpiDerm Skin Model (EPI-200, Lot no.: 23280 MatTek Corporation, Ashland MA, U.S.A).
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.
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Remarks:
Skin was moistened with 25 μl Milli-Q water to ensure close contact of the test item to the tissue
Details on test system:
After exposure tissues were washed with phosphate buffered saline to remove the test substance and tissues were incubated with MTT-medium for 3 hours to evaluate cell viability.
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.

Acceptability of the assay
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 3-minute exposure to the positive control should be ≤ 30%.
c) In the range of 20 – 100% viability, the maximum inter-tissue variability (in viability) is ≤ 30% between two tissues treated identically.
d) In the range of 20 – 100% viability, the maximum difference in percentage between the mean viability of two tissues and one of the two tissues is ≤ 15%.

Data evaluation and statistical procedures
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
other: concurrent MTT reduction control
Amount/concentration applied:
27.40 to 39.15 mg of the solid test item
Duration of treatment / exposure:
3 minutes or 1 hour
Duration of post-treatment incubation (if applicable):
none
Number of replicates:
2 replicates for 3 minute exposure and 2 replicates for 1 hour exposure

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
100
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure
Value:
90
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
One of the positive control tissues at the 1-hour treatment of this project was lost during the procedure, therefore a the positive control data from another project which was performed at same time was used. Evaluation: Since the positive control from the other project was treated identically with the same batch at the same time and the acceptance criteria of the positive control were met this has no influence on the study integrity.

Diona-R 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 Diona-R did not interfere with the MTT endpoint.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 15%.
The maximum inter-tissue variability in viability between two tissues treated identically was less than 14% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 7% 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 51% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 26%, 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:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the test substance is not irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin corrosion test using a human three dimensional epidermal model (EpiDerm (EPI-200)) the possible corrosive potential of Diona-R was tested through topical application for 3 minutes and 1 hour according to OECD/EC guidelines and GLP principles. The test substance was applied directly to 0.6 cm2 cultured skin (approximately 25 mg, in presence of 25 μl Milli-Q water). After 3 minutes or 1 hour, the substance was removed and the viability of the cells was tested by reduction of MTT. The positive control had a mean relative tissue viability of 15% after 3 minutes exposure. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Diona-R compared to the negative control tissues was 100% and 90% respectively. Because the mean relative tissue viability for Diona-R was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment Diona-R is considered to be not corrosive.