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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:
31 Jul 2018 - 14 Sep 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Appearance: Brown viscous liquid
- Storage condition of test material: At room temperature

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
Details on animal used as source of test system:
EpiDerm™ Reconstructed Human Epidermis
- All cells used to produce Eipderm™ are purchased or derived from tissue obtained by MatTek Corporation from acredited institutions.
- Cells are screened for potential biological contaminants (HIV-1, Hepatitis B, Hepatitis C, bacteria, yeast and fungi)
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Skin Model (EPI-200)
- Tissue lot number: 29404, kit J

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure (3 minutes): room temperature
- Temperature used during treatment / exposure (1 hour): in a controlled environment at 37.0 ± 1.0°C

REMOVAL OF TEST MATERIAL
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h (at 37.0 ± 1.0°C)
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 replicates per exposure duration

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- No color change was observed in aqueous conditions therefore it was concluded that the test item showed no color interference in aqueous conditions.
- Since a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint.
- Two freeze-killed tissues were therefore treated with test item and two freeze-killed non treated tissues were used per exposure time for the cytotoxicity evaluation with MTT
- N. of replicates: 2 replicates per exposure duration
- Method of calculation used: Correction was not needed, as the non-specific MTT reduction by the test item was <= 0%
(-1.48% and -1.75% of the negative control tissues after 3 minutes and 1 hour treatment, respectively).

ACCEPTANCE OF RESULTS:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the negative control tissues should be within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be < 15%.
c) In the range of 20 – 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
d) The non-specific MTT reduction should be <= 30% relative to the negative control OD.

DECISION CRITERIA
A test item is considered corrosive in the in vitro 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 control for MTT reduction by test item
Amount/concentration applied:
TEST MATERIAL
50 µL
Duration of treatment / exposure:
3-minute and 1-hour

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
77
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour exposure
Value:
8.5
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes (7.9%)
- Acceptance criteria met for variability between replicate measurements: Yes (≤16%)
- The non-specific MTT reduction was <=30% relative to the negative control (-1.48% and -1.75%)

Applicant's summary and conclusion

Interpretation of results:
other: Category 1 (corrosive) based on GHS and CLP criteria
Conclusions:
The in vitro skin corrosion test was conducted according to OECD 431 guideline and GLP principles.
It is concluded that this test is valid and that the substance is corrosive in the in vitro skin corrosion test.
Executive summary:

In an in vitro skin corrosion test using a human skin model (EpiDerm Skin Model), the influence of the substance on the viability of human skin was tested according to OECD 431 (2016) and GLP principles. The substance was applied undiluted directly to 0.6 cm2 cultured skin. Negative and positive controls were included. After 3 -minute and 1 -hour treatments the substance was removed and the viability of the cells was tested by reduction of MTT. In addition two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point, as it was found that the test item interacted with the MTT endpoint. Viability of unexposed skin was set at 100%. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the substance compared to the negative control tissues was 77% and 8.5%, respectively. Correction for non-specific MTT reaction by the test item was not needed. The positive control had a mean cell viability of 7.9% after 1 hour exposure. The acceptability criteria of the study were met. Since the mean relative tissue viability for the test item was below 15% after the 1-hour treatment, the substance is corrosive in the in vitro skin corrosion test. As based on the results of the study sub-categorization is not possible, the classification of Category 1 applies based on GHS and CLP criteria.