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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:
weight of evidence
Study period:
From 2017-05-30 to 2017-06-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Batch No.: 16023LS4B5
Purity: 98.3%

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g.OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model TM
- Tissue batch number(s): 17EKIN022
- Date of initiation of testing: 30 May 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 15 ± 0.5 minutes at room temperature.
- Temperature of post-treatment incubation (if applicable): the skin tissues were incubated for 42 hours at 37°C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 tissues each for test item treatment, negative and positive control.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is < 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 17.5 to 20.6 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL 5% SDS
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
93
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: the test item did not interfere with the MTT endpoint

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: the positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 8.1%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was ≤13%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

In vitro skin irritation test was performed using a human three dimensional epidermal model based on the most recent OECD 439 guidelines (2015).

Skin tissue was moistened with 5 μL of Milli-Q water and at least 10 mg of the test item was applied directly on top of the

skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 93%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

In conclusion, the test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.