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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:
key study
Study period:
from 05 December 2013 to 21 February 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: in-vitro study, performed according to internationally recognised guidelines and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
other: reconstructed human epidermis model

Test system

Amount / concentration applied:
undiluted
Duration of treatment / exposure:
15 minutes
Observation period:
42 hours
Details on study design:
TEST SYSTEM:
- Reconstructed human epidermis model Episkin small model (0.38 cm²) supplied by SkinEthic Laboratories, Lyon, France.
- Episkin kits were maintained in agar medium for transportation. They were delivered at day 13 and used at days 15 to 17.
- The test item was also tested on dead epidermis because of its interaction with MTT.

PROTOCOL:
- After preliminary tests (dilutions and staining tests), the colored test item was tested according to the specific coloring protocol.
- Application of the test substance: 5 µL of distilled water were first applied onto the tissues in order to moister the surface. 10 mg of the test material were then applied onto the epidermis (dead and alive).
- treatment and post-incubation: after 15 minutes treatment period at room temperature, tissues were rinsed with PBS+ and transferred on 2 mL/well of fresh maintenance medium and incubated for 42 hours at 37°C.
- Number of replicates: 3
- Negative control: 10 µL of PBS+
- Positive control: 10 µL of 50 mg/mL aqueous solution of Sodium Dodecyl Sulfate

MEASURED PARAMETERS:
- Cell viability measurement: MTT test. The tissue sample was placed in MTT solution (0.3 mg/mL) for 3 hours. The MTT is converted into blue formazan by the viable cells. The precipitated blue formazan product was then extracted from the tissue using acidic isopropanol as solvent, and the concentration of formazan was measured by determining the optical density (OD) at 570 nm.
- Determination of the IL-1α concentration in the culture medium (by a classic quantitative sandwich enzyme immunoassay technique).

ACCEPTANCE CRITERIA:
The OD of the negative control should be ≥0.600 and its standard deviation should be ≤18%.
The viability of the positive control should be <40% with regard to the negative control and the standard deviation should be ≤18%.
The OD raw material NSC on living epidermis should be ≤30% of the OD negative control.
The corrected OD of the product under test on dead epidermis should be ≤30% of the corrected OD of the living negative control.
The correlation coefficient of the calibration curve for IL-1α should be ≥0.99.
The IL-1α concentration of the negative control should be < 50 pg/mL.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: % viability
Value:
67.4
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 45 hours. Remarks: % SD = 17.6. (migrated information)
Irritation / corrosion parameter:
other: other: IL-1α (pg/mL)
Value:
21.5
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 hours. Remarks: % SD = 14.4. (migrated information)

In vivo

Irritant / corrosive response data:
As Mean viability value is < 50% and IL-1α is < 50 pg/mL, the test item is considered as potentially non irritant

Any other information on results incl. tables

Result summary: % viability and IL-1αconcentrations

Raw material

Run 1

Run 2

Run 3

Run 4

Mean

SD

 

Test item

(undiluted)

% viability

51.6

85.3

53.0

79.7

67.4

17.6

IL-1α (pg/mL)

42.7

10.6

16.6

16.0

21.5

14.4

This product presents results with a high variability due to the physical form of the raw material.

Applicant's summary and conclusion

Interpretation of results:
other:
Remarks:
Criteria used for interpretation of results: expert judgment
Conclusions:
Depending on the experimental conditions used, the in vitro evaluation of acute skin irritation by using the reconstructed human epidermis Episkin(SM) model, suggests that the test item tested undiluted is considered as potentially non irritant.
Executive summary:

In vitro evaluation of skin irritation was performed on the reconstructed human epidermis model Episkin small model (0.38cm²), with a method equivalent to OECD guideline 439 (GLP study, scored as validity 2 according to Klimisch criteria). The colored raw material was tested according to the specific coloring protocol. The test substance was qualified on 4 batches of reconstructed epidermis model and it was also tested on dead epidermis because of its interaction with MTT.

 

The tests item was tested undiluted. Negative and positive controls were tested in triplicate. The duration of treatement was 15 minutes, followed by an incubation period of 42h at 37°C. MTT test and determination of IL-1αconcentration were performed.

 

The mean of percentage of viability was 67.4% ± 17.6 and the mean of IL-1αconcentration 21.5 ± 14.4.

Depending on the experimental conditions used, the in vitro evaluation of acute skin irritation by using the reconstructed human epidermis Episkin(SM) model, suggests that the test item tested undiluted is considered as potentially non irritant.