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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:
Start of experimental phase: 29 June 2015 and End of experimental phase: 31 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-{octahydro-1H-4,7-methanoindene-2,5-diylbis[(2,1-phenylene)oxymethylene]}bis(oxirane)
EC Number:
943-519-6
Cas Number:
2149603-92-7
Molecular formula:
C28H32O4
IUPAC Name:
2,2'-{octahydro-1H-4,7-methanoindene-2,5-diylbis[(2,1-phenylene)oxymethylene]}bis(oxirane)
Test material form:
not specified

Test animals

Species:
other: Commercial reconstructed human epidermis (RhE) model named EPISKIN™.
Strain:
other: Not applicable
Details on test animals or test system and environmental conditions:
- Supplier: SkinEthic Laboratories (4, A. Fleming – 69007 Lyon – France).
- Batch number: 15-EKIN-034 (alive tissue)
- Arrived at RTC: on 25 August 2015

Functional controls:
- Quality controls: histology scoring, magnitude of viability and barrier function (IC50 determination).
- Biological safety: absence of HIV1 and 2, Hepatitis B and C antigens, absence of bacteria, fungi and mycoplasma.

Test system

Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Remarks:
The epidermis surfaces were moistened with 10 microL. sterile water before application of the test item.
Controls:
other: Positive control (SDS: Sodium Dodecyl Sulphate) diluted at the final concentration of 5% (v/v) in sterile water for injection and negative control is D-PBS.
Amount / concentration applied:
20 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/cm2)
Duration of treatment / exposure:
- An exposure time of 15 +/- 0.5 minutes was allowed in a ventilated cabinet at room temperature for the test item.
- Negative and postive control treatments were carried out staggering samples of approximately 1 minute +/- 0.5 minute, while test item treatments were performed staggering samples of approximately 2 minutes +/- 0.5 minute.
Observation period:
- A 42 1 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.
Number of animals:
Not applicable
Details on study design:
TEST SITE
- % coverage:20 mg/epidermis unit, each measuring 0.38 cm2
- Replicate: 3 replicates for positive,3 for negative control and 3 for the test item.

REMOVAL OF TEST SUBSTANCE
At the end of the exposure, tissue samples were rinsed with approximately 25 mL of sterile D-PBS filling and empting the tissue insert; only one treated sample was rinsed twice and the test item was mechanically removed by using sterile tweezers.
The excess liquid was carefully removed and the samples transferred into new wells pre-filled with 2 mL/well of maintenance medium.

SCORING SYSTEM:

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: Mean cell viability
Value:
93.5
Remarks on result:
other:
Remarks:
Time point: 15 minutes exposure and 42 hours recovery period. Max. score: 100.0. Remarks: Mean cell viability in %. Acceptable intra-replicate variability was obtained (SD of % viability = 15.4% lower than 18.). (migrated information)

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study, using a commercial reconstructed
human epidermis (RhE) model named EPISKIN™. The negative and positive controls gave the expected results and the study was accepted as valid.
The mean cell viability of the test item treated tissues was 93.5%. Based on the results obtained, the test item is classified as not irritant to the skin.
Executive summary:

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 +/- 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. The test item was tested as supplied

by the Sponsor.

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se; a colourless suspension, with yellow precipitate, was observed at the end of the incubation period, indicating that the test item could not interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se; a colourless suspension, without precipitate but with the presence of two different phases, was observed, which indicates that the test item does not have a colouring ability. Based on these results, no additional controls were

added in the Main Assay.

In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/cm2). Positive and negative controls [a 5% (v/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 μL/epidermis unit.

The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability. The positive control caused the expected cell death (6.2% of cell viability when compared to the negative control) and variability (SD of % viability

equal to 4.3). Based on the stated criteria (mean viability <= 40% and SD of % viability <=1 8), the assay was regarded as valid.

Only the OD-blank background subtraction was performed for the evaluation of irritant properties of the test item. The test item did not induce cell death in any replicate with a mean cell viability of 93.5%, when compared to the negative control.

Based on the results obtained, the test item is classified as not irritant to the skin.