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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:
2017-10-17 to 2018-03-02
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name: Vinylcyclohexane
- CAS No.: 695-12-5
- Batch No.: VCH/7/17K1
- Expiry date: 30 March 2019
- Purity: 99.8 %
- Appearance: Clear, colourless liquid

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:
Not applicable
Justification for test system used:
The SkinEthic reconstructed human tissue model EPISKIN^TM consists of an airlifted, living, multi-layered tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium, featuring normal ultra-structure and functionality equivalent to human tissue in vivo.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EPISKIN™ (SkinEthic);
- Tissue batch number(s): 17-EKIN-046

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (MTT incubation): 37 ± 1 °C (for 3 h ± 15 min, 5.0% CO2 / 95% air)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
At the end of the exposure, each tissue was rinsed with approximately 25 mL of sterile PBS, filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 mL/well of maintenance medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL/well of MTT ready-to-use solution
- Incubation time: 3 h ± 15 min at 37 ± 1 °C
- Spectrophotometer: plate spectrophotometer
- Wavelength: 595 nm

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA:
The test substance is considered to be non-corrosive to skin as the relative mean tissue viability after 4 h treatment was not decreased to less than 35% of the corresponding negative control tissues.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
Duration of treatment / exposure:
- Test item and negative control: 3, 60 and 240 minutes
- Positive control: 240 minutes

Duration of post-treatment incubation (if applicable):
3 hours +/- 5 minutes
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes /mean of two replicates
Value:
121
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes, mean of two replicates
Value:
56
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
240 minutes, mean of two replicates
Value:
7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
Pre-experiments: Before the main assay, a preliminary test was carried out to evaluated the compatibility of the test item with the test item. In a first step, the test item was assayed for the ability of reducing MTT per se. No interaction was recorded between the test item and MTT in the test conditons, thus no additional controls were added in the main phase for the evaluatoion of MTT non specific redction. In a second step, the test item was assayed for the ability of colouring water per se. An opaque suspension was obtained, indicating that the test item has no potential interfering ability.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Intra-replicate variability was acceptable with a difference of viability between the two replicates lower than 30 %, for all treatment times except with test samples at 60 minutes treatment time (35.7 %). However, this did not affect the integrity of the results because the viability of both replicates was higher than 35 % when compared to the concurrent negative control.

Any other information on results incl. tables

Table 2: Results of Main Assay after 240 minutes treatment time

Blank

OD (Blank)

OD- blank (Negative Control)

Viablity (%)

OD (Test Item)

Viablity (%) (Test Item)

Mean

0.038

0.910

100

0.065

7

SD

0.0014

0.026

 

0.007

 

CV (%)

3.72

2.8

0.5

10

13

* The mean cell viability of the test item treated tissues has been calculated after blank subtraction

Applicant's summary and conclusion

Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
In an in vitro skin corrosion test (OECD 431) using the human skin model EPISKIN™ the test item was tested as corrosive.
Executive summary:

The potential of Vinylcyclohexane (99.8% purity) to induce skin corrosion was analysed by using the three-dimensional human skin model EPISKIN™, comprising a reconstructed epidermis with a functional stratum corneum (OECD 431). The test item was applied topically to the EPISKIN™ tissue for 3 min, 60 min and 4 h followed by immediate determination of cytotoxic effects via MTT reduction assay.

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities compared to the corresponding negative control tissues concurrently treated with 0.9% NaCI (= 100% tissue viability). The positive control did induce the appropriate response. The controls confirmed the validity of the study. In this study under the given conditions the test item showed corrosive effects. The relative mean tissue viability after 4 h treatment was ≥35% (7%). Relative mean tissue viability was reduced to 56% after 60 min treatment and after 3 min treatment the relative mean tissue viability was 121%. The test item is therefore considered to be corrosive.