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Description of key information

The potential of Vinylcyclohexane to induce skin corrosion (OECD 431) and/or skin irritation (OECD 439) was tested in suitable in vitro test methods. Based on the results, Vinylcyclohexane (99.8% purity) can be considered as corrosive to the skin.

The eye irritancy potential of the target substance was assessed in an in vitro eye irritating assay (OECD 437). The result of the OECD 437 was that no prediction can be made for final classification. Based on the results from the in vitro skin irritation/corrosion tests the target substance is considered to cause severe skin burns and eye damage and classification as Skin Corr 1B, H314 is warranted. Thus, no further testing to assess the eye irritating potential was needed and the substance will also be classified as Eye Dam 1, H318.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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
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.

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

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.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-08-02 to 2018-01-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
The test system EPISKIN™ is a reconstructed human epidermis (RhE) model, which in its overall design (the use of human derived epidermis keratinocytes as cell source and use of representative tissue and cytoarchitecture) closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ reconstructed human epidermis model (SkinEthic)
- Tissue batch number(s): 17-EKIN-035

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT, exposure time of 15 ± 0.5 minutes
- Temperature of post-treatment incubation (if applicable): 37 °C for 42 ± 1 hours


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 D-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: each tissue insert was incubated with 2 mL/well of MTT ready-to-use solution.
- Incubation time: 3 h at 37 °C, 5% CO2 and saturated humidity.
- Wavelength: 595 nm

NUMBER OF REPLICATE TISSUES: 3

STUDY APPLICABILITY CRITERIA
The assay was considered valid if the following criteria were met:
– Blank controls: mean OD value < 0.1.
– Negative controls: mean OD value ≥ 0.6 and ≤ 1.5, SD of % viability ≤ 18.
– Positive controls: mean viability expressed as percentage of the negative control ≤ 40 % and SD of % viability ≤ 18.
– Test item: SD of % viability ≤ 18.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
Amount(s) applied (volume or weight with unit): 20 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 20 µL D-PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 20 µL 5% w/v) SDS solution
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 ± 1 hours
Number of replicates:
3 tissues per treatment group
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of three tissues
Value:
13
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:
For detailed results see Table 1 in box "Any other information on results" .

OTHER EFFECTS:
- Visible damage on test system: no
- Colour interference with MTT: yes

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: yes

Results of main experiment:

Table 1: Result of the Test Item Vinylcyclohexane

 

Negative Control

Positive Control

Test Item

Test Item (without MTT)

 

OD-blank

Viability (%)

OD-blank

Viability (%)

OD

Viability (%)

OD-blank

NSCliving(%)

Mean

0.882

100

0.023

3

0.115

13

0.009

1

SD

0.03

3.9

0.001

0.1

0.02

2.5

0.004

0.4

CV (%)

3.9

3.9

2.5

2.5

19.0

19.0

44.4

44.4

Interpretation of results:
study cannot be used for classification
Conclusions:
In conclusion, in this in vitro skin irritation study (OECD 439), Vinylcyclohexane is considerd to be irritant to the skin (UN GHS Category 2 or 1). Further testing is required.
Executive summary:

In an in vitro skin irritation study conducted according to OECD testing guideline 439, the skin irritant potential of Vinylcyclohexane (99.8 % purity) was analysed by using the three-dimensional human skin model EPISKIN^TM (Skin Ethic) comprising a reconstructed epidermis with a functional stratum corneum. Hereby, 20 µL of the test item was applied directly atop the EPISKIN ^TM tissue.

Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 minutes exposure and 42 hours post-incubation period and compared to those of the concurrent negative controls.

No colour change was noted in the MTT solution at the end of the incubation period, indicating that the test item could direct interact with MTT. In a second step the test item was assayed for the ability of colouring water per se. The analysis showed, that the test item has a potential interfering ability. Based on the results, an additional control was added in the main assay for the evaluation of non-specific colouring potential (NSC living)

The mean relative tissue viability (% negative control) was <= 50 % (13 %) after 15 minutes treatment and 42 hours post-incubation. Therefore, Vinylcyclohexane is considered to be irritating to the skin in accordance with UN GHS "Category 1 or 2" and further testing is required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Additional information

The potential of Vinylcyclohexane to induce skin corrosion (OECD 431) and/or skin irritation (OECD 439) was tested in suitable in vitro test methods. Based on the results, Vinylcyclohexane (99.8% purity) can be considered as corrosive to the skin.

The eye irritancy potential of the target substance was assessed in an in vitro eye irritating assay (OECD 437). The result of the OECD 437 was that no prediction can be made for final classification. Based on the results from the in vitro skin irritation/corrosion tests the target substance is considered to cause severe skin burns and eye damage and classification as Skin Corr 1B, H314 is warranted. Thus, no further testing to assess the eye irritating potential was needed and the substance will also be classified as Eye Dam 1, H318.

Justification for classification or non-classification

Based on the results from the in vitro skin irritation/corrosion tests the target substance is considered to cause severe skin burns and eye damage and classification as Skin Corr 1B, H314 is warranted.

Thus, no further testing to assess the eye irritating potential was needed and the substance will also be classified as Eye Dam 1, H318.