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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:
12 May - 16 Jun 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU B.40 bis (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
(1R)-1-isopropyl-4-methyl-cyclohex-3-en-1-ol
EC Number:
943-985-0
Cas Number:
20126-76-5
Molecular formula:
C10H18O
IUPAC Name:
(1R)-1-isopropyl-4-methyl-cyclohex-3-en-1-ol

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™ (EPI-200)
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23341
- Delivery date: 14 June 2016
- Date of initiation of testing: 14 June 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 ± 0.5 min exposure), 37 ± 1.5 °C (60 ± 5 min exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed using a wash bottle containing DPBS to remove any residual test material (20 times).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, SoftMax Pro Enterprise v.4.7.1)
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.908 ± 0.185 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.17 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce MTT, an additional test with freeze-killed or viable tissues was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is greater or equal than 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8 N
Duration of treatment / exposure:
3 ± 0.5 min and 60 ± 5 min
Number of replicates:
in duplicates for each treatment and control group

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
3 min exposure
Value:
114.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
60 min exposure
Value:
22
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS
- Direct-MTT reduction: Since the test substance did not directly reduce MTT, an additional test with freeze-killed or viable tissues was not performed.
- Colour interference with MTT: The test substance did not change colour, when mixed with deionised water and thus passed the colour interference pre-test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean negative control OD, both for the 3 and 60 min exposure period, was in the range of ≥ 0.8 and ≤ 2.8 for every exposure time thereby confirming the acceptable quality of the tissues.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 min exposure period (28.8%) and for the 60 min exposure period (7.4%) thus confirming the validity of the test system and the specific batch of tissue models.
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation in the range 20 – 100% viability between tissue replicates was < 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

Any other information on results incl. tables

Table 2. Results after treatment with the test substance and controls

Test group

Absorbance at 570 nm*

Mean absorbance of 2 tissues

Coefficient of variation (%)

Rel. absorbance (% of negative control)**

Tissue 1

Tissue 2

3 minutes treatment

Negative control

1.822

1.740

1.781

3.3

100.0

Positive control

0.412

0.615

0.513

27.8

28.8

Test substance

2.028

2.061

2.045

1.2

114.8

60 minutes treatment

Negative control

1.789

1.904

1.846

4.4

100.0

Positive control

0.141

0.132

0.136

4.8

7.4

Test substance

0.467

0.346

0.407

21.1

22.0

* Mean of three replicate wells after blank correction (blank after 3 and 60 min exposure was 0.039 and 0.036, respectively)

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the conducted test, the test substance did not possess corrosive properties towards reconstructed human epidermis tissue in the EpiDerm™ model.
Executive summary:

The skin corrosion potential of the test substance was assessed by an in vitro skin corrosion test using a human skin model according to OECD Guideline 431 and in compliance with GLP (2016). After treatment of the tissues with the test substance the mean relative absorbance value did not decrease (114.8%) after 3 min exposure compared to the negative control. After 1 h exposure the relative absorbance value was reduced to 22.0%. Both values did not exceed the threshold for corrosivity and therefore the test substance is not considered to be corrosive to the skin.