Registration Dossier

Diss Factsheets

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 April - 27 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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 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:
Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol
EC Number:
947-716-8
Molecular formula:
C15H30O
IUPAC Name:
Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-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™(EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23338
- Delivery date: 24 May 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 with DPBS 20 times in order to remove any residual test material.

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 2.078 ± 0.070 (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 5.64 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 showed no reducing capacity 1 h after MTT incubation, an additional test with freeze-killed tissues was not necessary.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than 50% and the viability after 1 hour exposure is greater 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:
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:
97.7
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:
105.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: The test substance showed no reducing capacity 1 hour after MTT incubation.
- 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 OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.627 and 1.755).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 hour, was < 15% (10.7%) compared to the negative control.
- Acceptance criteria met for variability between replicate measurements: The Coefficient of Variation (CV) in the range 20 - 100% viability between tissue replicates was ≤ 30% (values between 0.2% and 5.8%)

Any other information on results incl. tables

Table 2. Results after 3 and 60 min treatment with the test substance and controls

 

 

Exposure

interval (min)

Absorbance

of 3 wells

Aborbance

at 570 nm *

Mean absorbance of 2 tissues

CV

(%)

Rel. absorbance (% of negative control) **

Tissue 1

Tissue 2

Tissue 1

Tissue 2

Negative

control

 

 

 

3

1.738

1.636

1.701

1.598

1.649

4.4

100.0

Positive

control

0.276

0.335

0.239

0.297

0.268

15.4

16.2

Test

substance

1.624

1.672

1.587

1.635

1.611

2.1

97.7

Negative

control

 

 

 

60

1.655

1.661

1.619

1.625

1.622

0.2

100.0

Positive

control

0.234

0.185

0.198

0.148

0.173

20.2

10.7

Test

substance

1.816

1.677

1.780

1.640

1.710

5.8

105.4

* Mean of three replicate wells after blank correction

 ** Relative absorbance (rounded values): 100 × (mean absorbance test substance/positive control) / (mean 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 reconstructed human epidermis test the test substance does not possess corrosive properties.
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 (2017). Each of two human skin tissues (EpiDerm™) with a tissue size of 0.63 cm² were treated with 50 µL of the test substance, the negative control (deionised water) or the positive control (potassium hydroxide 8 N) for 3 and 60 min. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The relative mean tissue viability obtained after 3 and 60 min treatment with the test substance compared to the negative control tissues was 97.7% and 105.4%, respectively. Since, the mean relative tissue viability for the test substance was above the threshold for irritancy of 50% after 3 min and 15% after 60 min treatment the test substance was not considered to be corrosive to the skin. The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the substance in water did not lead to a change in colour. Additionally, optical evaluation of the MTT-reducing capacity of the test substance after 1 hour incubation with MTT-reagent did not show blue colour. The positive control, potassium hydroxide, revealed a mean cell viability of 10.7% (required ≤ 15%) after 60 min exposure and thus ensuring the validity of the test system. All other acceptability criteria were met. Based on the results of this study, the test substance was non-corrosive to the skin under the conditions of the test.

Categories Display