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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:
08 February 2018 to 23 March 2018
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(2015)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
D,L- Menthol / D,L-Isomenthol
Molecular formula:
C10H20O
IUPAC Name:
D,L- Menthol / D,L-Isomenthol

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Reconstructed epidermis of normal human keratinocytes, originating from adult donors
Source strain:
other: Keratinocyte strain: 00267
Justification for test system used:
The test is based on the experience that irritant chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin irritation potential of a test item by assessment of its effect on a three dimensional human epidermis model.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM and EPIDERMTM)
- Tissue batch number(s): 25888
- Expiration date: Not specified
- Shipping date: March 20th 2018
- Date of initiation of testing: March 20th 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): All incubations were carried out at 37.0 ± 1.5⁰ C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with DPBS to
remove residual test item after exposure period.
- Observable damage in the tissue due to washing: Not specified.
- Modifications to validated SOP: The tissues were rinsed with PBS instead of DPBS to remove any residual material.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: (1 mg/mL)
- Spectrophotometer: Microplate reader (Versamax® Molecular Devices) .
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- No. of replicates: 3

PREDICTION MODEL / DECISION CRITERIA
-The test substance is considered to be an irritant to skin if the relative mean tissue viability after a 15-minute exposure period followed by the 42-Hour post-exposure incubation period is less than 50% of the mean viability of the negative controls.
-The test substance is considered to be non-irritant to skin if the relative mean tissue viability after a 15-minute exposure period followed by the 42-Hour post-exposure incubation period is greater than 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 +/- 2mg (approx. 39 mg/cm2) wettd with 25µL DPBS
- Concentration (if solution): Not applicable.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25µL
- Concentration (if solution): Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL Sodium dodecyl sulphate
- Concentration (if solution): 5 %
Duration of treatment / exposure:
15 Minutes
Duration of post-treatment incubation (if applicable):
Subsequently the skin tissues were incubated for 42 hours at 37°C.
Number of replicates:
3 test replicates.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
4.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
3.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
7.9
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:
- Acceptance criteria met for negative control: The negative control value from the study had a mean relative tissue viability of 100% and a mean OD570 of 1.718 which is within the historic control data
range.
- Acceptance criteria met for positive control: The mean relative tissue viability of the positive control was ≤50% (2.9%) and the mean OD570 was 0.051 which is within the historic control data range.
- Acceptance criteria met for variability between replicate measurements: The standard deviation for the negative control was within the acceptance criteria of ≤18 for the percentage viability (8.5%). The standard deviation for the positive control was within the acceptance criteria of ≤18 for the percentage viability (0.1%). All validity criteria were met.

Any other information on results incl. tables

Table 1- Results of treatment with test item and controls

Treatment Group Tissue number   OD 570 nm well 1   OD 570 nm well 2   OD 570 nm well 3 Mean OD of 3 wells Mean OD of 3 wells blank corrected  Mean OD of 3 tissues blank corrected     Rel. viability [%] Tissue 1,2 +3*   Standard deviation Mean Rel. viability [%]**
 Blank   0.039  0.038  0.038  0.038           

Negative

control

1

2

3

1.947

1.668

1.750 

1.888

1.598

1.717 

1.908

1.614

1.714 

1.914

1.627

1.727 

1.876

1.589

1.689 

 1.718

109.2

92.5

98.3 

8.5

 

100.0

 

Postive control

1

2

0.089

0.091

0.088 

0.088

0.088

0.086 

0.088

0.093

0.087 

0.089

0.091

0.087 

0.050

0.053

0.049 

0.051

 

2.9

3.1

2.8 

0.1   2.9
 Test item

1

2

0.090

0.097

0.177 

0.090

0.095

0.171 

0.090

0.096

0.174 

0.090

0.096

0.174 

0.052

0.058

0.136 

0.082

 

3.0

3.4

7.9 

 2.7  4.8

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In conclusion, the test item is a skin irritant according to CLP (H315) and GHS (category 2).

Executive summary:

The study is assigned a reliability score of 1 (reliable without restrictions) as it followed OECD Guideline 439: In Vitro Skin irritation: Reconstructed Human Epidermis (RHE) Test and it is compliant with GLP. 25 +/-2 mg of the test item (approx 39 mg/cm2 according to guideline) was wetted with 25 µL of DPBS and applied to the EpiSkin tissue for 15 minutes followed by a 42-hour post-exposure incubation period. Negative and positive controls were also tested. The positive control had a mean cell viability of 2.9%. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 7%.

Therefore, all validity criteria were met. The substance under test conditions had a mean tissue viability of 4.8% compared to the negative control. This value is below the threshold for irritancy (50%). This concludes the test item is a skin irritant (Category 2).