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Skin irritation / corrosion

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skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 26 April 2017 and 09 June 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. QA statement)

Test material

Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:
Specific details on test material used for the study:
Identification: FRET 12-0492
Chemical Name: 4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran
Appearance: Yellow, liquid
Storage Conditions: In the refrigerator
Stability in Solvent: Not indicated by the Sponsor
Purpose of Use: Industrial chemical

In vitro test system

Test system:
human skin model
Source species:
Cell type:
other: Normal Human-Derived Epidermal Keratinocytes
Cell source:
other: no information
Source strain:
other: not applicable
Details on animal used as source of test system:
EpiSkin™ Kit
Supplier : SkinEthic Laboratories (69007 Lyon, France).
Date received : 07 June 2017
EpiSkinTM Tissues (0.38cm2) lot number : 17-EKIN-023
Justification for test system used:
In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin models EpiSkin™ and EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential .
unchanged (no vehicle)
Details on test system:
Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 07 June 2017. On the day of experiment the pre-incubation phase of the EpiSkin™ tissues started.

Test for Direct MTT Reduction and Colour Interference
Prior to the start of the test, the colour interference potential of the test item had to be evaluated. For this purpose 10 µL of the test item was mixed with 90 µL of deionised water in a pre-experiment. The mixture was gently shaken for 15 minutes at room temperature.
The colour of the mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, the measurement of the OD of the test item in water at 570 nm was not required and consequently not performed.
For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability 10 µL of the test item was added to 2 mL of MTT solution (0.3 mg/mL) and the mixture was incubated in the dark at
37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution was used as the control. If the MTT solution colour turns blue/purple, the test item is presumed to have reduced the MTT.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.


Pre-warming of EpiSkin™ Tissues
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for approximately 3 hours.

The negative control, positive control and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

IL-1 α Immunoassay
Samples of all treatment groups were taken from the wells. The plates were shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well was taken and was stored in the freezer at –20 °C. Since the results derived from the MTT assay were not unclear or borderline, the IL-1 α concentration in the medium was not determined and the taken samples were discarded after report finalization.

MTT Assay
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for about 2.5 hours at room temperature with gentle agitation.
Per tissue sample 2 x 200 µL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- Amount(s) applied (volume or weight with unit): 10µl

- Amount(s) applied (volume or weight): 10µl

POSITIVE CONTROL (SLS, Fluka, Sigma-Aldrich)
- Amount(s) applied (volume or weight): 10µl
- Concentration (if solution): 5% solution in deionised water
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hrs
Number of replicates:

Test system

unchanged (no vehicle)

Results and discussion

In vitro

Irritation / corrosion parameter:
other: Relative absorbance (% of negative control)
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test item in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 30.9% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.
Concerning acceptance criteria:
• The mean OD of the three negative control exposed tissues is  0.6 till ≤ 1.5 (range: 1.384 to 1.468).
• The rel. standard deviations between tissues of the same treatment group were ≤ 18% (3.2% (negative control) and 2.3% (test item)). For the positive control, the acceptance criteria were not met (28.7%). Reason: extremely low absorbance values of 0.014, 0.014 and 0.022, where small differences cause high rel. standard deviations. The OECD guideline 439 and the SkinEthic protocol request only the simple standard deviation being ≤ 18% as acceptance criteria between tissues of the same treatment group. If the simple standard deviation is considered (0.3%), the acceptance criteria are met. This deviation from the acceptance criteria is not considered to have an impact on the outcome of the study.
• The mean relative tissue viability of the positive control was ≤ 40% (1.2%).
• The acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (1.8 mg/mL).
• Historical Data for the negative control are not available. Reason: Envigo CRS GmbH changed the negative control for the test system “In vitro Skin Irritation Assay using RHE supplied by SkinEthic” from deionized water to PBS. The test is performed at Envigo CRS GmbH a long time prior to a former version of OECD 439 guideline, where deionized water or PBS are suggested as negative control. Originally, SkinEthic requested deionized water to be used as negative control in their protocol. All of Envigo CRS GmbH’s historical data are based on deionized water. In the current SkinEthic protocol, PBS is suggested as negative control. Therefore, Envigo CRS GmbH decided to change the negative control to be in accordance with the SkinEthic protocol, but the number of performed assays using PBS as negative control is still too low to issue historical data.
Historical Data for the positive control are available. But the determined viability value of 1.2% is not within the historical data (viability range: 3.90% - 32.73%). Since the historical data at Envigo CRS GmbH are updated at least once a year, after next update the present positive control viability of 1.2% will be within the historical range. Since neither the OECD guideline nor SkinEthic requests historical data for the positive control, the outcome of the study is not impacted by this.

Any other information on results incl. tables

Results after treatment with the test item and controls

Test Group

Absor-bance 570 nm
Tissue Well 1

Absor-bance 570 nm
Tissue Well 2

Mean Absor-bance 570 nm

Mean Absor-bance 570 nm*

Mean Absor-bance of 3 Tissues

Deviation of
 Tissue Absorbance

Relative Absor-bance [%] Tissue 1, 2, 3**

Standard Deviation [%]

Rel. Standard Deviation [%]****

Rel. Absorbance

[% of Negative Control]***







Negative Control





















Positive Control





















Test Item





















*         Mean of two replicate wells after blank correction

**       relative absorbance per tissue [rounded values]: (100 x (absorbance tissue)) / (mean absorbance negative control)

***     relative absorbance per treatment group [rounded values]: (100 x (absorbancetest item)) / (mean absorbancenegative control)


****      relative standard deviation per treatment group [rounded values]: (100 x standard deviation tissue absorbance) / mean absorbance tissue)


This in vitro study was performed to assess the irritation potential of FRET 12-0492 by means of the Human Skin Model Test.

The test item passed the MTT and colour interference pre-tests.

Each three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes.

The test item and the positive and negative controls were washed off the skin tissues treatment. After further incubation for approximately 42 hours the tissues were treated with the MTT solution for 3 hours following about 2.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

The acceptance criteria were met. Exceptions:

·        The rel. standard deviation between tissues exposed to the positive control was > 18% due to very low absorbance values.

·        Due to recent change of negative control at Envigo CRS GmbH for being in accordance with the tissue supplier’s test protocol, historical data for the negative control are not yet available.

The exceptions from the acceptance criteria did not have an impact on the outcome of the study.

After treatment with the test item the mean relative absorbance value was reduced to 30.9%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Criteria used for interpretation of results: EU
The relative mean tissue viability after 15 minutes treatment with the substance compared to the negative control tissue was 30.9%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment, the substance is considered to be a potential irritant.
Executive summary:

The possible skin irritation potential of the substance was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD TG 439. Skin tissue was treated by topical application of 10 µL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 30.9%.

Since the mean relative tissue viability for FRET 12 -0492 was below 50% after 15 minutes treatment the substance is considered to be irritant. The positive control had a mean cell viability of 1.2% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18% (with the exception of the positive control because extremely low absorbance values were recorded), indicating that the test system functioned properly.

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