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Toxicological information

Skin irritation / corrosion

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Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 17 October 2017. Experimental completion date 20 October 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model and in compliance with GLP.

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

Constituent 1
Chemical structure
Reference substance name:
4-(2-methylpropyl)benzyl propyl ether
EC Number:
Cas Number:
Molecular formula:
4-(2-methylpropyl)benzyl propyl ether
Test material form:
Specific details on test material used for the study:
Test item: FRET 11-0539
Test item identity: Benzene, 1-(2-methylpropyl)-4-(propoxymethyl)-, 4-(2-
methylpropyl)benzyl propyl ether
Storage conditions: Room temperature (15 - 25˚C), in the dark
Appearance: Clear, colorless liquid

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™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
unchanged (no vehicle)
Details on test system:
Reduction of MTT by Test Item
It is possible that a test item may reduce MTT, resulting in the production of a blue color without any involvement of cellular mitochondrial dehydrogenase. Since the test item is rinsed off the tissue before the MTT assay, this is usually avoided. However, it is possible that small amounts of test item may be present after washing or be released through the tissue into the MTT medium. If the mixture with the test item turns blue/purple after approximately 3 hours incubation at 37 ± 2ºC in a humidified atmosphere of 5% CO2 in air, MTT reduction may have occurred.
The MTT reducing capability of the test item, FRET 11-0539, was investigated by mixing 10 μL of the test item with 2 mL of 0.3 mg/mL MTT solution in duplicate. A control of 10 μL of purified water, mixed with 2 mL of 0.3 mg/mL MTT solution was also included in duplicate.

Check for Coloring Potential of Test Item
The test item, FRET 11-0539, was evaluated for its color or ability to become colored in contact with water (simulating a tissue humid environment). Evaluation was achieved by mixing 10 μL of the test item, FRET 11-0539, with 90 μL of purified water in a transparent micro-tube. 100 μL of purified water was included as a control. The solution was mixed for 15 minutes on a shaker. At the end of the shaking period the color of the solution was assessed by eye.

Receipt of Tissues
On receipt, the EpiSkin™ kit contents were checked and the inserts with tissues on agar were stored at room temperature until use. The kit was used within the expiry date indicated by the supplier (expiry date: 23 October 2017). The maintenance medium was pre-warmed to 37ºC. The tissues were removed from the agar and placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 24 hours at 37 ± 2ºC in a humidified atmosphere of 5% CO2 in air.

Preparation/Application of Samples
The positive and negative controls and the test item were in liquid form and were applied by dispensing a volume of 10 μL over each tissue using a positive displacement pipette.

Test Procedure
After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 ± 0.5 minutes with the test item, negative or positive control at room temperature. A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 μL, the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved flat spatula after 7 minutes application time.
After 15 ± 0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test item. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 ± 1 hour at 37 ± 2ºC in a humidified atmosphere of 5% CO2 in air.
After 42 ± 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours ± 5 minutes at 37 ± 2ºC in a humidified atmosphere of 5% CO2 in
At the end of 3 hours ± 5 minutes, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro-tube. When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration).
The tissues were extracted over 4 hours, protected from light at room temperature (vortexing after 2 hours).
After formazan extraction, duplicate 200 μL aliquots of the extractant from each tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.
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

- Amount(s) applied (volume or weight): 10μl
- Concentration (if solution): 5% solution in purified water
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 ± 1 hour
Number of replicates:

Test animals

other: EPIKSIN in vitro Reconstructed Human Epidermis (RHE) Model
other: Not applicable
Details on test animals or test system and environmental conditions:
Dissiminated (delete when editing)
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 11-11-1111
Dissiminated (delete when editing)
EpiSkinTM Tissues (0.38cm2) lot number : xx
Maintenance Medium lot number : xx
Assay Medium lot number : xx

Results and discussion

In vitro

Irritation / corrosion parameter:
other: other: relative mean viability
>= 103.2 - <= 110
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Possible Reduction of MTT by Test Item
There was no change in the test item, FRET 11-0539 /MTT solution or the water control/MTT solution after three hours incubation in the dark at 37 ± 2ºC in a humidified atmosphere of 5% CO2 in air. The test item had not interacted with the MTT.

Check for Coloring Potential of Test Item
The test item, FRET 11-0539 /water solution and water control were colorless after the 15 minute shaking period. The test item had not shown any potential for coloring water.

Assay Validity
Negative Control
The mean absorbance of the triplicate negative control values was 0.692 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 4.8 which was below the maximum value of 18.
Positive Control
The percentage mean viability of the positive control was 11.8 ± 3.2 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18%.

Any other information on results incl. tables

EpiSkin™ Results

Sample Tissue viability as percentage of mean OD negative
MTT endpoint
Replicate Tissues Mean ±
a b c
Negative control 94.6 101.6 103.7 100.0 ± 4.8 Not applicable
Positive Control 14.3 13 8.2 11.8 ± 3.2 Category 2
FRET 11-0539 102.9 109.4 107.5 106.6 ± 3.4 No category

Applicant's summary and conclusion

Interpretation of results:
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 106.6 ± 3.4%. Since the mean relative tissue viability for the substance above 50%
after 15 minutes treatment, the substance is considered not 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 106.6%.

Since the mean relative tissue viability for FRET 11 -0539 was higher than 50% after 15 minutes treatment the substance is considered to be non irritant. The positive control had a mean cell viability of 8.2% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.