2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

THe study was conducted between 16 May 2018 and 28 May 2018.

Reliability:

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

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

yes

Remarks:

These deviations were considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide

Cas Number:

2101947-22-0

Molecular formula:

C14H21NO2

IUPAC Name:

2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide

Test material form:

liquid

Specific details on test material used for the study:

Identification: FRET 15-0735
Physical state/Appearance: Clear colourless liquid
Storage Conditions: Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: 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: EpiSkin Laboratories, Lyon, France
Date received: 23 May 2018
EpiSkinTM Tissues (0.38cm2) lot number: 18-EKIN-021
Maintenance Medium lot number: 18-MAIN3-026
Assay Medium lot number: 18-ESSC-022

Justification for test system used:

The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 Hour post exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complementary end point can be used to either confirm a non-irritant result or will be used to override the non irritant result.

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 µL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.

Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 µL of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre labeled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12 well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 Minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42 Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
3.4.2.3 Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT 4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µL of the test item
10 µL of DPBS (negative control)
10 µL of SDS 5% w/v (positive controls)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: relative mean viability (%)

Run / experiment:

Mean

Value:

105.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test was repeated due to a failure to meet the assay acceptance criteria. The relative mean tissue viability for the positive control treated tissues was 10.5% relative to the negative control treated tissues and the standard deviation value of the viability was 7.7%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 1.065 and the standard deviation value of the viability was 10.5%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identical test item treated tissues was 8.8%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT. 

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

Test Item, Positive Control Item and Negative Control Item

It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

Mean OD₅₇₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of triplicate tissues	±SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	1.168	1.065	0.112	109.7	100*	10.5
	0.946			88.8
	1.081			101.5
Positive Control Item	0.206	0.112	0.082	19.3	10.5	7.7
	0.052			4.9
	0.078			7.3
Test Item	1.220	1.120	0.093	114.6	105.2	8.8
	1.105			103.8
	1.035			97.2

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

other:

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The relative mean viability of FRET 15-0735 treated tissues was 105.2% after the 15 Minute exposure period and 42 Hours post exposure incubation period. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment, the substance is considered to be non- 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 viability of FRET 15-0735 treated tissues was 105.2% after the 15 Minute exposure period and 42 Hours post exposure incubation period. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment, the substance is considered to be non- irritant. The relative mean tissue viability for the positive control treated tissues was 10.5% relative to the negative control treated tissues and the standard deviation value of the viability was 7.7%. The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 1.065 and the standard deviation value of the viability was 10.5%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identical test item treated tissues was 8.8%. The test item acceptance criterion was therefore satisfied.