2-Propenamide, 3-(1,3-benzodioxol-5-yl)-N,N-diphenyl-, (2E)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2009-09-08 to 2009-11-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted 2004-04-13

Deviations:

yes

Remarks:

MTT concentration used was not stated; supporting information for the specific skin model used incl. its validity was not stated

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

adopted 2008

Deviations:

yes

Remarks:

MTT concentration used was not stated; supporting information for the specific skin model used incl. its validity was not stated

Principles of method if other than guideline:

Minor deviations from the guideline OECD 431 (2015) occurred:
- according to the guideline complete information for the specific RhE model used including e.g. viability, barrier function, morphology, and tissue quality control should be stated. This is missing in this study report. - colour interference test was not conducted
- acceptability criteria are different from guideline
- MTT and isopropanol concentrations used were not stated

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-10-22

Test material

Test material information

Constituent 1

Reference substance name:

(2E)-3-(2H-1,3-benzodioxol-5-yl)-N,N-diphenylprop-2-enamide

Cas Number:

1309389-73-8

Molecular formula:

C22H17NO3

IUPAC Name:

(2E)-3-(2H-1,3-benzodioxol-5-yl)-N,N-diphenylprop-2-enamide

Test animals

Details on test animals or test system and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:

other: highly de-ionized water

Controls:

other: Control (50 µL highly de-ionised water) skin cultures were used.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μL of the test item

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL of the vehicle

Duration of treatment / exposure:

3 minutes and 1 hour

Observation period:

not applicable

Number of animals:

Number of skin tissue replicates per dose and per exposure time:
Test item: duplicates
Negative control: duplicates
Positive control: duplicates

Details on study design:

THREE DIMENSIONAL HUMAN EPIDERMIS MODEL:
- EpiDerm™ 200 kit were obtained from MatTek Corporation, Ashland MA, USA
- EpiDermTM tissues (surface 0.6 cm²) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, 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.

DIRECT MTT REDUCTION:
- approximately 25 μL bulk volume of the test item was added to 0.9 mL of the MTT solution.
- mixture was incubated in the dark at about 37 °C for 55 to 65 minutes.
- negative control (50 μL of vehicle) was tested concurrently.
- if the MTT solution colour or, in case of water-insoluble test items the border to the water-phase, turned blue / purple, the test item was presumed to directly reduce MTT.
- in case that direct MTT reduction occurred, one freeze-killed control tissue per exposure time was treated with, each, the test article and the negative control, in the same way as described below.

CORROSION TEST:
- tissues were kept in the refrigerator.
- at least 1 hour but not more than 1.5 hours before test-item application, tissues were transferred to 6-well plates with assay medium and preconditioned in the incubator at 37 °C.
- preincubation medium was replaced with fresh medium before application.
- two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator) and test material, negative control (50 μL highly de-ionized water) and positive control (50 μL 8 n potassium hydroxide) were used.
- for the test item, the vehicle was applied first followed by the test material.
- control tissues were concurrently applied.
- tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application.
- tissues were incubated in MTT solution for 3 hours.
- after incubation, tissues were washed with PBS
- formazan produced by the tissues was extracted with isopropanol over night at room temperature.
- optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically.
- blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

DATA EVALUATION.
- Principle: the OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the negative control (percent of control) is used for evaluating whether or not a test material is corrosive.
- Calculation of individual and mean optical densities: the individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way is calculated.
- Tissue viability: the quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 negative control value in percent for each exposure time.

EVALUATION OF RESULTS:
Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 minutes treatment compared to the negative control tissues. A chemical is considered as "corrosive" or "non-corrosive" according to table 1 in the field "Any other information on materials and methods incl. tables" below.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure (mean of 2 tissue replicates)

Value:

104

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure (mean of 2 tissue replicates)

Value:

123

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

HISTORICAL DATA

Table 1: Historical range of negative control (OD570)

Exposure time	Historical period	Mean OD	Standard deviation	Mean + 2 Standard deviation	Mean – 2 Standard deviation
3 minutes	Mar 2008 – Nov 2009	1.891	0.257	2.40	1.38
60 minutes	Mar 2008 – Nov 2009	1.897	0.230	2.36	1.44

Table 2: Historical range of positive control (OD570)

Exposure time	Historical period	Mean OD	Standard deviation	Mean + 2 Standard deviation	Mean – 2 Standard deviation
3 minutes	Mar 2008 – Nov 2009	0.408	0.110	0.63	0.19
60 minutes	Mar 2008 – Nov 2009	0.189	0.051	0.29	0.09

Table 3: Viability (%)

Exposure time	Historical period	Mean %	Standard deviation	Mean + 2 Standard deviation	Mean – 2 Standard deviation
3 minutes	Mar 2008 – Nov 2009	21.84	5.21	32.26	11.42
60 minutes	Mar 2008 – Nov 2009	9.97	2.11	14.18	5.75

RESULTS

Table 4: Result of corrosion test with the test item

Test article		Exposure: 3 minutes			Exposure: 1 hour
		tissue 1	tissue 2	mean	tissue 1	tissue 2	mean
Negative control	mean OD570	1.2662	1.4622	1.3642	1.6587	1.7942	1.7264
	viability [% of negative control]	92.8	107.2	100	96.1	103.9	100
Test item	mean OD570	1.6172	1.7452	1.6812	1.7767	1.8022	1.7894
	viability [% of negative control]	118.5	127.9	123	102.9	104.4	104
Positive control	mean OD570	0.3322	0.3452	0.3387	0.2282	0.1947	0.2114
	viability [% of negative control]	24.3	25.3	25	13.2	11.3	12

The test item is not able to reduce MTT directly.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

For classification purposes, results of this in vitro skin corrosion study need to be assessed together with the results from an in vitro skin irritation study.

Conclusions:

In an in vitro skin irritation study according to OECD guideline 431, the substance was not corrosive to the skin.

Executive summary:

In this in vitro study the substance was tested for its skin corrosive potential according to the OECD guideline 431 (2004) by the means of the human skin model test using EpiDerm™. Duplicates of EpiDerm™ tissues were exposed to either the substance (25 µL) with the vehicle (25 µL), the positive control (8 n potassium hydroxide; 50 µL) or the negative control (highly de-ionised water; 50 µL) for each of two different exposure periods: 3 minutes and 1 hour. Afterwards the test and the control items were rinsed off the tissues, and a 3 hour incubation period with MTT solution followed.

After incubation, tissues were washed and the formazan produced by the tissues was extracted with isopropanol overnight. This was followed by the measurement of the optical density (OD570).

After an exposure period of 3 minutes the relative tissue viability increased to 123 % (threshold for corrosivity: 50 %). After an exposure period of 1 hour the relative tissue viability increased to 104 % (threshold for corrosivity: 15 %). Therefore, the substance was not considered to be corrosive. The controls confirmed the validity of the study.

According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not corrosive to the skin.