Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 November 2019 - 28 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 18 June 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

Principles of method if other than guideline:

DEviation:
Cell Viability Measurement
• The start time for the MTT incubation was not noted correctly. The correct time cannot be
traced.
Evaluation: The responses for the negative and positive control indicated that an
appropriate incubation time was used. Therefore this deviation has no impact on the study
result..

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

EC Number:

951-458-1

Molecular formula:

Not applicable - UVCB

IUPAC Name:

Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

Test material form:

liquid

Details on test material:

Substance name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
Test item handling: Use amber glassware or wrap container in aluminum foil
EC Number: 951-458-1
Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light

Specific details on test material used for the study:

Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Purity/Composition correction factor: No correction factor required
EC Number: 951-458-1
Substance name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
Test item handling: Use amber glassware or wrap container in aluminum foil

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Reconstructed human epidermis

Source strain:

not specified

Details on animal used as source of test system:

The model consists of normal, human-derived epidermal keratinocytes 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 EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Model used: EpiDerm Reconstucted Human Epidermmis
Tissue batch numbers: 30928 and 32126
Date of initiation of testing: 25 November 2019
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM.

TEST ITEM PREPARATION
No correction was made for the purity/composition of the test item. The liquid test item was applied undiluted (50 µL) directly on top of the tissue. To protect the test item from light glassware was wrapped in tin-foil.

TEMPERATURE USED FOR TEST SYSTEM
Temperature used during treatment / exposure: 36.5 - 37.2°C

ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 63 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data, these deviations are considered not to affect the study integrity.

APPLICATION/TREATMENT OF THE TEST ITEM
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test item was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 µL of the undiluted test item was added into the 6-well plates on top of the skin tissues. In addition, since the test item reacted with the MTT medium, two freeze-killed tissues were treated with test item and two freeze-killed non treated tissues were used per exposure time for the cytotoxicity evaluation with MTT.For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point. Negative and positive controls were shared with parallel studies. All information pertaining to shared tissues are archived in the raw data.

REMOVAL OF TEST MATERIAL AND CONTROLS
Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Incubation time: The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm
- Filter/Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

The amount of extracted formazan was determined spectrophotometrically in triplicate.

Test for the Interference of the Test Item with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Test for Color Interference by the Test Item:
The test item was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the color interference, 50 µL of the test item or 50 µL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the
exposure time the mixture was shaken and it was checked if a blue / purple color change was observed.

Test for Reduction of MTT by the Test Item:
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 µL of the test item or 50 µL Milli-Q water as a negative control were added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline.


CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Fresh tissues / killed tissues: Killed tissues
N. of replicates : since the test item reacted with the MTT medium, two freeze-killed tissues were treated with test item and two freeze-killed non treated tissues were used per exposure time for the cytotoxicity evaluation with MTT.

Procedure used to prepare the killed tissues
Freeze-killed. Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to ≤-15°C. The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 mL DMEM. Further use of killed tissues was similar to living tissues. DMEM (Dulbecco’s Modified Eagle’s Medium) Supplemented DMEM, serum-free.

Cell Viability Measurement
The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature.


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.


ACCEPTABILITY CRITERIA
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8).

b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.

c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

d) The non-specific MTT reduction should be ≤ 30% relative to the negative control OD. All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.


INTERPRETATION
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Table 1 presents the data interpretation and optional sub-categorisation in case a test item will be corrosive
See Table 1: Data interpretation and sub-categorisation of test items in 'Any other information on materials and methods' incl. tables

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL liquid test item (undiluted)

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8.0 normal solution

Duration of treatment / exposure:

3-minute and 1-hour treatment/exposures

Number of replicates:

Two (two tissues per exposure period and two tissues each for the positive and negative controls)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute treatment with the test item

Value:

80

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour treatment with the test item

Value:

84

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

Any other information on results incl. tables

The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint.  

In addition to the normal 3-minute and 1-hour procedure, two tissues were treated with test item. Instead of MTT solution these tissues were incubated with DMEM. The non-specific reduction of MTT by the test item was 0.63% and 1.62% of the negative control tissues after 3 minutes and 1 hour respectively.  

The mean absorption at 570 nm measured after treatment with the test item and controls are presented in Appendix 1, Table 1. The individual OD570 measurements are presented in Appendix 2.  

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative Control	1.787	2.106	1.946	±	0.226	1.837	1.986	1.946	±	0.226
Test Item^(1)	1.565	1.559	1.562	±	0.004	1.741	1.480	1.562	±	0.004
Positive Control	0.187	0.178	0.183	±	0.007	0.129	0.187	0.183	±	0.007
SD = Standard deviation
Duplicate exposures are indicated by A and B.
^(1)The test item values are corrected for the non-specific MTT reaction (0.63 and 1.62 at the 3 minute and 1 hour treatment, respectively).

The individual OD570 measurements are presented in the following table (attached in Appendix 2):

	3-minute application (OD570)		1-hour application (OD570)
	A	B	A	B
Negative control
OD570measurement 1	1.8243	2.1438	1.9075	2.0896
OD570measurement 2	1.8348	2.1432	1.8636	1.9975
OD570measurement 3	1.8304	2.1618	1.8709	2.0008
Test item
OD570measurement 1	1.6544	1.6057	1.8401	1.5761
OD570measurement 2	1.6024	1.6116	1.7981	1.5495
OD570measurement 3	1.6047	1.6261	1.8085	1.5357
Test item on freeze killed tissue
OD570measurement 1	0.1645	0.1743	0.1560	0.1557
OD570measurement 2	0.1623	0.1689	0.1554	0.1519
OD570measurement 3	0.1642	0.1859	0.1552	0.1544
Non - treated freeze killed tissue
OD570measurement 1	0.1214	0.2338	0.1231	0.1284
OD570measurement 2	0.1207	0.2157	0.1203	0.1251
OD570measurement 3	0.1228	0.1324	0.1212	0.1249
Positive control
OD570measurement 1	0.2307	0.2255	0.1762	0.2416
OD570measurement 2	0.2301	0.2177	0.1702	0.2284
OD570measurement 3	0.2312	0.2204	0.1701	0.2209
OD = Optical density
Duplicate exposures are indicated by A and B.

The following table (Table 2, Appendix 1) shows the mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues:

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	80	84
Positive control	9.4	8.3

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 80% and 84% respectively.  Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit≤2.8) and the laboratory historical control data range (See Appendix 3). The mean relative tissue viability following the 1-hour exposure to the positive control was 8.3%.  

In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was≤15%, indicating that the test system functioned properly (Appendix 1, Table 3).

	3 minute	1 hour
Negative control	15	7.5
Test item	0.4	15
Positive control	5.1	31
CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the test item for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item was a light orange liquid. The test item was applied undiluted (50 µL) directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 8.3% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤15%, indicating that the test system functioned properly.

Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint.  

In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point. The non-specific reduction of MTT by the test item was 0.63% and 1.62% of the negative control tissues after 3 minutes and 1 hour respectively.  

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 80% and 84%, respectively.  Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.