2-ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 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:

2019

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:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate

EC Number:

278-051-5

EC Name:

2-ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate

Cas Number:

75005-95-7

Molecular formula:

C28H30O3

IUPAC Name:

2-ethylhexyl 2-{[1,1'-biphenyl]-4-carbonyl}benzoate

Test material form:

liquid

Details on test material:

Colourless to pale yellow liquid (determined by Charles River Den Bosch).
Stored at room temperature.

Specific details on test material used for the study:

Physical Description: Colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: 96.27%
Storage Conditions: At room temperature
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm Skin Model (EPI-200, Lot no.: 32126 Kit A and kit B)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: All cells used to produce EpiDerm are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions.

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 Skin Model (EPI-200)
Tissue batch number: Lot no.: 32126 Kit A and kit B
Production date: November 2019
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.
DMEM (Dulbecco’s Modified Eagle’s Medium) - Supplemented DMEM, serum-free supplied by MatTek Corporation.


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.


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). Both supplied by MatTek Corporation.

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 (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. 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.


TEST ITEM PREPARATION/APPLICATION
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.

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 (see figure 1). 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.

Fifty µL of the undiluted test item was added into the 6-well plates on top of the skin tissues. 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.

After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) 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.


CELL VIABILITY MEASUREMENT
The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours (see deviation) at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.


ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following 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%.

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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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.

Duration of treatment / exposure:

The test consists of topical application of the test item on the skin tissue for 3-minute and 1-hour.

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application viability (percentage of control)

Value:

101

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 application viablilty (percentage of control)

Value:

98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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 the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

Any other information on results incl. tables

The mean absorption at 570 nm measured after treatment with the test item and controls are presented below (and in appendix 1, Table 1 in attached background material):

Mean Absorption in the in vitro Skin Corrosion Test with the test item

	3 minute application					1 hour application
	A (OD570)	B (OD570)	Mean(OD570)	SD		A (OD570)	B (OD570)	Mean (OD570)	SD
Negative control	1.643	1.773	1.708	±	0.092	1.643	1.773	1.708	±	0.092
Test item	1.786	1.674	1.73	±	0.079	1.786	1.674	1.73	±	0.079
Positive control	0.288	0.368	0.328	±	0.056	0.288	0.368	0.328	±	0.056

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.043). Isopropanol was used to measure the background absorption.

The individual OD570 measurements are presented in Appendix 2 (in attached backrgound material).  

The below table (appendix 1, table 2 in attached background material) shows the mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues. 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 101% and 98% 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.

Mean Tissue Viability in the in vitro Skin Corrosion Test with C991

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	101	98
Positive control	19	7.3

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 (Appendix 3 in attached background material). The mean relative tissue viability following the 1-hour exposure to the positive control was 7.3%.  

In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 8%, indicating that the test system functioned properly (below table, appendix 1, table 3 in attached background material).

Coefficient of Variation between Tissue Replicates

	3 minute	1 hour
Negative control	7.3	5.6
Test item	6.3	4.1
Positive control	22	8

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

2 -ethylhexyl 2 -{[1,1'-biphenyl]-4 -carbonyl}benzoate 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 colourless to pale yellow 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 7.3% 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 < 8%, indicating that the test system functioned properly.

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 101% and 98%, 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.