4-Ethoxy-2,3-difluorphenylboronic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 10, 2016 - April 15, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(4-ethoxy-2,3-difluorophenyl)boronic acid

EC Number:

606-729-6

Cas Number:

212386-71-5

Molecular formula:

C8 H9 B F2 O3

IUPAC Name:

(4-ethoxy-2,3-difluorophenyl)boronic acid

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

standard model

Vehicle:

unchanged (no vehicle)

Remarks:

No vehicle used in this study; The test item was applied neat to the tissues.

Details on test system:

CELL CULTURE
- Supplier: EpiSkin/SkinEthic Laboratories, Lyon, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 16-RHE-025
- Expires: March 21, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritating to skin if the viability is greater than 50%.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg of solid test material
- Concentration (if solution): n/a

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL (Dulbecco`s Phosphate-Buffered Saline)
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% aqueous solution of sodium dodecyl sulfate in deionised water

Duration of treatment / exposure:

42 min (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1

Value:

101.18

Vehicle controls validity:

not applicable

Remarks:

The test item was applied neat to the tissues

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: yes
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:
Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
The negative control OD values were 1.575, 1.555 and 1.698 and, thus, in the range of ≥0.8 and ≤3.0.

Acceptability of the Positive and Negative Control:
After treatment with the negative control (DPBS-buffer) the mean OD was 1.609 (standard deviation: 4.84%) and, thus, higher than the historically established boundary of 1.436.
After treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) the mean viability value was 1.28% (standard deviation: 13.56%) and, thus, lower than the historically established boundary of 3.28%.

The standard deviation of the negative control and the positive control was ≤18%, respectively.

Variability of the Data:
The standard deviation of the three tissues treated with the test item was 1.07% and, thus, ≤18%.
Therefore, the study fulfilled the validity criteria.

Any other information on results incl. tables

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	1.609	100
Positive Control	42	0.021	1.28
Test Material	42	1.708*	101.18

*Corrected optical density after true metabolic conversion

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

Executive summary:

Objective

This in vitro study was performed to assess the skin irritation potential of the test item by means of the Reconstructed Human Epidermis (RHE) Test.

 

Study Design

The test consisted of a topical exposure of the test item to a human reconstructed skin model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.

 

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before adding the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to the tissues.

 

The test item has the ability to directly reduce MTT. To evaluate the extent of non-specific interaction, three killed tissues were treated with the test item or the negative control, respectively. The treatment and MTT assay of the killed tissues was similar to the handling of the living tissues. The obtained OD for a non-specific reduction was subtracted from OD-values obtained after treatment of living tissues with the test item to calculate the cell viability.

 

Results

After treatment with the negative control (DPBS-buffer) the mean OD was 1.609 (study acceptance criteria: >1.436). Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.28% (study acceptance criteria: <3.28%). Therefore, the study fulfilled the validity criteria.

 

The tissue viability after treatment with the test item was 101.18% and, thus, higher than 50%,i.e.according to UN GHS classification the test item is considered as non-irritant to skin (UN GHS: No Category).

 

Conclusion

Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).