3-Fluorphenylboric acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 Oct 2016 - 2 Dec 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:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

476-720-8

EC Name:

-

Cas Number:

768-35-4

Molecular formula:

C6 H6 B F O2

IUPAC Name:

(3-fluorophenyl)boronic acid

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

SkinEthic™ RHE-model RHE/S/17
- Batch no.: 16-RHE-114

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with minimum 25 mL DPBS; excess DPBS removed by shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Modifications to validated SOP: none

DYE BINDING METHOD
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin category 2 if the viability is less than or equal to 50 %.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Application volume: 16 ± 2 mg

NEGATIVE CONTROL
- Application volume: 16 ± 0.5 µL

POSITIVE CONTROL
- Application volume: 16 ± 0.5 µL

Duration of treatment / exposure:

42 minutes (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

The test item as well as the positive and negative control were tested in batch-triplicates.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 1

Value:

1.45

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 2

Value:

2.01

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 3

Value:

1.69

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

1.72

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The pre-test for direct MTT-reducing capacity of the test item did not result in blue colour, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. After treatment with the negative control (DPBS-buffer) the mean OD was 1.954 (standard deviation: 1.37 %) and, thus, higher than the historically established threshold of 1.431.
- Acceptance criteria met for positive control: Yes. After treatment with the positive control (5 % aqueous solution of sodium dodecyl sulfate) the mean viability value was 1.48 % (standard deviation: 2.30 %) and, thus, lower than the historically established threshold of 3.14 %.
- Acceptance criteria met for variability between replicate measurements: The standard deviation between the three tissues replicates treated with the test item was 16.45 % and, thus. <18 %. The standard deviations between the three tissue replicates of the negative control and the positive control were 1.37 % and 2.30 %, respectively, and, thus, <18 %.
- Range of historical values if different from the ones specified in the test guideline: The negative control OD values were 1.925, 1.978 and 1.959 and, thus, in the range of >0.8 and <3.0.

Any other information on results incl. tables

Table 1 Optical density and Tissue viability

Group	Tissue 1		Tissue 2		Tissue 3		Mean		SD
	OD	viability	OD	viability	OD	viability	OD	viability	viability
Negative Control	1.925	98.52%	1.978	101.22%	1.959	100.26%	1.954	100.0%	1.37%
Positive Control	0.029	1.48%	0.028	1.45%	0.030	1.52%	0.029	1.48%	2.30%
Test item	0.028	1.45%	0.039	2.01%	0.033	1.69%	0.034	1.72%	16.45%

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of the present study, the test item is considered to possess an irritant potential to skin.

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model.

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the 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). The test item, negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. After treatment with the negative control (DPBS-buffer) the mean OD was 1.954 (study acceptance criterion: > 1.433). Treatment with the positive control (5 % aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.48 % (study acceptance criterion: <3.14 %). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was lower than 50 %, i.e. according to OECD 439 the test item is considered as irritant to skin (Category 2).