4-Butoxy-2,3-difluorophenylboronic Acid (contains varying amounts of Anhydride)

Endpoint summary

Description of key information

For the assessment of irritation / corrosion in vitro methods have been applied. The following results have been obtained:

Skin irritation

OECD 439: Category 2 or 1

OECD 431: not corrosive

Based on the weight of evidence approach the test item is considered to be irritating to skin (Category 2).

Eye irritation

OECD 492: Category 2 or 1

OECD 437: Causes serious eye damage (UN GHS: Category 1).

The test item causes serious eye damage and must be classified as UN GHS Category 1.

Respiratory tract irritation

There are currently no validated tests on respiratory tract irritation, however, it is a reasonable precaution to assume that corrosive (and severely irritating) substances would also cause respiratory tract irritation. Since the substance induced serious eye damage, it is assumed as a precautionary measure that the test item cause also respiratory tract irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 21, 2018 - July 31, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006. B.40.bis. In vitro skin corrosion: human skin model test

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 29, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol SkinEthic Skin Corrosivity Test, April 2012.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: 18-RHE-073
- Expires: July 9, 2018

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
At the end of the exposure periods, the test item, positive and negative control was removed immediately by gently rinsing with a minimum volume of 20 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg ± 3 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): 40 µL ± 3 µl (deionised water )
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL ± 3 µl
- Concentration (if solution): An 8N potassium hydroxide solution dissolved deionised water pure was used as positive control.

Duration of treatment / exposure:

3 min & 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1 (3 min)

Value:

101.6

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:

other: Non-corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1 (1 hour)

Value:

116.3

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:

other: Non-corrosive

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

Acceptance Criterion Result
Negative control OD ≥ 0.8 and ≤ 3.0 1.572 to 1.765

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

Acceptance Criterion Result
Mean OD negative control ≥ 0.8 and ≤ 3.0 1.742 (3 min)
1.637 (1 hour)

Mean viability positive control < 15 % after 1-hour exposure 0.7 %

Range between identically treated < 30 % 8.4 % (3 min)
tissues with test item 0.7 % (1 hour)

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

Acceptance Criterion Result
Mean OD negative control ≥ 1.596 (3 min) 1.742 (3 min)
≥ 1.467 (1 hour) 1.637 (1 hour)

Mean viability positive control ≤ 0.94 % 0.7 %

Negative Control, Positive Control and Test Substance Data Acceptance Criteria stated by the Testing Laboratory:

Group Acceptance Criterion Result
Range between identically Negative control < 30 % 2.6 % (3 min)
treated tissues 8.2 % (1 hour)
Positive control < 30 % 16.7 % (1 hour)
Test substance < 30 % 8.4 % (3 min)
0.7 % (1 hour)


The study met all acceptance criteria.

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	3	1.742	100.0
Negative Control	60	1.637	100.0
Positive Control	60	0.011	0.7
Test Material	3	1.770	101.6
Test Material	60	1.904	116.3

Interpretation of results:

GHS criteria not met

Remarks:

Non-corrosive

Conclusions:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 431. Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 431. Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 18, 2018 - May 14, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No. 640/2012 amending, for the purpose of its adaption to technical progress, Regulations (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No pre-treatment; The test item was applied neat to the tissues.

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:

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 ECV AM in 2008.

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: 18-RHE-044
- Expires: April 30, 2018

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.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL: 16 mg ± 2 mg
NEGATIVE CONTROL: 16 µL (Dulbecco`s Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1

Value:

2.8

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:

positive indication of irritation

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

Acceptance Criterion Result
Negative control OD ≥ 0.8 and ≤ 3.0 1.638 to 1.726

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

Acceptance Criterion Result
Mean OD negative control ≥ 1.2 1.672
Mean viability positive control < 40 % 1.4 %
SD of group-mean value ≤ 18 % 7.1 % (positive control)
2.8 % (negative control)

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

Acceptance Criterion Result
Mean OD negative control ≥ 1.455 1.672
Mean viability positive control ≤ 2.97 % 1.4 %

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18 % 14.3 %

The study met all acceptance criteria.

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	1.672	100
Positive Control	42	0.023	1.4
Test Material	42	0.047	2.8

Interpretation of results:

other: UN GHS: Category 2 or Category 1

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 considered to possess an irritant potential to skin (UN GHS: Category 2 or Category 1).

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. 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 human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (+/- 1 minute). 10 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 application of 16 mg of 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. All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5 % aqueous solution of sodium dodecyl sulfate) were met. Following treatment with the test item, the tissue viability was 2.8 % and, thus, lower than 50 %, i.e. according to OECD 439 the test item is considered as irritant to skin (UN GHS: Category 2 or Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jul 09, 2018 - Oct 19, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaption to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

PREPARATION OF THE TEST MATERIAL
The test item was prepared as a 20 % (w/v) suspension in a 0.9 % sodium chloride solution. The stability in the vehicle was not investigated. The test item preparation was administered within <1 hour after preparation.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL: 750 µL (i.e. 150 mg/750 µL)

NEGATIVE / VEHICLE CONTROL: 750 µL

Designation: 0.9 % sodium chloride solution
Supplier: B. Braun Melsungen AG, Germany
Batch: 17361013
Storage: 2 to 8 °C
Released until: August 2020


POSITIVE CONTROL: 750 µL

Designation: Art. 814223
Synonym: Imidazole
Supplier: Merck KGaA, Germany
Batch: S5798623
Purity (GC) 99.8 % (a/a)
Storage: At room temperature
Released until: April 30, 2023

Imidazole was dissolved with 0.9 % sodium chloride solution to a concentration of 20 % (w/v).

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

in vitro: triplicate design

Irritation parameter:

in vitro irritation score

Run / experiment:

Run 1 / Experiment 1

Value:

72.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 0.2 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 – 3.1).
- Acceptance criteria met for positive control: After treatment with the positive control (20 % Imidazole) the calculated IVIS was 103.8 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS: 83 – 131.8).

Therefore, the study fulfilled the acceptance criteria.

 

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	Standard deviation
Negative control	0.9 % sodium chloride solution	-0.5	0.002	-0.470	0.5	0.9
		0.7	0.004	0.760
		1.2	0.003	1.245
Positive control	Imidazole (20 %)	62.6	2.272	96.680	99.6	4.5
		59.2	2.536	97.240
		67.0	2.516	104.740
Test item		72.9	0.072	73.980	72.8	2.4
		69.5	0.043	70.145
		74.0	0.028	74.420

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under the conditions of this study, the test item did show an eye hazard potential. The test item is inducing serious eye damage (UN GHS: Category 1).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apr 05, 2018 - Jun 04, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular™ EIT,

Version / remarks:

September 14, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; For use with MatTek Corporation`s Reconstructed Human EpiOcular Model; MatTek Corporation

Version / remarks:

June 29, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No pre-treatment, the test item was applied neat to the tissues.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL: 50 mg per tissue

NEGATIVE / VEHICLE CONTROL: 50 µL per tissue

Sterile deionized water was used as negative control.


POSITIVE CONTROL: 50 µL per tissue

Designation: Methyl acetate
Supplier: MatTek In Vitro Life Science Laboratories
Lot-No.: 022118ISA
Catalog #: TC-MA
Purity (GC): 99.7 %
Appearance: Colorless liquid
Expiration date: February 21, 2019
Storage: 15 to 30 °C

Duration of treatment / exposure:

6 hours

Number of animals or in vitro replicates:

in vitro: duplicate design

Irritation parameter:

other: Viability %

Run / experiment:

Run 1 / Experiment 1

Value:

1.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

According to OECD 492 no prediction can be made regarding the eye irritating potential.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No observations

ACCEPTANCE OF RESULTS:
1. The negative control OD is > 0.8 and < 2.5 (1.671 and 1.474).
2. The mean relative viability of the positive control is below 50 % of the negative control viability (40.0 %).
3. The difference of viability between the two relating tissues of a single chemical is < 20 % (values between 1.1 % to 12.5 %) in the same run (for positive and negative control tissues and tissues of single chemicals).

The study met all acceptance criteria.

	Mean OD	Mean Viability
Negative Control	2.034	100.0 %
Positive Control	0.603	29.6 %
Test Item	0.033	1.6 %

Interpretation of results:

other: UN GHS Category 1 or Category 2

Conclusions:

Under the conditions of the present study, the test item did show an eye hazard potential. The test item is considered to be UN GHS Category 1 or Category 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the provided information the test item must be classified for skin irritation Category 2 and for eye irritation Category 1 according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.