3-Fluorphenylboric acid

Endpoint summary

Description of key information

Based on the results of an in vitro skin corrosion study according to OECD 431, the test substance showed skin corrosion properties (reference 7.3.1 -1).

Based on the results of an in vitro skin irritation study according to OECD 439, the test substance showed skin irritation properties (reference 7.3.1 -2).

Based on the results of an in vitro eye irritation study according to OECD 437, the test item is considered to have eye damaging potential (reference 7.3.2-1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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 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.

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

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

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

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.

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%

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).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 Dec 2016 - 02 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS. (IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

The SkinEthic(TM) RHE model RHE/S17 was obtained form EpiSkin/SkinEthic Laboratories, Lyon, France

Justification for test system used:

The reconstructed human epidermis model is an accepted in vitro method to replace animal testing. The model closely mimics the biochemical and physiological properties of the upper parts of human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number: 16-RHE-130

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ambient temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing step: using minimum volume of 20 mL DPBS, Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50 %, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Application volume: 20 ± 3 mg per tissue

NEGATIVE CONTROL
- Application volume: 40 ± 3 µL per tissue

POSITIVE CONTROL
- Application volume: 40 ± 3 µL per tissue

Duration of treatment / exposure:

3 minutes or 1 hour

Number of replicates:

The test item as well as the negative control were tested with two replicate tissues per time point (3 min and 1 hour exposure). The positive control was tested with two replicate tissues only for 1 hour.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 1; 3 minutes

Value:

58.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 2; 3 minutes

Value:

57.11

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean; 3 minutes

Value:

57.86

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 1; 1 hour

Value:

4.99

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 2; 1 hour

Value:

6.56

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean; 1 hour

Value:

5.77

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The pre-test for direct MTT reducing capacity did not result in blue color, 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. The negative control OD values were 2.019, 2.059, 1.675 and 1.699 and, thus, in the range of >0.8 and <3.0.
- Acceptance criteria met for positive control: Yes. After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.59 % after 1 hour exposure and, thus, lower than 15 %.
- Acceptance criteria met for variability between replicate measurements: Yes. The range between identically treated tissues was less than 30 % after 3 minutes exposure (2.62 %). The range between identically treated tissues after 1 hour exposure was 31.50 %, but the optical densities measured were <0.3.

Table 1 Optical density and Tissue viability

		Tissue 1		Tissue 2		Mean		CV
Group	time	OD	viability	OD	viability	OD	viability	viability
negative control	3 min	2.019	99.02 %	2.059	100.98 %	2.039	100.00 %	1.39 %
neagtive control	1 h	1.675	99.30 %	1.699	100.70 %	1.687	100.00 %	0.99 %
positive control	1 h	0.009	0.51 %	0.011	0.66 %	0.010	0.59 %	17.90 %
test item	3 min	1.195	58.60 %	1.164	57.11 %	1.180	57.86 %	1.83 %
test item	1 h	0.084	4.99 %	0.111	6.56 %	0.097	5.77 %	19.24 %

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

Under the conditions of the present study, the test item is considered to possess a corrosive potential to skin (combination of optional sub-categories 1B and 1C).

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin corrosion 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 corrosion potential.

Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 20 ± 3 µg of the liquid test item, and 40 +/- 3 µL of the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues.

After treatment with the negative control (deionised water) the mean OD per tissue replicate was 2.019 and 2.039 after 3 minutes exposure and 1.675 and 1.687 after 1 hour exposure (study acceptance criterion: >0.8 and <3.0). Treatment with the positive control (potassium hydroxide, 8N) revealed a mean viability of 0.59 % after 1 hour (study acceptance criterion: <15 %). Therefore, the study fulfilled the validity criteria.

Following treatment with the test item, the tissue viability was >50 % after 3 minutes exposure and <15 % after 1 hour exposure, i.e. according to OECD 431 the test item is considered as corrosive to skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Oct 2016 - 06 Dec 2016

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:

2013

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:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Age at study initiation: 20-43 months
- Corneal diameter: 25 - 27 mm
- Storage, temperature and transport conditions of ocular tissue: cooled on ice
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Volume applied: 150 mg/750 µL
- Concentration: 20 % (w/v) in a 0.9 % sodium chloride solution

VEHICLE
- Volume applied: 750 µL

Duration of treatment / exposure:

240 minutes at 32 +/- 1 °C

Duration of post- treatment incubation (in vitro):

90 minutes (Corneal permeability)

Number of animals or in vitro replicates:

Three corneas were used per group (negative control, positive control and test item group).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9 % sodium chloride solution

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 750 µL of a 20 % solution were applied for 240 minutes

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 times with washing medium

- POST-EXPOSURE INCUBATION: in fresh medium

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: baseline opacity was determined with a calibrated opacitometer, the light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values).
- Corneal permeability: corneas were incubated again in an incubator in a horizontal position at 32 ± 1°C for 90 minutes, amount of fluorescein that crossed the cornea was measured spectrophotometrically 490 nm

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula (referring to OECD Guideline 437) was used to determine the In Vitro Irritancy Score (IVIS) of the negative control:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The following formula was used to determine the In Vitro Irritancy Score (IVIS) of the positive control and the test item:
IVIS = corrected opacity value + (15 x corrected permeability OD490 value)
- The In Vitro Irritancy Score (IVIS) was calculated for each individual treatment and positive control cornea. The mean In Vitro Irritancy Score (IVIS) value of each treated group was calculated from the individual In Vitro Irritancy Score (IVIS) values.

DECISION CRITERIA:
IVIS ≤ 3 No Category (according to GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Serious eye damage, Category 1 (according to GHS)

ACCEPTANCE CRITERIA:
A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean (IVIS positive control: 78.6 - 135.0).
The negative control responses should result in an IVIS that falls within three standard deviations of the current historical mean (IVIS negative control: -1.4 - 3.4).
A single test run with three corneas should be sufficient for a test item when the resulting classification is unequivocal. In cases of the following borderline results in the first testing run, a second test run should be considered.
- 2 of the 3 corneas give discordant predictions from the mean of all 3 corneas or
- 1 of the 3 corneas give discordant predictions from the mean of all 3 corneas, and the discordant result is >10 IVIS units from the cut-off threshold of 55.

Irritation parameter:

in vitro irritation score

Remarks:

replicate 1

Run / experiment:

1

Value:

247.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

replicate 2

Run / experiment:

1

Value:

262.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

replicate 3

Run / experiment:

1

Value:

247.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Remarks:

mean

Run / experiment:

1

Value:

252.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: yes


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, IVIS 1.5
- Acceptance criteria met for positive control: yes, IVIS 105.4
- Range of historical values if different from the ones specified in the test guideline: negative control IVIS: -1.4 - 3.4, positive control IVIS: 78.6 - 135.0

The resulting classification of the test item in this study is unequivocal and no borderline results were obtained. Therefore, a single testing run composed of three corneas per group was considered sufficient.

Table 1 Opacity, permeability and IVIS results

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	Standard deviation
Negative control	0.9% sodium chloride solution	3.0	0.000	3.000	1.5	1.8
		1.9	0.001	1.915
		-0.5	-0.001	-0.515
Positive control	20 % Imidazole in 0.9 % NaCl solution	69.6	2.422	105.930	105.4	8.6
		49.1	3.168	96.620
		72.9	2.720	113.700
Testitem	Test item 20 % in 0.9 % NaCl solution	223.9	1.597	247.855	252.7	8.6
		242.4	1.345	262.575
		220.5	1.810	247.650

Interpretation of results:

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

Conclusions:

Under the conditions of the present study the test item induces serious eye damage.

Executive summary:

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas according to OECD test guideline 437. The corneas were exposed to the test item as a 20 % (w/v) suspension in a 0.9 % sodium chloride solution. As negative control 0.9 % sodium chloride solution and as positive control 20 % (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group).

After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again.

After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 1.5 (study acceptance criteria range: -1.4 - 3.4). Treatment with the positive control (20 % Imidazole) revealed an IVIS of 105.4 (study acceptance criteria range: 78.6 - 135.0). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with the test item was 252.7 and, thus, higher than 55, i.e. according to OECD 437 the test item induces serious eye damage (UN GHS: Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

 

To determine the corrosion or irritation potential of the test item for the skin a weight of evidence approach was used.

 

Studies

OECD 431 (reference 7.3.1 -1)

The objective of the present study was to investigate the potential of the test item to induce skin corrosion 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 corrosion potential.

Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 20 ± 3 µg of the liquid test item, and 40 +/- 3 µL of the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues.

After treatment with the negative control (deionised water) the mean OD per tissue replicate was 2.019 and 2.039 after 3 minutes exposure and 1.675 and 1.687 after 1 hour exposure (study acceptance criterion: >0.8 and <3.0). Treatment with the positive control (potassium hydroxide, 8N) revealed a mean viability of 0.59 % after 1 hour (study acceptance criterion: <15 %). Therefore, the study fulfilled the validity criteria.

Following treatment with the test item, the tissue viability was >50 % after 3 minutes exposure and <15 % after 1 hour exposure, i.e. according to OECD 431 the test item is considered as corrosive to skin.

 

OECD 439 (reference 7.3.1 -2)

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).

 

Conclusion

Using the weight of evidence approach the results of the two in vitro studies on skin corrosion (OEDC 431) and skin irritation (OECD 439) demonstrate that the test item is considered to possess a corrosive potential to skin. The test item requires classification for skin corrosion (UN GHS Category 1, combination of optional sub-categories 1B and 1C).

 

Eye irritation

OECD 437

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas according to OECD test guideline 437. The corneas were exposed to the test item as a 20 % (w/v) suspension in a 0.9 % sodium chloride solution. As negative control 0.9 % sodium chloride solution and as positive control 20 % (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group).

After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again.

After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 1.5 (study acceptance criteria range: -1.4 - 3.4). Treatment with the positive control (20 % Imidazole) revealed an IVIS of 105.4 (study acceptance criteria range: 78.6 - 135.0). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with the test item was 252.7 and, thus, higher than 55, i.e. according to OECD 437 the test item induces serious eye damage (UN GHS: Category 1).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available data for skin irritation/corrosion are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is considered to be classified for skin corrosion (UN GHS: Category 1B/C, H314) under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EC) 2019/521.

The available data for eye irritation are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is considered to be classified for causing serious eye damage (UN GHS: Category 1, H318) under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EC) 2019/521.