4-hydroxybenzaldehyde

Endpoint summary

Description of key information

Skin Irritation:

In a study according to OECD TG 431 the test item is not identified to possess a corrosive potential to skin (reference 7.3.1 -1). In a further study according OECD TG 439 the test item is not considered to possess an irritant potential to skin (reference 7.3.1 -2).

Eye Irritation:

In a study according to OECD TG 437 the test item is inducing serious eye damage (reference 7.3.2 -1).

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:

weight of evidence

Study period:

May 2, 2018 - June 28, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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: 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:

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:

The reconstituted human epidermis (RHE)-model is a three-dimensional human skin model and is histologically comparable to the in vivo human tissue. It consists of a main basal, supra basal, spinous and granular layer and a functional stratum corneum. The RHE-model is an accepted in vitro alternative model for skin corrosion testing. It involves topical application of test item to the surface of the epidermis and the subsequent assessment of the effects on cell viability. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin/SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 18-RHE-060

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours ± 15 min
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedenshall, Germany
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 1.2 (CV =7.5%)
- Barrier function: 4.9 h
- Morphology: 5 cell layers, absence of significant histological abnormalities, well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Contamination: no

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%.

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
The negative control OD values for the SkinEthic RHE-model have to be in the range of ≥ 0.8 and ≤ 3.0 at 570 nm as given in OECD Guideline 431.

Acceptance Criteria stated by Episkin/SkinEthic Laboratories:
Negative control acceptance criteria: The negative control data meet the acceptance criteria if the mean OD value of the 2 tissues is≥ 0.8 and ≤ 3.0 at 570 nm.
Positive control acceptance criteria: The positive control data meet the acceptance criteria if it classified as corrosive (% viability < 15% after 1 hour exposure).
Test substance data acceptance criteria: The range between identically treated tissues has to be less than 30%, with the exception of cases with OD ≤ 0.3 and for viabilities out the range 20 – 100%.

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is three standard deviations above the current historical mean (0.94%). The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundary at 570 nm. The boundary is two standard deviations below the current historical mean (3 minutes exposure: 1.596; 1 hour exposure: 1.467).

Negative Control, Positive Control and Test Substance Data Acceptance Criteria stated by the Testing Laboratory:
The range between identically treated tissues has to be less than 30%, with the exception of cases with OD ≤ 0.3 and for viabilities out the range 20 – 100%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg ± 3 mg of solid test material
Before adding 20 ± 3 mg of the test item, 20 ± 2 µL of deionised water was spread to the epidermis surface to improve further contact between the test item and the epidermis.

NEGATIVE CONTROL
- Amount(s) applied: 40 µL ± 3 µL (deionised water )

POSITIVE CONTROL
- Amount(s) applied: 40 µL ± 3 µL
- Concentration: An 8N potassium hydroxide solution dissolved deionised water pure

Duration of treatment / exposure:

3 min & 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / 3 min

Value:

100.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / 1 hour

Value:

23.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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 1.563 to 1.703

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

Acceptance Criterion Result
Mean OD negative control ≥ 0.8 and ≤ 3.0 1.694 (3 min)
1.608 (1 hour)

Mean viability positive control < 15% after 1-hour exposure 0.9%

Range between identically treated < 30% 1.4% (3 min)
tissues with test item 17.6% (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) 1.694 (3 min)
≥ 1.467 (1 hour) 1.608 (1 hour)

Mean viability positive control ≤ 0.94% 0.9%

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% 1.0% (3 min)
treated tissues 5.8% (1 hour)
Positive control < 30% 83.3% (1 hour)
Test substance < 30% 1.4% (3 min)
17.6% (1 hour)

The range between identically treated tissues with the positive control was higher than 30% after 1 hour exposure (83.3%), but the optical densities measured were <0.3.
The study met all acceptance criteria.

Table 1: Results

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	3	1.694	100.0
Negative Control	60	1.608	100.0
Positive Control	60	0.014	0.9
Test Material	3	1.701	100.4
Test Material	60	0.379	23.5

Table 2: Historical Data

Positive Control		Negative Control   3min		Negative Control 1 hours
Mean Viability [%]	0.58	Mean Absorption [OD570]	1.898	Mean Absorption [OD570]	1.963
Standard Deviation [%]	0.12	Standard Deviation	0.151	Standard Deviation	0.248

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 431. Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

Executive summary:

A study according OECD TG 431 was conducted 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. 40 ± 3 µL of either the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. Before application of 20 ± 3 mg of the solid test item, 20 ± 2 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.9% and, thus, lower than the historically established threshold of 0.94%. After treatment with the negative control (deionised water) the mean ODs were 1.694 (3 minutes exposure) and 1.608 (1 hour exposure) and, thus, higher than the historically established thresholds of 1.596 and 1.467, respectively. Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was ≥50% after 3 minutes exposure (mean viability: 100.4%) and ≥15% after 1 hour exposure (mean viability: 23.5%), i.e. according to OECD 431 the test item is not considered as corrosive to skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May 18, 2018 - July 2, 2018

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)

Version / remarks:

July 28, 2015

Deviations:

no

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:

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

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 HCVAM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin/SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 18-RHE-068

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours ± 15 min
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedenshall, Germany
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 1.2 (CV =3.1%)
- Barrier function: 4.5 h
- Morphology: 5 cell layers, absence of significant histological abnormalities, well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
A test item is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is > 50%. A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is < 50%. Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
The negative control OD values for the RHE-model have to be in the range of ≥0.8 and ≤3.0 as given in OECD Guideline 439.

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:
The negative control data meet the acceptance criteria if the mean OD value of the 3 tissues is ≥ 1.2 at 570 nm.The standard deviation value is considered valid if ≤ 18% of group mean-value.
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is < 40%.The standard deviation value is considered valid if ≤ 18% of group mean-value.

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:
The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundaryat 570 nm. The boundary is two standard deviations below the current historical mean (1.450).
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is three standard deviations above the current historical mean (2.87%).

Test Substance Data Acceptance Criteria
The standard deviation of the three tissues treated with the test item should be ≤18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 16 mg ± 2 mg

NEGATIVE CONTROL
- Amount(s) applied: 16 µL

POSITIVE CONTROL
- Amount(s) applied: 16 µL
- Concentration: 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

Value:

97.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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 1.594 to 1.794

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

Acceptance Criterion Result
Mean OD negative control ≥ 1.2 1.665
Mean viability positive control < 40% 1.4%
SD of group-mean value ≤ 18% 7.1% (positive control)
6.7% (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 1.665
Mean viability positive control ≤ 2.97% 1.4%

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18% 6.5%

The study met all acceptance criteria.

Table 1: Results

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	1.665	100
Positive Control	42	0.022	1.4
Test Material	42	1.629	97.8

Table 2: Historical Data

Positive Control		Negative Control
Mean Viability [%]	1.42	Mean Absorption [OD570]	1.987
Standard Deviation [%]	0.48	Standard Deviation	0.268

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.

Executive summary:

A study according OECD TG 439 was performed 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). 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 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 97.8% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

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:

March 28, 2018 - May 24, 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)

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL (150 mg/750 µL)

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

in vitro: triplicate design

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice. The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded. Each cornea was mounted in a cornea holder (CiToxLAB, Veszprém, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
For equilibration, the corneas in the holder were incubated (BSS 160, Grumbach Brutgeräte GmbH, Asslar, Germany) in a vertical position at 32 ± 1°C for about one hour. At the end of the incubation period, the incubation medium was replaced by fresh pre-warmed (32 ± 1°C) incubation medium in both compartments. The baseline opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). 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). Any corneas that showed macroscopic tissue damage (e.g.scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. Three corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control group.

NUMBER OF REPLICATES
Three corneas were used per group (negative control, positive control or test item group). Therefore, a total number of 9 corneas was used in this study.

NEGATIVE CONTROL USED
Negative/Vehicle Control: 0.9% sodium chloride solution, B. Braun Melsungen AG, Batch: 16455011,

POSITIVE CONTROL USED
Imidazole, Merck KGaA, Batch S6746923, Purity (GC): 99.8% (a/a)

APPLICATION DOSE AND EXPOSURE TIME
Fresh incubation medium was filled into the posterior compartment, while the surface of the cornea in the anterior compartment was treated with 750 µL of either the test item preparation, negative or positive control. The negative and positive control preparations were introduced with the closed-chamber method through the dosing holes of the chamber, whereas the test item was applied to the anterior chamber with the open-chamber method. For this, the window locking-ring and the glass window were removed from the anterior chamber prior to treatment. After application, the corneas were incubated in an incubator in a horizontal position at 32 ± 1°C for 240 minutes.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: none

REMOVAL OF TEST SUBSTANCE
After the incubation period, the negative and positive control preparations were removed from the anterior chamber without opening the chamber. The corneal surface was washed three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. Fresh incubation medium was replaced in both compartments prior to reading the opacity value after treatment. To remove the test item from the epithelium, the window locking-ring and the glass window from the anterior chamber were removed. The corneas were gently rinsed with wash medium using a syringe. Before measurement of the opacity value after treatment, fresh incubation medium was replaced in both compartments.

- POST-EXPOSURE INCUBATION: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: An increased permeability of the cornea is indicative for an impairment of the integrity of the corneals’ epithelial cell layers. To determine the permeability of the treated corneas, fresh incubation medium was added to the posterior compartments and 1 mL of a fluorescein solution was administered to the anterior compartments. The corneas were incubated again in an incubator in a horizontal position at 32 ± 1°C for 90 minutes. The amount of fluorescein that crossed the cornea was measured spectrophotometrically in the medium from the posterior chamber.

- Others: Each cornea was observed visually and pertinent observations were recorded (e.g.tissue peeling, residual test chemical, non-uniform opacity patterns).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
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: Decision criteria as indicated in the TG was used.
IVIS ≤ 3 results in no classification, IVIS >55 results in GHS-classification as “inducing serious eye damage, category 1”, IVIS between >3 and ≤55 results in ‘no prediction can be made’

Definition of Study 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: 82.4 – 132.9).
The negative control responses should result in an IVIS that falls within three standard deviations of the current historical mean (IVIS negative control: -1.5 – 3.1). 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

Run / experiment:

Experiment 1

Value:

64

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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: Yes
After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.5 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.5 – 3.1).
- Acceptance criteria met for positive control: Yes
After treatment with the positive control (20% Imidazole) the calculated IVIS was 118.9 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS: 82.4 – 132.9).

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	SD
Negative control	0.9% NaCl Solution	0.6	0.001	0.615	0.5	0.2
		0.3	0.000	0.300
		0.5	-0.001	0.485
Positive control	20% Imidazole solution	83.8	2.301	118.315	118.9	8.8
		56.2	3.619	110.485
		85.4	2.842	128.030
Test item	Test item	75.7	0.012	75.880	64.0	10.4
		54.7	0.106	56.290
		59.8	0.010	59.950

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 did show an eye hazard potential. The test item is inducing serious eye damage.

Executive summary:

A study according OECD TG 437 was conducted to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure 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 0.5 (study acceptance criteria range: -1.5– 3.1). Treatment with the positive control (20% Imidazole) revealed an IVIS of 118.9 (study acceptance criteria range: 82.4 – 132.9). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 64.0 and, thus, higher than 55, i.e.according to OECD 437 the test item is inducing serious eye damage (UN GHS: Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation/corrosion

A study according OECD TG 431 was conducted 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. 40 ± 3 µL of either the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. Before application of 20 ± 3 mg of the solid test item, 20 ± 2 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.9% and, thus, lower than the historically established threshold of 0.94%. After treatment with the negative control (deionised water) the mean ODs were 1.694 (3 minutes exposure) and 1.608 (1 hour exposure) and, thus, higher than the historically established thresholds of 1.596 and 1.467, respectively. Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was ≥50% after 3 minutes exposure (mean viability: 100.4%) and ≥15% after 1 hour exposure (mean viability: 23.5%), i.e. according to OECD 431 the test item is not considered as corrosive to skin.

A study according OECD TG 439 was performed 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). 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 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 97.8% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Eye irritation/corrosion

A study according OECD TG 437 was conducted to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure 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 0.5 (study acceptance criteria range: -1.5– 3.1). Treatment with the positive control (20% Imidazole) revealed an IVIS of 118.9 (study acceptance criteria range: 82.4 – 132.9). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 64.0 and, thus, higher than 55, i.e.according to OECD 437 the test item is inducing serious eye damage (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.

Justification for classification or non-classification

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

The available data for skin and eye irritation are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The substance is considered to be classified for eye irritation Cat.1 (H318) and for STOT SE Cat. 3 (H335) according to the Regulation (EC) No 1272/2008, as amended for fourteenth time in Regulation (EU) No 2020/217. The test item is not considered to be classified for skin irritation/corrosion according to EU Regulation (EC) No 1272/2008.