1,3-bis(hydroxymethyl)urea

Endpoint summary

Description of key information

Skin:

Based on the results of an a skin irritation study according to OECD guideline 439, the test item was determined to be non-irriating to skin (UN GHS: No Category) (reference 7.3.1-1).

Eye:

BCOP:

Based on the results of an a skin irritation study according to OECD guideline 437, the test item should not be considered for classification as UN GHS Category 1 (severely eye damaging) due to the observed IVIS of 6.2 according to UN GHS classification (reference 7.3.2-1). No prediction could be made for eye irritating potential UN GHS Category 2 or no classification for eye damaging potential.

RhCE:

In an in vitro skin irritation test (RhCE) according to OECD Guideline 492, the test item showed an eye irritating potential (UN GHS Category 1 or 2) (reference 7.3.2-2)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March 2017 to 31 March 2017

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:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number: 17-RHE-038

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: min. 25 mL DPBS, no number provided for washing steps.
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: no

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

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: positive control: mean: 1.45 ± 0.53 %

NUMBER OF REPLICATE TISSUES: 3

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

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg ± 2 mg per tissue

NEGATIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue

POSITIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue
- Concentration: 5 %

Duration of treatment / exposure:

42 minutes (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1

Value:

98.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2

Value:

85

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3

Value:

92

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: not specified
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Positive control: 1.45 ± 0.53 % mean viability. The threshold is two standard deviations below the current historical negative control mean (1.459); negative control: 2.025 ± 0.283 mean OD570. The threshold is three standard deviations above the current historical positive control mean (3.03 %).

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin irritation assay (RhE) according to OECD Guideline 439, the test substance is not considered to possess an irritant potential to skin under the conditions of the present study.

Executive summary:

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability in a skin irritation study according to OECD 439. 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. After treatment with the negative the mean OD was 1.778 (study acceptance criterion: > 1.459). Treatment with the positive control revealed a mean viability value of 1.4 % (study acceptance criterion: < 3.03 %). Thus, the acceptance criteria were met. Following treatment with the test item the tissue viability was 92.0 % and, thus, higher than 50 %. Therefore it can be concluded that under the conditions of the present study, the test is not considered to possess an irritant potential to skin (UN GHS: No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

31 March 2017 to 28 April 2017

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:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Number of animals: not specified, nine corneas
- Characteristics of donor animals: age 15 - 42 months (cornea diameter 25-27 mm)
- Storage, temperature and transport conditions of ocular tissue: The eyes were kept and transported in transport medium cooled on ice.
- Time interval prior to initiating testing: not specified
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin was added for the transport (5 mL/500 mL HBSS).

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied : 150 mg
- Concentration: 20% (w/v), 150 mg/750 µL

VEHICLE
- Amount applied : 750 µL
- Concentration: 0.9 % sodium chloride solution

Duration of treatment / exposure:

240 min

Duration of post- treatment incubation (in vitro):

90 min with fluorescein

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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. Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

SOLVENT/NEGATIVE CONTROL USED: 0.9 % sodium chloride solution

POSITIVE CONTROL USED: 20 % (w/v) Imidazole in 0.9 % sodium chloride solution

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: three times with wash medium, once with incubation medium

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- 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)

DECISION CRITERIA: <= 3: No category, >3 - <= 55: no prediction, > 55: Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Tissue 1

Value:

5.885

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Tissue 2

Value:

9.115

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Tissue 3

Value:

3.55

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: A study with reference substances (Feb. 2017) confirmed the accuracy and reliability of the test method used in the laboratory.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Interpretation of results:

other: Not Category 1 (irreversible effects on the eye) based on GHS

Conclusions:

The test substance should not be considered for classification as Category 1 (severely eye damaging) due to the observed IVIS of 6.2 according to UN GHS classification.

Executive summary:

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 according to OECD 437. 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 the calculated IVIS was 0.9 (study acceptance criteria range: -1.4 - 3.3). Treatment with the positive control revealed an IVIS of 105.5 (study acceptance criteria range: 79.8 - 133.4). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 6.2 and, thus higher than 3 and lower than 55. Therefore according to UN GHS classification the test substance should not be considered for classificaion as UN GHS Category 1 (severely eye damaging).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 May 2017 to 01 August 2017

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:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: see "Any other information on materials and methods"

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used: The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) 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 eye irritation potential.
Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.
- RhCE tissue construct used, including batch number: EpiOcular™ Tissue (OCL-200, OCL-212), Lot No: 23787, Keratinocyte strain: 4F1188, MatTek In Vitro Life Science Laboratories
- Doses of test chemical and control substances used:
test substance 50 mg
pos./neg. Control: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
Exposure: 37 °C and 5 % CO2
Post-exposure: room temperature
Post-exposure incubation: 37 °C and 5 % CO2
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
For correct interpretation of results it is necessary to assess the ability of a test item to directly reduce MTT. For this purpose a 1.0 mg/mL MTT solution (in DMEM) was prepared. 50 mg of the test item were added to 1 mL of the MTT solution in a 6-well plate and the mixture was incubated at 37 ± 1°C and 5 % CO2 and protected from light for 3 hours (± 5 minutes). Sterile deionised water (50 µL) was used as negative control concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT. Colored test items or test items which become colored after application to the tissues may interfere with the quantitative photometric MTT measurement if the colorant binds to the tissue and is extracted together with MTT. Therefore, the test item was checked for its colorant properties. The non-colored test item was tested for its ability to become colorant after contact with water or isopropanol. For this purpose, 50 mg of the test item were added to 1.0 mL of water in a 6-well plate and the mixture was incubated for 1 hour at 37 ± 1 °C and 5 % CO2 (HERACELL C02-Inkubator VA, Kendro Laboratory Products GmbH, Langenselbold, Germany) protected from light. Furthermore, 50 mg were added to 2 mL isopropanol, the same amount as used for MTT extraction, and was incubated in 6-well plates for 2 to 3 hours at room temperature.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan: After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37 °C and 5 % CO2.
The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 mL isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically.
The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate.

Irritation parameter:

other: % - cell viability

Run / experiment:

Tissue 1

Value:

0.025

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: % - cell viability

Run / experiment:

Tissue 2

Value:

0.026

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: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

The pre-test for direct MTT-reducing capacity of the test item 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.

The results obtained after treatment of the reconstructed human cornea-like epithelium (RhCE) model with Art. D4879 (N.N'-Bis(hydroxymethyl)urea) are given in the following table:

Table 1: Cell Viability

Group	Tissue 1		Tissue 2		Mean		SD	Difference between tissue replicates (%)
	OD	Viability (%)	OD	Viability (%)	OD	Viability (%)	Viabiliy
Negative Control	1.415	99.2	1.437	100.8	1.426	100.0	1.13	1.6
Positive Control	0.153	10.7	0.150	10.5	0.152	10.6	0.14	0.2
Test Substance	0.025	1.8	0.026	1.8	0.026	1.8	0.00	0.0

Interpretation of results:

other: Category 1 or 2

Conclusions:

In an in vitro skin irritation test (RhCE) according to OECD Guideline 492, the test substance showed an eye irritating/damaging potential.

Executive summary:

In an in vitro skin irritation test (RhCE) according to OECD Guideline 492, the potential of N,N'-Bis(hydroxymethyl)urea to induce eye irritation in an in vitro human cornea model was determined. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) 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 eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 1.426 (study acceptance criterion: > 0.8 and < 2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 10.6 % (study acceptance criterion: < 50 %). Thus, the acceptance criteria were met.

Following treatment with the test substance, the tissue viability was 1.8 % and, thus, lower than 60 %, i.e. according to OECD 492 the test item is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or Category 2).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

OECD 439

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability in a skin irritation study according to OECD 439 (reference 7.3.1-1). 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. After treatment with the negative the mean OD was 1.778 (study acceptance criterion: > 1.459). Treatment with the positive control revealed a mean viability value of 1.4 % (study acceptance criterion: < 3.03 %). Thus, the acceptance criteria were met. Following treatment with the test item the tissue viability was 92.0 % and, thus, higher than 50 %. Therefore it can be concluded that under the conditions of the present study, the test is not considered to possess an irritant potential to skin (UN GHS: No Category).

Eye irritation

OECD 437

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 according to OECD 437 (reference 7.3.2-1). 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 the calculated IVIS was 0.9 (study acceptance criteria range: -1.4 - 3.3). Treatment with the positive control revealed an IVIS of 105.5 (study acceptance criteria range: 79.8 - 133.4). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 6.2 and, thus higher than 3 and lower than 55. Therefore according to UN GHS classification the test substance should not be considered for classificaion as Category 1 (severely eye damaging).

OECD 492

To assess the potential of the test substance to induce eye irritation, a follow-up test was performed. In an in vitro skin irritation test (RhCE) according to OECD Guideline 492 (reference 7.3.2-2), the potential of N,N'-Bis(hydroxymethyl)urea to induce eye irritation in an in vitro human cornea model was determined. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) 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 eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 1.426 (study acceptance criterion: > 0.8 and < 2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 10.6 % (study acceptance criterion: < 50 %). Thus, the acceptance criteria were met. Following treatment with the test substance, the tissue viability was 1.8 % and, thus, lower than 60 %, i.e. according to OECD 492 the test item is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or Category 2).

Conclusion:

According to the OECD IATA for serious eye damage and eye irritation, a top-down approach for the in vitro assessment of the potential eye hazard of the test substace was performed. The initial test according to OECD 437 (BCOP) allowed no classification as eye damging (not UN GHS Category 1). The following test according to OECD 492 (RhCE) indicated a classification of the test substance (UN GHS Category 1 or 2). Consequently, the test substance should be considered as eye irritating (UN GHS Category 2).

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the test substance is not considered to be classified for skin irritation/corrosion under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776. In contrast, the test substance should be considered as eye irritating (UN GHS Category 2, H319) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.