Registration Dossier

Administrative data

Description of key information

Skin irritation:

Based on the results of the OECD 404 study, the test item is considered to be not irritating to skin (reference 7.3.1 -1).

Eye irritation:

Based on the results of an in vitro eye irritation assay according to OECD 437, the test item is not considered to have eye damaging potential (UN GHS: Category 1). No prediction can be made for eye damaging potential UN GHS Category 2 or no classification for eye damaging potential (reference 7.3.2-1).

Based on the results of an in vitro eye irritation assay according to OECD 492, the test substance is considered to possess eye damaging 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 vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 September 2008 - 20 October 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
2002
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TETRABBIT Kft. 2173 Kartal, Csaszar ut 135, HUNGARY
- Age at study initiation: 11 weeks
- Weight at study initiation: 3376-3404 g
- Housing: single, in metal cages
- Diet: P. Strengthened Female Hare (Herba) diet ad libitum
- Water: tap water from watering bottles ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
- From: 30 September 2008
- To: 14 October 2008
Type of coverage:
occlusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5 g
Duration of treatment / exposure:
initial test: 3 minutes, 1 hour and 4 hours
confirmatory test: 4 hours
Observation period:
In the initial test, the degree of irritation was evaluated at 3 minutes, 1 and 4 hours after the treatment, then at 1, 24, 48, 72 hours and one and two weeks following the patch removal.
In the confirmatory test, the degree of irritation was evaluated at 1, 24, 48 and 72 hours following the patch removal.
Number of animals:
3, males only
Details on study design:
TEST SITE
- Area of exposure: approximately 6 cm2 area of intact skin
- Type of wrap: plastic wrap

REMOVAL OF TEST SUBSTANCE
- Washing: After each treatment period (initial test: 3 min., 1 hour and 4 hours, confirmatory test: 4 hours) the rest of the test item was removed using body temperature water.

SCORING SYSTEM: Draize, 1959
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
6320/M
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
2549/M
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
6325/M
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
6320/M
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
2549/M
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
6325/M
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
In the initial test any irritant response was found in animal No.: 6320/M after 3 minutes exposition. Very slight erythema (score 1) was observed in this animal after 1 hour exposition, as well as well defined erythema (score 2) occurred in same animal after 4 hours exposition. Since corrosive effect was not observed in the initial test, two additional animals were treated with one patch, for an exposure period of four hours.
In the confirmatory test, one hour after the patch removal well defined erythema (score 2) was observed in animal No.: 6320/M. In the others (No.: 2549/M, 6325/M) very slight erythema (score 1) appeared.
24 hours after the patch removal all animals became free of symptom.
48 hours after the patch removal all animals were symptom-free.
72 hours after the treatment the confirmatory study was terminated, since there were no primary irritation symptoms. The observation of the first animal continued for two weeks.
1 week after the patch removal the animal of initial test was symptom-free.
2 weeks after the patch removal the initial test was terminated, since there were no any primary irritation symptoms.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item is considered to be not irritating to skin under the test conditions chosen.
Executive summary:

The acute skin irritation study of the test item was performed in New Zealand White rabbits. The irritation effects of the test item were evaluated according to the Draize method (OECD No.: 404, 2002).

The study consisted of initial and confirmatory test. The initial test was needed, because the substance was suspected to have corrosion potential on the basis of pH value (1.76). In initial test, three patches were applied sequentially to the hairless skin of one experimental animal. The dose was 0.5 g per patches.

In confirmatory test, the test item was administered in pure state, in a single dose of 0.5 g to the hairless skin of all experimental rabbits. The untreated skin of each animal served as control.

After each patch removal, the rest of the test item was removed with water of body temperature.

The irritation symptoms were examined at 1, 24, 48 and 72 hours then one and two weeks after the patch removal in first animal and at 1, 24, 48 and 72 hours after the patch removal in second and third animals. For the initial test, in the first animal the test site was also examined after the treatment (3 min., 1 and 4 hours exposition time).

In the initial test very slight erythema was observed in first animal after 1 hour exposition. Well defined erythema appeared in same animal after 4 hours exposition. Since corrosive effect was not found in the initial test, two additional animals were treated with one patch, for an exposure period of four hours.

One hour after the patch removal, very slight erythema occurred in two animals. Well defined erythema was observed in first animal.

24 hours after the patch removal all animals were symptom-free.

72 hours after the treatment the confirmatory study was terminated, since there were no primary irritation symptoms. The observation of the first animal continued for two weeks.

2 weeks after the patch removal the initial test was terminated, since there were no any primary irritation symptoms.

The animals' individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema and oedema were 0.00, 0.00 and 0.00 respectively.

During the study the behaviour and general state of animals were normal.

There were no notable body weight changes during the contact and observation period.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10 July 2017 - 16 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to
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. certificate)
Species:
other: isolated bovine cornea
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Characteristics of donor animals: 11 - 135 months old
- 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 vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL of the dissolved test item (i.e. 0.15 g/0.75 mL 0.9% sodium chloride solution)

VEHICLE
- Amount applied: 0.75 mL
- Concentration: 0.9%

Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3 corneas per group
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. 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, 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. 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
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. At the end of the incubation period, the basal 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.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: No, vehicle control used

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL of the dissolved test item (i.e. 0.15 g/0.75 mL 0.9% sodium chloride solution) were applied for 240 min

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE:
After the incubation period the corneal surface was washed three times with washing medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The light transmission through the corneas was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany).
- Corneal permeability: The amount of fluorescein that crossed the cornea was measured spectrophotometrically with a microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)

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 according, 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: 80.7 - 132.7). 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.).
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:
48.5
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
replicate 2
Run / experiment:
1
Value:
46.2
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
replicate 3
Run / experiment:
1
Value:
47.2
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes
Interpretation of results:
other: not Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Based on the results of the OECD 437 study, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (no Category).
Executive summary:

The objective of the present study was to examine the potential of the test item to induce serious eye damage in the BCOP assay. 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) solution 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 dissolved 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 0.4 (study acceptance criteria range: -1.4 - 3.2). Treatment with the positive control (20% Imidazole) revealed an IVIS of 120.4 (study acceptance criteria range: 80.7 - 132.7). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 47.3 and, thus, higher than 3 and lower than 55. Therefore, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (no Category).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
8 August 2017 - 2 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: reconstructed human cornea-like epithelium
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.

Characterisation of the test system:
- Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
- Lot No.: 27002
- Keratinocyte strain: 4F1188
- Supplier: MatTek In Vitro Life Science Laboratories
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Volume applied: 50 mg/tissue
Duration of treatment / exposure:
6 hours (± 15 minutes)
Duration of post- treatment incubation (in vitro):
25 minutes (± 2 minutes) at room temperature and 18 hours (± 15 minutes) at 37°C
Number of animals or in vitro replicates:
The test item as well as the positive and negative control were tested in batch-duplicates.
Details on study design:
- Details of the test procedure used:

Preparation:
On day of receipt, the tissues were equilibrated in their 24-well shipping container to room temperature for about 15 minutes. Afterwards the tissues were removed from the shipping container using sterile forceps and transferred to 6-well plates containing 1 mL pre-warmed (37°C) assay medium. Any agarose adhering to the inserts was removed by gentle blotting on gauze or paper towel. Afterwards, the tissues were incubated at 37°C and 5% CO2 overnight (16-24 hours) without medium exchange.

Pre-Treatment:
After the overnight incubation, the tissues were pre-wetted with 20 µL DPBS and incubated at 37°C and 5% CO2 for 30 minutes (± 2 minutes).

Exposure and Post-Treatment:
After the 30 minute DPBS pre-treatment, the negative and the positive control were tested by applying 50 µL topically on the EpiOcularTM tissues. The solid test item was tested by evenly applying 50 mg topically on the EpiOcularTM tissues. The tissues were placed back into the culture medium after dosing and incubated at 37°C and 5% CO2 for 6 hours (± 15 minutes).
At the end of the 6 hours treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (room temperature) DPBS. Three clean beakers, containing a minimum of 100 mL each of DPBS were used per group. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the two tissues per group were rinsed simultaneously by holding the replicate inserts together by their collars using forceps. The test item or control articles were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed at least two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS at least three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material.
After rinsing, the tissues were immediately transferred in 5 mL of pre-warmed (room temperature) assay medium in a 12-well plate for 25 minutes (± 2 minutes) at room temperature.
After the 25 minutes incubation, each insert was removed from the assay medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material and transferred in 6-well plates filled with 1 mL of pre-warmed (37°C) assay medium for 18 hours (± 15 minutes) at 37°C and 5% CO2.

- RhCE tissue construct used, including batch number:

Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 27002
Keratinocyte strain: 4F1188
Supplier: MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used: test chemical: 50 mg, control substances: 50 µL

- Duration and temperature of exposure, post-exposure: exposure: 6 hours (± 15 minutes) at 37°C; post-exposure: 25 minutes (± 2 minutes) at room temperature and 18 hours (± 15 minutes) at 37°C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No. 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.

- 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.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:

If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category). If the test item-treated tissue viability is <60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).

- Acceptance Criteria:
The results are acceptable if:
1. The negative control OD >0.8 and <2.5,
2. The mean relative viability of the positive control is:
a) 30 minute exposure: below 50% of control viability
b) 6 hour exposure: below 50% of control viability
3. Acceptable variability between tissue replicates: < 20 %
Irritation parameter:
other: tissue viability %
Remarks:
tissue 1
Run / experiment:
1
Value:
4.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
other: tissue viability %
Remarks:
tissue 2
Run / experiment:
1
Value:
5.4
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:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
Interpretation of results:
other: Category 1 (irreversible effects on the eye) or Category 2 (irritating to eyes) 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 identified as potentially requiring classification and labelling according to UN GHS (Category 1 or Category 2).
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcularTM) 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 EpiOcularTM-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 1.185 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 17.3% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the mean tissue viability was 5.1% 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 404 (reference 7.3.1 -1):

The acute skin irritation study of the test item was performed in New Zealand White rabbits. The irritation effects of the test item were evaluated according to the Draize method (OECD No.: 404, 2002).

The study consisted of initial and confirmatory test. The initial test was needed, because the substance was suspected to have corrosion potential on the basis of pH value (1.76). In initial test, three patches were applied sequentially to the hairless skin of one experimental animal. The dose was 0.5 g per patches.

In confirmatory test, the test item was administered in pure state, in a single dose of 0.5 g to the hairless skin of all experimental rabbits. The untreated skin of each animal served as control.

After each patch removal, the rest of the test item was removed with water of body temperature.

The irritation symptoms were examined at 1, 24, 48 and 72 hours then one and two weeks after the patch removal in first animal and at 1, 24, 48 and 72 hours after the patch removal in second and third animals. For the initial test, in the first animal the test site was also examined after the treatment (3 min., 1 and 4 hours exposition time).

In the initial test very slight erythema was observed in first animal after 1 hour exposition. Well defined erythema appeared in same animal after 4 hours exposition. Since corrosive effect was not found in the initial test, two additional animals were treated with one patch, for an exposure period of four hours.

One hour after the patch removal, very slight erythema occurred in two animals. Well defined erythema was observed in first animal.

24 hours after the patch removal all animals were symptom-free.

72 hours after the treatment the confirmatory study was terminated, since there were no primary irritation symptoms. The observation of the first animal continued for two weeks.

2 weeks after the patch removal the initial test was terminated, since there were no any primary irritation symptoms.

The animals' individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema and oedema were 0.00, 0.00 and 0.00 respectively.

During the study the behaviour and general state of animals were normal.

There were no notable body weight changes during the contact and observation period.

Eye irritation

OECD 437 (reference 7.3.2 -1):

The potential of the test item to induce serious eye damage was invesitigated in the BCOP assay according to 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) solution 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 dissolved 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 0.4 (study acceptance criteria range: -1.4 - 3.2). Treatment with the positive control (20% Imidazole) revealed an IVIS of 120.4 (study acceptance criteria range: 80.7 - 132.7). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 47.3 and, thus, higher than 3 and lower than 55, i.e. according to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.

OECD 492 (reference 7.3.2 -2):

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcularTM) 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 EpiOcularTM-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 1.185 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 17.3% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 5.1% 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: Since one in vitro test alone can not predict reliably the eye irritating/damaging potential of the test item, an integrated test strategy, consisting of the tests according to OECD Guideline 437 (eye damage, reference 7.3.2-1) and OECD Guideline 492 (eye irritation, 7.3.2-2) was performed. According to the results of the performed tests the test item is considered to be eye irritating (UN GHS Category 2, H319).

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 substance is not considered to be classified for skin irritation and is considered to be classified for eye irritation (Category 2, H319) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.