Registration Dossier

Administrative data

Description of key information

Skin corrosion

- Key study: According to OECD 430 and EU Method B.40. GLP study. The test item is not skin corrosive when it is tested in skin discs of Wistar rats. The mean TER results for the skin discs treated with the test item were equal to 19.57 kΩ (animal no. 1) and 17.78 kΩ (animal no.2).

Skin irritation

- Key study: According to OECD 439 and EU Method B.46. GLP study. The relative mean tissue viability (% negative control) after 15 min of exposure and 42 h post-incubation was > 50% (101.2%). The test item is therefore considered as non-irritant to the skin.

Eye irritation

- Weight of evidence: OECD 438 and EU Method B. 48. GLP study. The combinations of the 3 endpoints for the test item were 2 x II, 1 x I. Therefore, no prediction can be made.

- Weight of evidence: According to OECD 405 and EU Method B.5. GLP study. The corneal opacity in rabbits was less than 1, the iritis less than 1, the conjunctival redness less than 2 and the conjunctival oedema (chemosis) less than 2, calculated as the mean scores following grading at 24, 48 and 72 hours after application of the test material and which fully reverses within an observation period of 7 days. Therefore, the test item is not irritating to eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24 February 2016 to 24 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Purity: 92%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature and humidity avoided
Test system:
isolated skin discs
Source species:
rat
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
Wistar
Details on animal used as source of test system:
SOURCE ANIMAL
- Source: Centre for Experimental Medicine at the Medical University in Katowice
- Sex: females
- Age at study initiation (in days): 21 days old.
- Housing: plastic cage (58 x 37 x 21 cm) covered with a wire bar lid.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 3 days
Justification for test system used:
A transcutaneous electrical resistance test (TER) is an important step when trying to predict and evaluate toxic properties of a given test item. It is performed in order to obtain information on risks resulting from skin contact with this test item, whereas the obtained results may be used to classify it. The in vitro skin corrosivity study was performed in order to obtain information on health hazards resulting from skin contact with the test item. Corrosive materials are identified on the basis of their ability to produce a loss of normal stratum corneum integrity and barrier function, which is measured as a reduction in the TER below a threshold level for this method (5 kΩ).
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Procedure used: The animals were euthanized and the dorso-lateral skin of each animal was removed and stripped of excess subcutaneous fat. The skin discs were cut out using a scalpel. Each skin disc was placed over one of the ends of a PTFE (polytetrafluoroethylene) tube. The skin disc were fully submerged in a MgSO4 solution.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was greater than 10 kΩ

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 21-22°C.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps:1
- Modifications to validated SOP: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: The dye binding procedure was not necessary in this case since all TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 (two animals 3 skin disc per animal)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the mean TER value is less than or equal to 5 kΩ and the skin disk is obviously damaged.
- The test substance is considered to be non-corrosive to skin if the mean TER value obtained for the test substance is greater than 5 kΩ, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 150 μL
- Concentration (if solution): undiluted (100%)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 150 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 150 μL
- Concentration (if solution): 10M hydrochloric acid
Duration of treatment / exposure:
24 hours
Duration of post-treatment incubation (if applicable):
Not applicable
Number of replicates:
3
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
animal 1 skin disc 1
Value:
19.01
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
animal 1 skin disk 2
Value:
19.75
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
animal 1 skin disc 3
Value:
19.96
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
mean animal 1
Value:
19.57
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
animal 2 skin disc 1
Value:
17.31
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
animal 2 skin disc 2
Value:
17.95
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
animal 2 skin disc 3
Value:
18.09
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
mean animal 2
Value:
17.78
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: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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

Before the beginning of the experiment, the electrical resistance of two skin discs was measured for each animal skin (animals no. 1 and 2). In each case, the skin discs gave the resistance values greater than 10 kΩ; therefore, the remainder of the skin discs of the animals could have been used in the experiment.

Results of the control transcutaneous electrical resistance test (TER):

Animal number

Skin disc number

TER value (kQ)

1

1

11.37

2

13.95

2

1

12.36

2

14.07

Experiment: results of the transcutaneous electrical resistance test (TER):

Animal

number

Tested substance

Skin disc number

TER value (kQ)

Mean TER valueSD (kO)

1

Positive control - 10M HCl

1

0.92

0.92 ± 0.01

2

0.91

3

0.92

Negative control - distilled water

1

16.21

16.77±0.49

2

17.13

3

16.98

Test item

1

19.01

19.57±0.50

2

19.75

3

19.96

2

Positive control - 10M HCl

1

0.93

0.92 ± 0.01

2

0.92

3

0.92

Negative control - distilled water

1

16.75

16.77±0.25

2

17.03

3

16.54

Test item

1

17.31

17.78±0.42

2

17.95

3

18.09

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not skin corrosive when it is tested in skin discs of Wistar rats.
Executive summary:

The skin corrosion potential of the test substance was determined in accordance with the OECD nº 430 with GLP. A transcutaneous electrical resistance test (TER) was performed on skin discs of Wistar female rats in order to obtain information on health hazards resulting from skin contact with the test item. At the beginning of the experiment, the animals were 21 days old. In order to control the procedure quality, the electrical resistance of two skin discs obtained from each test animal was measured before the start of the experiment. In each case, the skin disc resistance values were greater than 10 kΩ; therefore, the remainder of the animals’ skin discs could have been used in the experiment. The undiluted test item was uniformly applied to the epidermal surface of the skin disc placed inside a tube. Concurrent positive (10M hydrochloric acid) and negative (distilled water) controls were used. Three skin discs obtained from each animal were used for the test item and three for each control item. The test item and the control items were evenly applied to the discs for 24 hours and kept at 21-22°C. Then, they were removed by washing with a jet of tap water. LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin in kΩby placing the databridge electrodes on either side of the skin disc. After the transcutaneous electrical resistance test (TER), the skin discs were subjected to a gross examination in order to reveal possible damage. The dye binding procedure was not necessary in this case since all TER values for the test item were higher than 5 kΩand there were not any visible changes on the skin discs. The mean TER results for the skin discs treated with the test item were equal to 19.57 kΩ (animal no. 1) and 17.78 kΩ (animal no. 2). They can be accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations of the skin discs treated with the test item did not reveal any pathological changes. On the grounds of the study, it may be stated that the test substance does not lead skin corrosion/severe irritation.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23 December 2015 to 17 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: L’Oréal Standard Operating Procedure: “EpiSkin™ Skin Irritation Test Method“
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM DB-ALM Protocol No. 131 “EpiSkin™ Skin Irritation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch:13 October 2017
- Purity: 92%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability: Instable after repeated contact to air, hydrolizes slowly in contact with humidity
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
not specified
Details on animal used as source of test system:
Not applicable
Justification for test system used:
This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model
- Tissue batch number(s): 16-EKIN-024
- Date of initiation of testing: 13 June 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 +/- 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Washing: the tissues were washed with DPBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter bandwidth: band pass of maximum ± 30 nm
- Linear OD range of spectrophotometer: 570 ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
Relative viability [%] positive control: Mean 11.8, SD 8.3, n=65
SD of viability [%]: mean 8.5, SD 8.2, n=286

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues treated with 10 μL of the test item (KT) and with the negative control (KU) respectively.
- N. of replicates: 3
- Method of calculation used: non-specific reduction of MTT (NSMTT) was calculated relative to the negative control of living tissues (NK) per treatment period according to the following formula:
NSMTT [%] = [(OD KT - OD KU)/OD NK] * 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment, with 3 replicates and 2 OD lectures.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin f the tissue viability after exposure and post-treatment incubation is higher than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 μL
- - Concentration (if solution): 100% (undiluted)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL DPBS
Concentration (if solution): 100% (undiluted)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL SDS
- Concentration (if solution): 5% solution
Duration of treatment / exposure:
15 min +/- 0.5 min
Duration of post-treatment incubation (if applicable):
42 h +/- 1h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
ca. 114.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
ca. 88
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
ca. 100.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
101.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: not observed
- Colour interference with MTT: not observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for blank: mean OD570 nm of the blank is < 0.1
- Acceptance criteria met for negative control: mean absolute OD570 nm of the three negative control tissues is ≥ 0.6 and ≤ 1.5
- Acceptance criteria met for positive control: mean relative tissue viability of the three positive control tissues is ≤ 40%
- Acceptance criteria met for variability between replicate measurements: standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.

Result of the Test Item VL3

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

0.687

0.709

0.848

0.887

0.757

0.770

0.067

0.074

0.067

0.072

0.066

0.066

0.877

0.891

0.682

0.694

0.771

0.794

OD570(Blank- Corrected)

0.646

0.668

0.807

0.846

0.716

0.729

0.026

0.033

0.026

0.031

0.025

0.025

0.836

0.850

0.641

0.653

0.730

0.753

Mean OD570Of The Duplicates (Blank- Corrected)

0.657

0.826

0.722

0.029

0.029

0.025

0.843

0.647

0.741

Total Mean OD570Of

3 Replicate Tissues (Blank-Corrected)

0.735*

0.028

0.744

SD OD570

0.085

0.002

0.098

Relative Tissue Viabilities [%]

89.4

112.4

98.2

4.0

3.9

3.4

114.7

88.0

100.8

Mean Relative Tissue Viability [%]

100.0

3.8**

101.2

SD Tissue Viability [%]***

11.6

0.3

13.4

CV [% Viability]

11.6

8.5

13.2

Test Acceptance Criteria

 

Value

Cut off

pass/fail

Mean OD570nm Blank

0.041

<0.1

pass

Mean Absolute OD570nm NK

0.776

0.6 < NK <1.5

pass

Mean Relative Viability [%] PC

3.8

<40%

pass

Max. SD of%Viability [%]

13.4

<18%

pass

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is no irritant when it is tested on in vitro human skin model.
Executive summary:

The skin irritation potential of the test substance was determined in accordance with the OECD Nº 439 with GLP. This substance was tested on the in vitro EPISKIN-Standard Model™, a reconstituted three-dimensional human epidermis model. Hereby, the test item was applied topically. Pre-experiments were conducted in order to determine interferences on results, such as non-specific reduction of MTT and coloring potential of the test item. After that, the main experiment was conducted with three replicates, a negative control and a positive control. The volume of test item applied was 10μL (undiluted, 26.3μL/cm2). Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls. Under these given conditions the test item showed no irritant effects. The relative mean tissue viability (% negative control) after 15 min of exposure and 42 h post-incubation was > 50% (101.2%). The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 8 April 2016 to 19 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Analytical purity: 92%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature and humidity avoid
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: from the conventional husbandry of the Institute of Occupational Medicine in Łódź
- Age at study initiation:5-month-old
- Weight at study initiation: At the beginning of the experiment, rabbit no. 1 weighed 3.85 kg, rabbit no. 2 weighed 4.72 kg and rabbit no. 3 weighed 4.71 kg.
- Housing: The animals were individually housed in metal cages. The dimensions of the cages were 73 x 70 x 45 cm (length x width x height). To environmental enrichment in each cage were placed wooden blocks for laboratory animals
- Diet (e.g. ad libitum): ad libitum access to the “LSK” standard granulated laboratory fodder (Batch no. 8/15, 1/16 and 2/16) produced by Wytwórnia Koncentratów i Mieszanek Paszowych AGROPOL, Motycz
- Water (e.g. ad libitum): ad libitum drinking tap water
- Acclimation period: The animals were quarantined and observed for 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): air-conditioned rooms, 20 – 23ºC
- Humidity (%): 33 – 53%
- Air changes (per hr): about 13 times/h
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml of the test item
- Concentration (if solution): 100%
Duration of treatment / exposure:
Applied once
Observation period (in vivo):
Detailed clinical observations for changes in the cornea, iris, and conjunctiva were performed 1, 24, 48 and 72 hours and 7 days after the application of the test item
Number of animals or in vitro replicates:
3
Details on study design:
SCORING SYSTEM:

Corneal opacity score:
0- No ulceration or opacity
1- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible
2- Easily discernible translucent area, details of iris slightly obscured
3- Nacreous area, no details of iris visible, size of pupil barely discernible
4- Opaque cornea, iris not discernible through the opacity

Iris score
0-Normal
1-Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia or injection, iris reactive to light (a sluggish reaction is considered to be an effect)
2-Hemorrhage, gross destruction, or no reaction to light

Conjuntive redness score
0-Normal
1-Some blood vessels hyperaemic (injected)
2-Diffuse, crimson colour, individual vessels not easily discernible
3-Diffuse beefy red

Chemosis score (swelling)
0-Normal
1-Some swelling above normal
2-Obvious swelling, with partial eversion of lids
3-Swelling, with lids about half closed
4-Swelling, with lids more than half closed

Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 1
Reversibility:
fully reversible within: 7d
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 1.3
Max. score:
2
Reversibility:
fully reversible within: 7d
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 1.3
Max. score:
2
Reversibility:
fully reversible within: 7d
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 0.3
Max. score:
1
Reversibility:
fully reversible within: 7d
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 0.7
Max. score:
1
Reversibility:
fully reversible within: 7d
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 1
Max. score:
2
Reversibility:
fully reversible within: 7d
Irritant / corrosive response data:
No irritant

Table 1: Evaluation of the animals' ocular lesions

Animal number

 

Part of the eye

 

Readings after

 

Average after 24, 48, and 72 hours

 

1 hour

 

24 hours

 

48 hours

 

72 hours

 

7 days

 

 

 

 

1

Cornea

 

0

0

0

0

0

0.0

Iris

 

0

0

0

0

0

0.0

Conjunctiva

erythema

 

2

1

1

1

0

1.0

swelling

 

2

1

0

0

0

0.3

 

 

 

2

Cornea

 

 

0

0

0

0

0

0.0

Iris

 

 

0

0

0

0

0

0.0

Conjunctiva

erythema

 

2

2

1

1

0

1.3

swelling

 

2

1

1

0

0

0.7

 

 

 

3

Cornea

 

 

0

0

0

0

0

0.0

Iris

 

 

0

0

0

0

0

0.0

Conjunctiva

erythema

 

2

2

1

1

0

1.3

swelling

 

2

2

1

0

0

1.0

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not classified as irritant to the rabbit eye. After application of the test item, slightly changes in the conjunctiva of three rabbits were observed, but these changes were transient.

Executive summary:

The eye irritation potential of the test substance was determined in accordance with the OECD Nº 405 with GLP. VL3 was tested on three New Zealand female White rabbits (0.1ml/eye). Animals were observed at 24h, 48, 72 and 7 days after the test item was applied. The corneal opacity score, the iris score and the conjunctive score were recorded. No corneal opacity or iris damage were observed but a transient pathological changes in the conjunctiva of three rabbits were observed.. However, this effect was fully reversible within 7 days. The substance is not classified according to CLP Regulation since the corneal opacity was <1, the iritis <1, the conjunctival redness <2 and the conjunctival oedema (chemosis) <2, calculated as the mean scores following grading at 24, 48 and 72 hours after instillation of the test material and which fully reverses within an observation period of 7 days.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 24 February 2016 to 24 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Analytical purity: 92%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature and humidity controlled
- Solubility and stability of the test substance in the solvent/vehicle: The substances hydrolises in contact with water.



Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: licensed slaughterhouse, i.e. Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice
- Characteristics of donor animals (e.g. age, sex, weight): 7 week-old, 1.5-2.5 kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container. All eyeballs used in the test came from the same group of eyes collected on a specific day. Till the moment of their transfer, the eyeballs were kept in special containers at temperature of -18°C.
- Time interval prior to initiating testing: The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 30 minutes.
- indication of any existing defects or lesions in ocular tissue samples: corneal integrity was evaluated with a 2% fluorescein solution (w/v) for a few seconds. After the removal of the dye by rinsing the corneal surface with a physiological salt solution, fluorescein retention and corneal opacity scores were determined using the slit-lamp microscope BP 900 LED (HAAG STREIT) to ensure that the cornea was undamaged. Only eyeballs without damage were analyzed (fluorescein retention and opacity profusion scores fell below 0.5). After the insertion of the eyeballs to the apparatus, another evaluation of corneal opacity and swelling was performed. Eyeballs with the corneal opacity and/or fluorescein retention score higher than 0.5 or some additional signs of damage were replaced. Eyeballs with corneal thickness deviating more than 10% from the mean value for all eyeballs were also replaced.



Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): in a volume of 0.03 mL
- Concentration (if solution): 100%

VEHICLE
- Amount(s) applied (volume or weight with unit): in a volume of 0.03 mL
- Concentration (if solution): 100%
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse
Duration of post- treatment incubation (in vitro):
Quick rinsing with 20 mL of physiological salt at ambient temperature
Number of animals or in vitro replicates:
three eyeballs each group.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Only undamaged eyes were selected. Evaluation was performed with a 2% fluorescein solution (w/v), corneal thickness was also evaluated as mentioned before. The corneal thickness was determined using the depth measuring device no. 1 on the slit-lamp microscope and an SP-100 pachymeter. The slit-width on slit-lamp microscope was set at 0.095 mm. The SP-100 pachymeter measures the period of time from emitting ultrasounds (velocity 1640) to receiving the same ultrasound, reflected by corneal posterior part.

EQUILIBRATION AND BASELINE RECORDINGS
Prior to the application of the test item and the control items, all examined and approved eyeballs were incubated at temperature of 32°C ± 1.5°C for 45-60 minutes in order to equilibrate them to the test system. During the incubation, the eyeballs were continuously supplied with physiological salt at constant temperature of 32 ± 1.5°C and in the average volume of 0.10-0.15 mL/min. After that, a zero reference measurement for corneal thickness and opacity was recorded. It served as a baseline (i.e. time = 0). The fluorescein score determined at dissection served as the baseline measurement for that endpoint.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : physiological salt

POSITIVE CONTROL USED: 10 % acetic acid

APPLICATION DOSE AND EXPOSURE TIME : 0.03 mL, the test item and the control items were uniformly applied to the corneal surface for 10 seconds.

OBSERVATION PERIOD : pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse. The quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: hey were rinsed from the eye with 20 mL of physiological salt at ambient temperature. Next, the eyeballs in their holders were placed in the superfusion apparatus in the original upright position. The additional rinsing was not necessary.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Before the start of the experiment, a control corneal opacity measurement was carried out. Eyeballs with the corneal opacity score higher than 0.5 were replaced. Corneal opacity was determined by assigning appropriate values to opaque areas.

- Damage to epithelium based on fluorescein retention: Before the start of the experiment, a control fluorescein retention measurement was carried out. Eyeballs with fluorescein retention scores higher than 0.5 were replaced. The quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure.

- Swelling: Measured with optical pachymeter on a slit-lamp microscope; slit-width setting: The degree of corneal swelling was determined by measuring corneal thickness using a slit-lamp microscope and an SP-100 pachymeter (TOMEY). Before the start of the experiment, a control corneal swelling measurement was carried out. Eyeballs with corneal thickness deviating more than 10% from the mean value for all eyeballs were replaced.
- Macroscopic morphological damage to the surface: It was evaluated if any morphological effects, e.g. pitting of corneal epithelial cells, roughening of the corneal surface, and sticking of the test item to the cornea were visible.

- Others (e.g, histopathology): Following the final evaluation of the treated eyeballs (240 minutes after the application of the test item and the control items), the eyeballs were fixed in a 4% solution of formaldehyde. Next, specimens were collected (one specimen in the plane including the cornea, lens, and optic nerve). The tissue material was dehydrated and prepared using a paraffin technique. Paraffin blocks were cut into smaller parts, whose thickness was 5 μm, with a microtome and stained using Hematoxylin and Eosin. The following layers of the cornea were evaluated: anterior epithelium, anterior elastic lamina (Bowman’s membrane), corneal stroma, posterior elastic lamina (Descemet’s membrane), and posterior epithelium. All treated eyeballs were subject to this evaluation

SCORING SYSTEM:
- Mean corneal swelling (%) : It was expressed as a percentage and calculated according to the following formula:
corneal swelling = (corneal thickness at time t – corneal thickness at time t = 0)/corneal thickness at time t = 0 x 100
The mean percentage of corneal swelling for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, the final category score for the test item and the control items was given.

- Mean maximum opacity score : The mean corneal opacity value for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, the final category score for the test item and the control items was given.
Score
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris are clearly visible
2: Easily discernible translucent areas; details of the iris are slightly obscured
3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4: Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment : The mean fluorescein retention value for all test eyeballs was calculated on the grounds of the observations made 30 minutes after the end of the exposure. This value was used to determine the general category score for the test item and the control items.
Score
0: No fluorescein retention
0.5: Very minor single cell staining
1: Single cell staining scattered throughout the treated area of the cornea
2: Focal or confluent dense single cell staining
3: Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: Yes, The results from corneal opacity, swelling, and fluorescein retention were evaluated separately in order to generate an ICE class for each endpoint. The ICE classes for each endpoint were then combined in order to generate an Irritancy Classification for the test item and the control items.
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean
Value:
ca. 1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE class II
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
ca. 1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE Class II
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean
Value:
ca. 1.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
ICE class I
Irritation parameter:
morphological effects
Run / experiment:
mean
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Fluorescein retention – results

The mean fluorescein retention values for the concurrent positive and negative controls were 3.0 (ICE class IV) for acetic acid and 0.0 (ICE class I) for physiological salt. These values fell within the acceptable ranges for the method.

Corneal opacity - results

The mean corneal opacity values for the concurrent positive and negative controls were 4.0 (ICE class IV) for acetic acid and 0.0 (ICE class I) for physiological salt. These values fell within the acceptable ranges for the method.

Corneal swelling - results

The mean corneal swelling values for the positive control (10 % acetic acid) were from 10.5 % (ICE class II) to 39.7 % (ICE class IV). As for the concurrent negative control samples (physiological salt), the maximal mean corneal swelling values was equal 3.2 % (ICE class I).These values fell within the acceptable ranges for the method.

Gross evaluation of the treated corneas - results

During the gross examination of the positive control eyeballs, roughening of the corneal surface was observed. The negative control eyeballs and the ones treated with the test item did not exhibit any changes of the corneal surface.

Histopathological evaluation of the treated corneas - results

- Histopathological examination of the negative control corneas revealed: wrinkling of the Bowman’s membrane (eyeballs no. 8, no. 9), dissection of the corneal stroma (eyeball no. 8). All changes which were observed in eyeball no. 8 and 9 are associated with physiological process of replacement of the epithelial cells, and / or changes in ocular pressure during the experiment, and / or technical processing of the material.

- Histopathological examinations of the positive control corneas revealed: slight exfoliation, slight erosions (eyeball no. 4), erosions (eyeball no. 5), coagulation of the superficial layer of the anterior corneal epithelium (eyeballs no. 4, no. 5), karyolysis, karyorrhexis (eyeballs no. 4, no. 5, no.6), karyopyknosis (eyeball no. 5, no. 6), cell vacuolation (eyeball no. 5, no. 6), necrosis, cell swelling (eyeballs no. 4, no. 5, no. 6), vacuolation (eyeballs no. 5, no. 6), exfoliation, and slight erosions of the anterior corneal epithelium (eyeball no. 6), loosening of collagen fibers (eyeballs no. 4, no. 6), dissection of the corneal stroma (eyeball no. 5). These changes confirmed corrosive properties of acetic acid.

- Histopathological examinations of the corneas treated with the test item revealed: wrinkling of the Bowman’s membrane (eyeballs no. 2, no. 3), very slight exfoliation of the superficial layer of the anterior corneal epithelium (eyeball no. 3).

Table 1. Fluorescein retention

observation after

time t

(minutes)

Test item

positive control

10 % acetic acid

negative control

physiological saline

eyeball no.

eyeball no.

eyeball no.

 

1

2

3

4

5

6

7

8

9

0

0

0

0

0

0

0

0

0

0

30

1.0

2.0

1.0

3.0

3.0

3.0

0.0

0.0

0.0

Table 2. Corneal opacity

observation after

time t

(minutes)

Test item

positive control

10 % acetic acid

negative control

physiological saline

eyeball no.

eyeball no.

eyeball no.

 

1

2

3

4

5

6

7

8

9

0

0

0

0

0

0

0

0

0

0

30

1.0

1.0

1.0

4.0

4.0

4.0

0.0

0.0

0.0

75

1.0

1.0

1.0

4.0

4.0

4.0

0.0

0.0

0.0

120

1.0

1.0

1.0

4.0

4.0

4.0

0.0

0.0

0.0

180

1.0

1.0

1.0

4.0

4.0

4.0

0.0

0.0

0.0

240

1.0

1.0

1.0

4.0

4.0

4.0

0.0

0.0

0.0

Table 3. Corneal swelling (%)

observation after

time t

(minutes)

Test item

positive control

10 % acetic acid

negative control

physiological saline

eyeball no.

eyeball no.

eyeball no.

 

1

2

3

4

5

6

7

8

9

0

-

-

-

-

-

-

-

-

-

30

-1.1

-4.5

-5.9

11.2

10.3

10.1

-4.0

0.0

-1.1

75

-2.9

-2.2

-5.4

9.0

15.0

14.8

-3.7

-0.5

0.5

120

-0.5

-2.7

-5.4

12.0

21.7

21.5

-3.9

0.3

0.6

180

1.9

-1.8

-5.2

26.4

33.4

31.3

-3.1

3.7

1.9

240

3.0

3.5

-1.7

31.7

43.8

43.6

0.0

3.9

5.6

“-” = decrease in corneal thickness, no swelling

Table 4. Gross evaluation of the treated corneas

observation after

time t

(minutes)

Test item

positive control

10 % acetic acid

negative control

physiological saline

eyeball no.

eyeball no.

eyeball no.

 

1

2

3

4

5

6

7

8

9

0

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

30

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

75

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

120

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

180

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

240

NC

NC

NC

SIGNS

SIGNS

SIGNS

NC

NC

NC

NC = no changes

SIGNS = roughening of the corneal surface

Table 5. Evaluation of fluorescein retention

observation after

time t

(minutes)

 

Test item

 

positive control

10 % acetic acid

 

negative control

physiological saline

 

average

ICE class

average

ICE class

average

ICE class

30

1.3

II.

3.0

IV

0.0

I

Table 6. Evaluation of corneal opacity

observation after

time t

(minutes)

 

Test item

 

positive control

10 % acetic acid

 

negative control

physiological saline

 

average

ICE class

average

ICE class

average

ICE class

0

1.0

II

4.0

IV

0.0

I

30

1.0

II

4.0

IV

0.0

I

75

1.0

II

4.0

IV

0.0

I

120

1.0

II

4.0

IV

0.0

I

180

1.0

II

4.0

IV

0.0

I

240

1.0

II

4.0

IV

0.0

I

Table 7. Evaluation of corneal swelling (%)

observation after

time t

(minutes)

 

Test item

 

positive control

10 % acetic acid

 

negative control

physiological saline

 

average

ICE class

average

ICE class

average

ICE class

30

-3.9

I

10.5

II

-1.7

I

75

-3.5

I

12.9

III

-1.2

I

120

-2.9

I

18.4

III

-1.0

I

180

-1.7

I

30.4

III

0.9

I

240

1.6

I

39.7

IV

3.2

I

“-” = percentage of corneal thickness decrease, no swelling

See Illustration: Histopathological evaluation of the cornea - test item. H&E, 100x (very slight exfoliation of the superficial layer of the anterior corneal epithelium, wrinkling of the Bowman’s membrane).

Interpretation of results:
study cannot be used for classification
Conclusions:
The combinations of the 3 endpoints for the test item were 2 x II, 1 x I. Therefore, no prediction can be made.
Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs isolated from chickens killed for human consumption were examined and incubated at temperature of 32 ± 1.5°C for 45-60 minutes in order to equilibrate them to the test system. The test item, positive control (10 % acetic acid) and the negative control (physiological salt) were applied in a volume of 0.03 mL during 10 seconds. Three eyeballs were used for each condition. Then, they were rinsed, placed in the superfusion apparatus and evaluated (pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse). At all observation time points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure. Finally, the eyeballs were fixed in a 4% solution of formaldehyde for histopathological examinations. The mean fluorescein retention value for the eyeballs treated with the test item was equal to 1.3 (ICE

class II), the mean corneal opacity was equal 1.0 (ICE class II), and the maximal mean corneal swelling value was equal 1.6 % (ICE class I). These results can be accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations of the eyeballs treated with the test item did not exhibit any changes of the corneal surface. Histopathological examinations of the corneas treated with the test item revealed: wrinkling of the Bowman’s membrane (eyeballs no. 2, no. 3), very slight exfoliation of the superficial layer of the anterior corneal epithelium (eyeball no. 3). According to UN GHS classification criteria "no prediction can be made", since the ICE Class combination of the 3 endpoints were 2 x II and 1 x I.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion:

The skin corrosion potential of the test substance was determined in accordance with the OECD nº 430 with GLP. A transcutaneous electrical resistance test (TER) was performed on skin discs of Wistar female rats. In order to control the procedure quality, the electrical resistance of two skin discs obtained from each test animal was measured before the start of the experiment (they greater than 10 kΩ). The undiluted test item was uniformly applied to the epidermal surface of the skin disc placed inside a tube. Concurrent positive (10M hydrochloric acid) and negative (distilled water) controls were used. Three skin discs obtained from each animal were used for the test item and three for each control item. The test item and the control items were evenly applied to the discs for 24 hours and kept at 21-22°C. Then, they were removed by washing with a jet of tap water. LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin in kΩby placing the databridge electrodes on either side of the skin disc. After the transcutaneous electrical resistance test (TER), the skin discs were subjected to a gross examination in order to reveal possible damage. The dye binding procedure was not necessary in this case since all TER values for the test item were higher than 5 kΩand there were not any visible changes on the skin discs. The mean TER results for the skin discs treated with the test item were equal to 19.57 kΩ (animal no. 1) and 17.78 kΩ (animal no. 2). They can be accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations of the skin discs treated with the test item did not reveal any pathological changes. On the grounds of the study, it may be stated that the test substance does not lead skin corrosion/severe irritation.

Skin irritation:

The skin irritation potential of the test substance was determined in accordance with the OECD Nº 439 with GLP. This substance was tested on the in vitro EPISKIN-Standard Model™, a reconstituted three-dimensional human epidermis model. Hereby, the test item was applied topically. Pre-experiments were conducted in order to determine interferences on results, such as non-specific reduction of MTT and coloring potential of the test item. After that, the main experiment was conducted with three replicates, a negative control and a positive control. The volume of test item applied was 10μL (undiluted, 26.3μL/cm2). Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls. Under these given conditions the test item showed no irritant effects. The relative mean tissue viability (% negative control) after 15 min of exposure and 42 h post-incubation was > 50% (101.2%). The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Eye irritation in vitro:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs isolated from chickens killed for human consumption were examined and incubated at temperature of 32 ± 1.5°C for 45-60 minutes in order to equilibrate them to the test system. The test item, positive control (10 % acetic acid) and the negative control (physiological salt) were applied in a volume of 0.03 mL during 10 seconds. Three eyeballs were used for each condition. Then, they were rinsed, placed in the superfusion apparatus and evaluated (pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse). At all observation time points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure. Finally, the eyeballs were fixed in a 4% solution of formaldehyde for histopathological examinations. The mean fluorescein retention value for the eyeballs treated with the test item was equal to 1.3 (ICE class II), the mean corneal opacity was equal 1.0 (ICE class II), and the maximal mean corneal swelling value was equal 1.6 % (ICE class I). Gross examinations of the eyeballs treated with the test item did not exhibit any changes of the corneal surface. Histopathological examinations of the corneas treated with the test item revealed: wrinkling of the Bowman’s membrane (eyeballs no. 2, no. 3), very slight exfoliation of the superficial layer of the anterior corneal epithelium (eyeball no. 3). According to UN GHS classification criteria "no prediction can be made", since the ICE Class combination of the 3 endpoints were 2 x II and 1 x I.

Eye irritation in vivo:

The eye irritation potential of the test substance was determined in accordance with the OECD Nº 405 with GLP. VL3 was tested on three New Zealand female White rabbits (0.1ml/eye). Animals were observed at 24h, 48, 72 and 7 days after the test item was applied. The corneal opacity score, the iris score and the conjunctive score were recorded. No corneal opacity or iris damage were observed but a transient pathological changes in the conjunctiva of three rabbits were observed.. However, this effect was fully reversible within 7 days. The substance is not classified according to CLP Regulation since the corneal opacity was <1, the iritis <1, the conjunctival redness <2 and the conjunctival oedema (chemosis) <2, calculated as the mean scores following grading at 24, 48 and 72 hours after instillation of the test material and which fully reverses within an observation period of 7 days.

Justification for classification or non-classification

Skin irritation. Based on the available information, mean tissue viability >50% in the OECD 439 test, the substance is non-irritant to the skin according to the CLP Regulation (EC) no. 1272/2008.

Eye irritation: Based on the available information, corneal opacity <1, iritis <1, conjuntival redness <2 and conjuntival oedema <2 in the OECD 405 test, the substance is not irritating to eyes according to CLP Regulation (EC) no. 1272/2008.