Registration Dossier

Administrative data

Description of key information

- Skin irritation/corrosion: not irritating (similar to OECD 404, GLP, Rel. 2)

- Eye irritation: irritant (OECD 438, OECD 492, GLP, Rel. 1)

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:
4 December 1984 - 14 January 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
the grading scale is different from the OECD guideline (Draize); 3-day observation period; the same animals are tested with different test items
GLP compliance:
yes
Specific details on test material used for the study:
Extract of elemi gum with benzyl benzoate, containing 30% benzyl benzoate
Name of test material: Elemi resinoid P
Appearance: thick pale yellow jelly
Storage: ambiant temperature
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: commercial supplier
- Age at study initiation: 9-12 weeks
- Weight at study initiation: 1.62-2.34 kg
- Housing: individually in suspended cages with wire mesh floor
- Diet (e.g. ad libitum): pelleted, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 3-4 days
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Remarks:
3 or 4 days prior to testing the backs and flanks of rabbits are clipped. The hair is given a final clip about 2h prior to treatment
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
0.5 mL
Duration of treatment / exposure:
4 h
Observation period:
72 h
Number of animals:
7
Details on study design:
Each patch consists of a 25x25 mm, 16-ply gauze pad backed by a 20x30 mm strips of thin polythene and stuck to a 25x75 mm strip of zinc oxide plaster.
Up to 6 patch tests are applied on each animal
Rabbits are immobilised in a canvas body sleeve and placed in a quiet place for the 4-h application period, at the end of which the animals are removed from the body sleeves and the corners of each treatment site marked. Patches are then removed and excess substance wiped from the skin with a damp tissue.

Assessment:
Treatment sites are assessed immediately after removal of the patches and 24, 48 and 72 h after treatment.

Reactions are assessed for erythema, edema, cracking and scaling on a 9-point scale ranging from slight (a) to severe (h). Any other features of responses are described and recorded.

-: no reaction
a: marginal (very slight)
b: slight
c: fairly distinct
d: quite distinct
e: becoming well developped
f: well developped
g: becoming severe
h: severe
Irritation parameter:
overall irritation score
Basis:
other: all animals
Time point:
24/48/72 h
Reversibility:
other: reversible
Remarks on result:
probability of weak irritation
Other effects:
No signs of cracking or scaling were observed.

Table 1: Individual scores

Rabbit No

4 hours

24 hours

48 hours

72 hours

 

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

1

-

-

b

a

b

a

-

-

2

b

a

b

a

-

-

a

-

3

b

a

b

a

a

-

a

a

4

b

a

a

-

a

-

-

-

5

-

-

-

-

-

-

-

-

6

b

a

-

-

a

-

-

-

7

a

a

-

-

a

-

-

-

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions, only reversible slight signs of edema and erythema, equivalent to maximum score 1 in Draize grading scale, were observed therefore the substance can be considered as slightly irritant and does not require classification according to Regulation EC N° 1272/2008 (CLP) criteria.
Executive summary:

7 New Zealand White rabbits were applied 0.5 mL of undiluted test item for 4 hours under semi-occlusive conditions. Reactions were recorded immediately, 24, 48 and 72 h after treatment.

Under the experimental conditions, partly reversible slight signs of edema and erythema, equivalent to maximum score 1 in Draize grading scale, were observed therefore the substance can be considered as slightly irritant and does not require classification according to Regulation EC N° 1272/2008 (CLP) criteria.

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:
key study
Study period:
02-10 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 492 without any deviation
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:
adopted 28 July 2015
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: As the test item was a paste considered as a solid, it was administered after being directly applied on a nylon mesh in order to cover the entire surface of the epidermis (corresponding to 50 mg).
Species:
human
Details on test animals or tissues and environmental conditions:
- Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation]
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): 50 mg
- Concentration: Undiluted
Duration of treatment / exposure:
30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)
Duration of post- treatment incubation (in vitro):
- Post-exposure immersion period: 25 minutes at room temperature
- Post-exposure incubation period: 17 hours and 45 minutes at standard culture conditions
Number of animals or in vitro replicates:
Test item, negative and positive controls were applied on duplicate tissues.
Details on study design:
MAIN TEST
- Pre-incubation of the tissues:
On the day of receipt, the 12 tissues in their 24-well shipping container were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 21 hours and 05 minutes at standard culture conditions.
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
- Treatment and post-treatment incubation of the tissues:
As the test item was a paste considered as a solid, it was administered after being directly applied on a nylon mesh in order to cover the entire surface of the epidermis (corresponding to 50 mg). The test item was applied to 2 DPBS pre-treated RhCE (EpiOcularTM tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were used. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 viable RhCE tissue replicates during 6 hours at standard culture conditions.
After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS. The rinsed tissues were checked for any colouration and noted for comparable colour with the negative control. This rinsing step was followed by a 25-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue. The RhCE constructs were then incubated for a 17-hour and 45-minute post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.
- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD was proportional to the number of living cells.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 17 hours and 05 minutes at 6±3°C in the dark. The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2). The OD measurement of formazan extracts at 570 nm was measured in triplicate samples using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
Irritation parameter:
other: % mean viability of the tissues
Run / experiment:
Main test
Value:
29.43
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 29.43% versus 9.92% in the positive control (Methyl acetate). Refer Tables 7.3.2/1 for more details.

Table 7.3.2/1: Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

Tissue

OD

Mean OD/disc (#)

Mean OD/product

Viability %

Mean viability %

Difference of viability %

Negative control

1

0.780

0.799

0.726

110.13

100.00

20.26

0.797

0.821

2

0.652

0.652

89.87

0.657

0.648

Positive control

1

0.076

0.074

0.072

10.20

9.92

0.55

0.074

0.072

2

0.070

0.070

9.65

0.071

0.071

Test item

1

0.195

0.169

0.214

23.29

29.43

12.27

0.159

0.155

2

0.265

0.258

35.56

0.259

0.252

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Interpretation of results:
other: Category 2 (irritating to eyes) or Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance was identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).
Executive summary:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

As the test item was a paste considered as a solid, it was administered after being directly applied on a nylon mesh in order to cover the entire surface of the epidermis (corresponding to 50 mg). The test substance was applied to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with PBS at room temperature, a 25-minute post-exposure immersion period at room temperature and a 17-hour and 45-minute post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay.

The mean percent tissue viability of the RhCE replicates treated with the test substance was 29.43% versus 9.92% in the positive control (Methyl acetate).

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance was identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2017- February 2018
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)
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay.
- Characteristics of donor animals (e.g. age, sex, weight): The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
- Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 4 October 2017 at 8:25 am.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were enucleated at Phycher on 4 October 2017 at 9:55 am.
- Indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: None
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): 380 mg
- Concentration (if solution): Test item was used as supplied
Duration of treatment / exposure:
Test item was applied for 10 seconds to the cornea
Number of animals or in vitro replicates:
1, 3 and 3 eyes for negative & positive control and test item, respectively.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
- The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus were at a controlled temperature between 31.4 °C and 32.3 °C.
- After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.
- Once all eyes had been examined and approved, the eyes were incubated between 45 and 57 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES
- 1, 3 and 3 eyes for negative & positive control and test item, respectively.

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: 5% Benzalkonium chloride

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, the eye (in its holder) was removed from the superfusion apparatus, placed in a horizontal position, and 380 mg of the test item was applied, as supplied, to the cornea for 10 seconds such that the entire surface of the cornea was evenly covered with the test item.

REMOVAL OF TEST SUBSTANCE
- After exposure, test item was rinsed from the eye with 20 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
- Treated corneas were evaluated before the pre-treatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

METHODS FOR MEASURED ENDPOINTS:
- All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm.
- The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which was determined only at pretreatment and 30 minutes after exposure to the test item) were determined at each of the above time points.

SCORING SYSTEM:
- Mean corneal swelling (%): Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope.
Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100
The mean percentage of corneal swelling for all tested eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for the test item.
- Mean maximum opacity score: Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all tested eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item.
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris clearly visible
2: Easily discernible translucent area; 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 tested eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.
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:
- Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for the test item.
- Once each endpoint was evaluated, ICE classes were assigned based on a predetermined range. Interpretation of corneal thickness, opacity, and fluorescein retention using four ICE classes was done according to the table 7.3.2/1, 7.3.2/2, 7.3.2/3.
Irritation parameter:
cornea opacity score
Run / experiment:
maximal mean score
Value:
0.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
mean score
Value:
1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
maximal mean score
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OCULAR REACTIONS:
- maximal mean score of corneal opacity: 0.2, corresponding to ICE class I;
- mean score of fluorescein retention: 1.3, corresponding to ICE class II;
- maximal mean corneal swelling: 2%, corresponding to ICE class I.
The combination of the three endpoints for test item was 1 x II, 2 x I.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.
- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.
Interpretation of results:
GHS criteria not met
Conclusions:
In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to category “no category”, as defined by OECD guideline No.438.
Executive summary:

An ex vivo eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

 

Test item was applied, as supplied, at the dose of 380 mg, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 0.2 , corresponding to ICE class I;

- mean score of fluorescein retention: 1.3, corresponding to ICE class II;

- maximal mean corneal swelling: 2%, corresponding to ICE class I. 

The combination of the three endpoints for test item was1 x II, 2x I.

The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to category “no category", as defined by OECD guideline No.438. Therefore, test item does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category)

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

7 New Zealand White rabbits were applied 0.5 mL of undiluted test item for 4 hours under semi-occlusive conditions. Reactions were recorded immediately, 24, 48 and 72 h after treatment.

Under the experimental conditions, partly reversible slight signs of edema and erythema, equivalent to maximum score 1 in Draize grading scale, were observed therefore the substance can be considered as slightly irritant and does not require classification according to Regulation EC N° 1272/2008 (CLP) criteria.

Eye irritation:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

As the test item was a paste considered as a solid, it was administered after being directly applied on a nylon mesh in order to cover the entire surface of the epidermis (corresponding to 50 mg). The test substance was applied to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 6 hours at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with PBS at room temperature, a 25-minute post-exposure immersion period at room temperature and a 17-hour and 45-minute post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay.

The mean percent tissue viability of the RhCE replicates treated with the test substance was 29.43% versus 9.92% in the positive control (Methyl acetate).

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test substance was identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Eye corrosion

An ex vivo eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

 

Test item was applied, as supplied, at the dose of 380 mg, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 0.2 , corresponding to ICE class I;

- mean score of fluorescein retention: 1.3, corresponding to ICE class II;

- maximal mean corneal swelling: 2%, corresponding to ICE class I. 

The combination of the three endpoints for test item was1 x II, 2x I.

The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to category “no category", as defined by OECD guideline No.438. Therefore, test item does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category)

Justification for classification or non-classification

Self - classification :

According to the results of OECD TG 438 and OECD 492 , the registered substance should be classified as eye irritant according to Regulation EC N° 1272/2008 (CLP) and GHS.

According to the results of an in vivo study conducted similarly to OECD guideline 404, the registered substance should not be classified for skin irritation according to Regulation EC No 1272/2008 (CLP) and GHS.