Registration Dossier

Administrative data

Description of key information

In an experimental study according to OECD test guideline 439 under GLP conditions, the test substance did not reduce cell viability. Therefore, it is considered to be not skin irritating (reference 7.3.1 -1).

In an experimental study according to OECD test guideline 438 under GLP conditions, no ocular corrosion or severe irritation potential of the test item was observed (reference 7.3.2 -1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-06-29 to 2016-07-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult donors, not further specified
Details on animal used as source of test system:
Not applicable
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM) (from EPISKIN SNC Lyon, France)
- Tissue batch number: 16-EKIN-026
- Production date: not provided
- Expiry date: 04 July 2016
- Certification date: 28 June 2016
- Shipping date: not provided
- Delivery date: not provided
- Date of initiation of testing: 29 June 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C in an incubator with 5 % CO2, ≥95 % humidified atmosphere

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 washing with approximately 25 mL PBS 1x solution
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Qualitiy control was performed at manufacturer for batch and showed historical scoring (22.7 -/+ 0.5) and IC50 (2.6 mg/mL) determinations in the expected ranges (>= 19.5 and >= 1.5- <=3 mg/mL respectively).
- Contamination: Blood and epidermal samples from same donors were free of HIV1+2 antibodies, hepatitis C antibodies, hepatitis B antigen HBs, bactieria, fungus and mycoplasma

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not needed as no colour change was observed after three hours of incubation.

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

PREDICTION MODEL / DECISION CRITERIA
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control. If the viability is > 50 % the test item is non-corrosive.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 mg (to moistened skin)

NEGATIVE CONTROL
- Amount applied: 10 µL
- Concentration: 1x

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % aq.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
104
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
18
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is white and therefore considered to be not able to significantly stain the tissues and lead false estimate of viability. Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method the laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES (mean OD of 0.762 in the range from 0.6 to 1.5; SD of 10.23 therefore < 18)
- Acceptance criteria met for positive control: YES (mean viability of 18 % in the range from 0 to 40 %; SD of 7.55 therefore < 18)
- Acceptance criteria met for variability between replicate measurements: YES (test substance: SD of 14.82 therefore < 18)
- Range of historical values if different from the ones specified in the test guideline: NC: OD mean 0.802 (min 0.555, max. 1.32); PC: OD mean 0.1 (min 0.015, max. 0.299), Viability mean 13 % (min. 2 %, max. 39 %)

Table 1: Results

Substance

 

Optical Density (OD)

Viability (%)

Negative Control: 1x PBS

1

0.672

88

2

0.798

105

3

0.815

107

mean

0.762

100

standard deviation (SD)

10.23

Positive Control: SDS (5 % aq.)

1

0.077

10

2

0.151

20

3

0.190

25

mean

0.139

18

standard deviation (SD)

7.55

Test item

1

0.773

102

2

0.691

91

3

0.914

120

mean

0.793

104

standard deviation (SD)

14.82

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained using the EPISKIN model showed that the test item reveals no skin irritation potential.
Executive summary:

An EpiSkinTM SM test with the test item was performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015.

Triplicates of the human skin model EPISKIN (Reconstructed Human Epidermis, SkinEthic Laboratories) were treated with test item and incubated for 15 minutes at room temperature. Exposure of the reconstructed human epidermis skin units with test item was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5 % aq.) and 1×PBS treated epidermis (three units / positive and negative control) were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean value: 104 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-06-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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)
Version / remarks:
26 July 2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
08 December 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Chicken heads for obtained from TARAVIS KFT, 9600 Sárvár, Rábasömjéni út 129, Hungary
- Age at study initiation: eyes for approx. 2 hours old (after head removal)
- Weight at study initiation: not relevant


SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Number of animals: not specified
- Characteristics of donor animals: not specified
- Storage, temperature and transport conditions of ocular tissue: within 2 hours from collection at ambient temperature of 19.4 ºC to 20.3 ºC, heads were wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box)
- Time interval prior to initiating testing: not specified
- indication of any existing defects or lesions in ocular tissue samples: No. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. Any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced.
- Indication of any antibiotics used: not specified
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.03 g (per eye)

CONTROLS
- Amount applied: 0.03g Imidazole (positive control) or 30 µL NaCl (in saline; negative control) per eye
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
4 hours
Number of animals or in vitro replicates:
3 eyes (test substance and positive control)
1 eye (negative control)
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 5 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm. Any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

OBSERVATION PERIOD
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.
The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.
Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation, but cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse.
The gentle rinsing with 20 mL saline was performed in all (three eyes) test item treated eyes after the 30 and 75 of observation. All test item treated eyes were totally cleared at 120 minutes after the post-treatment rinse.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: was performed
- Damage to epithelium based on fluorescein retention: One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope.
- Swelling: depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm
- Macroscopic morphological damage to the surface: was performed
- Others: no histopathology evaluation was performed

SCORING SYSTEM:
based on the OECD guideline.

DECISION CRITERIA: the decision criteria as indicated in the TG was used.
Irritation parameter:
percent corneal swelling
Run / experiment:
mean up to 75 and 240 min
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0 and 0
Positive controls validity:
valid
Remarks:
23 and 26
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
0.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.0
Positive controls validity:
valid
Remarks:
4.0
Irritation parameter:
fluorescein leakage
Run / experiment:
mean
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.0
Positive controls validity:
valid
Remarks:
3.0
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: -

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not applicable

Table 1: Results for the test item

 Observation

  Value

  ICE Class

 Mean maximum corneal swelling at up to 75 min

0%

I

 Mean maximum corneal swelling at up to 240 min

0%

I

 Mean maximum corneal opacity

0.2

I

  Mean fluoresin retention

0.5

I

 Other Observations

None    

Overall ICE class

3xI

 

Table 2: Results for positive control Imidazole

 Observation

  Value

  ICE Class

 Mean maximum corneal swelling at up to 75 min

23%

III

 Mean maximum corneal swelling at up to 240 min

26%

III

 Mean maximum corneal opacity

4.0

IV

  Mean fluoresin retention

3.0

IV

 Other Observations

Cornea opacity score 4 was observed in three eyes at 75 minutes after the post-treatment rinse.

Overall ICE class

1xIII, 2xIV

The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1. 

 

Table 3: Results for the negative control NaCl (9 g/L saline)

 Observation

  Value

  ICE Class

 Mean maximum corneal swelling at up to 75 min

0% 

 Mean maximum corneal swelling at up to 240 min

 0%

 Mean maximum corneal opacity

 0.0

I

  Mean fluoresin retention

 0.0

 I

 Other Observations

None    

Overall ICE class

3xI  

 

Based on the overall ICE Class the negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

Positive and negative control values were within the corresponding historical control data ranges.

 

Table 4: Historical control data of Positive control (Period of 2011 - 2015)

IMIDAZOLE HISTORICAL CONTROL Dose level: 30 mg / eye

n=168

Relative
obobservation time
(m(min)

Corneal thickness

Corneal opacity score

Fluorescein retention

30

75

120

180

240

 

 30

75

120

180

240

 

 ∆FR

Maximium

swelling (%):

34

45

49

54

55

Max. OS:

4.0

4.0

4.0

4.0

4.0

Max. FR:

3.0

Minimum swelling (%):

3

9

12

14

15

Min. OS:

2.8

3.3

3.5

3.5

3.5

Min. FR:

2.7

Average:

19

27

31

35

37

Average:

3.7

3.9

3.9

3.9

3.9

Average:

3.0

 

Table 5: Historical control data of Negative control (Period of 2011 - 2015)

NaCl (9 g/L saline) SOLUTION HISTORICAL CONTROL Dose level: 30 µL / eye

n=120

Relative
obobservation time
(m (min)

Corneal thickness

Corneal opacity score

Fluorescein retention

30

75

120

180

240

 

30

75

120

180

240

 

 ∆FR

Maximum

swelling (%):

3

3

3

4

3

Max. OS:

0.5

0.5

0.5

0.5

0.5

Max. FR:

0.5

Minimum swelling (%):

0

0

0

0

0

Min. OS:

0

0

0

0

0

Min. FR:

0.0

Average:

0.1

0.3

0.3

0.3

0.3

Average:

0.0

0.0

0.0

0.0

0.0

Average:

0.0

Remark:

n = number of examined eyes

∆FR = Difference between fluorescein retention and fluorescein retention reference value

OS = Opacity score

Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed.
Executive summary:

An Isolated Chicken Eye Test (ICET) according to OECD 438 was used to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes and rinsed after 10 seconds with saline. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

The Imidazole (positive control) was ground before use in the study. The test item and positive control were applied in an amount of 30 mg/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study.

One negative control eye was treated with 30 μL saline solution.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 3xI.

Positive and negative controls showed the expected results. The experiment was considered to be valid.

In this in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 3xI.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

An EpiSkinTM SM test with the test item was performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015.

Triplicates of the human skin model EPISKIN (Reconstructed Human Epidermis, SkinEthic Laboratories) were treated with test item and incubated for 15 minutes at room temperature. Exposure of the reconstructed human epidermis skin units with test item was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5 % aq.) and 1×PBS treated epidermis (three units / positive and negative control) were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean value: 104 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions.

Eye irritation

An Isolated Chicken Eye Test (ICET) according to OECD 438 was used to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes and rinsed after 10 seconds with saline. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

The Imidazole (positive control) was ground before use in the study. The test item and positive control were applied in an amount of 30 mg/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study.

One negative control eye was treated with 30 μL saline solution.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 3xI.

Positive and negative controls showed the expected results. The experiment was considered to be valid.

In this in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 3xI.

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. Based on available data on skin irritation, the test item does not require classification for skin irritation or corrosion according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on the potential to cause damage to the eye, the test item does not require classification for eye irritation or damage according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.