Registration Dossier

Administrative data

Description of key information

Skin:

In an in vitro skin irritation assay (EpiSkin SM) according to OECD guideline 439, the test item did not show a skin irritating potential.

Eye:

In an in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model according to OECD guideline 438, the test substance no final conclusion on the eye irritating potential could be drawn.

In an in vitro eye irritation assay (BCOP) according to OECD 437 the test item did not show eye irritating properties.

These two in vitro studies lead to the overall conclusion that the test item has no eye damaging potential (no classification for eye irritation according to UN GHS no Category).

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:
December 12, 2016- March 01, 2017
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)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
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
non-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: EpiSkin SM
- Tissue batch number: 16-EKIN-050

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure: roomt temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time the EpiSkin SM units were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC)
- Wavelength: 570 nm (± 10 nm; Read out range: 0-3.5 Abs)

NUMBER OF REPLICATE TISSUES:
3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues and killed tissues
- Procedure used to prepare the killed tissues: freeting
- No of replicates: 2 replicates color controls and 2 replicates non-specific colour control were used. Furthermore, 3 killed treated tissues and 3 killed negative control tissues were used for the MTT evaluation.
- Method of calculation used:
Data calculation for normal test items
Blank
– The mean of the 6 blank OD values is calculated:
Negative control
– Individual negative control OD values are corrected with the mean blank OD: :
OD Negative Control (ODNC) = ODNCraw – ODblank mean
– The corrected mean OD of the 3 negative control values are calculated: this corresponds to 100% viability
Positive control
– Individual positive control OD values are corrected with the mean blank OD: :
OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The corrected mean OD of the 3 positive control values are calculated
– The % viability for each positive control replicate is calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100
% Positive Control 3 = (ODPC3 / mean ODNC) ×100
– The mean value of the 3 individual viability % for positive control is calculated:
Mean PC % = (%PC1 + %PC2 + %PC3) / 3
Test item
– Individual test item OD values are corrected with the mean blank OD: :
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The corrected mean OD of the 3 test item values are calculated
– The % viability for each test item replicate is calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
– The mean value of the 3 individual viability % for test item is calculated
Mean TT % = (%TT1 + %TT2 + %TT3) / 3

Data calculation for MTT-interacting items
Test items that interfere with MTT can produce non-specific reduction of the MTT. It is necessary to evaluate the OD due to non-specific reduction and to subtract it before calculations of viability %.
– Non-specific MTT reduction calculation (NSMTT):
NSMTT = [(ODKT - ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD (mean)
ODKT: test item treated killed tissues OD (mean)
ODNC: negative control OD (mean)
If NSMTT is > 50% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.
– True MTT metabolic conversion (TODTT) is undertaken if NSMTT is < 50%:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues
– The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / ODNC] × 100
% RV 2= [TODTT2 / ODNC] × 100
% RV 3 = [TODTT3 / ODNC] × 100
– The mean value of the 3 individual relative viability % for test item is calculated
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3

Data calculation for dyes and chemicals able to colour the tissue
For test items detected as able to stain the tissues the non-specific OD is evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability %.
– Non Specific Colour % (NSC %):
NSC % = (mean ODCT / mean ODNC) × 100
ODCT: test item treated tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
– True MTT metabolic conversion (TODTT) is undertaken if NSC % is > 5 % and ≤ 50 %.
TODTT = [ODTV – mean ODCT]
ODTV: test item-treated tissues (incubated with MTT)
ODCT: test item-treated tissues (not incubated with MTT)
– The % relative viability (% RV) for each test substance replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / ODNC] × 100
% RV 2= [TODTT2 / ODNC] × 100
% RV 3 = [TODTT3 / ODNC] × 100
– The mean value of the 3 individual relative viability % for test substance is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3
– If NSC % is ≤ 5 % then the normal calculation mode is used.
– If NSC % is > 50 % relative to the negative control, additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability exposure is or equal to or less than 50 %.
- The test substance is considered to be non-corrosive to skin if the viability after exposure is greater than 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
Positive and negative control
A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.

TEST MATERIAL
- Amount applied: 10 mg

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

POSITIVE CONTROL
- Amount applied: 10 µL, SDS
- Concentration: 5 % aq.
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Duration of post-treatment incubation (if applicable):
42 hours (± 1h)
Number of replicates:
Three replicates were used for the test item and controls, respectively.
Irritation / corrosion parameter:
% tissue viability
Value:
>= 95 - <= 103
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: The non-specific MTT reduction (NSMTT) was determined to be 1.476 %. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.
- Colour interference with MTT: As the test item has an intrinsic colour (bluish black), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.022. The Non Specific Colour % (NSC %) was calculated as 2.1 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

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

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:

 

Table 1: OD values and viability percentages of the controls:

Substance

Optical Density (OD)

Viability (%)

Negative Control:
1x PBS

1

1.135917

108

2

0.909867

87

3

1.096867

105

mean

1.047550

100

standard deviation (SD)

11.53

Positive Control:
SDS (5 % aq.)

1

0.053267

5

2

0.090567

9

3

0.203817

19

mean

0.115883

11

standard deviation (SD)

7.48

 

Table 2: OD values and viability percentages of the test item (including corrected values):

Test Item

Optical Density (OD)

TODTT

Viability (%)

Relative Viability (%)

Leuco Sulfur Blue 13

1

1.015117

0.999650

97

95

2

1.077467

1.062000

103

101

3

1.094567

1.079100

104

103

mean

1.062383

1.046917

101

100

standard deviation (SD)

3.99

3.99

 

Table 3: OD values of additional controls for MTT-interacting test item:

Additional controls

Optical Density (OD)

Negative control killed tissues:
1x PBS

1

0.022267

2

0.026317

3

0.030267

mean

0.026283

Test item treated killed tissues:
Leuco Sulfur Blue 13

1

0.049317

2

0.041667

3

0.034267

mean

0.041750

 

Table 4: OD values and NSC % of additional control:

Additional colour control

Optical Density (OD)

Non Specific Colour %(NSC %)

Leuco Sulfur Blue 13
(test item treated tissueswithout MTT incubation)

1

0.024067

2.1

2

0.020117

mean

0.022092


Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin irritation assay according to OECD 439, the test item did nit show skin-irritating properties and is therefore not classified (UN GHS No Category).
Executive summary:

An in vitro skin irritation assay (EpiSkin SM) has been performed according to OECD 439 to predict the skin irritation potential of the test item.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material 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 (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The test item has an intrinsic colour (bluish black), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

The test item is a possible MTT-reducer and has an intrinsic colour (bluish black). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

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.

In this in vitro skin irritation test using the EPISKIN model, the test item did not significantly reduce cell viability in comparison to the negative control (mean viability: 100 %). All obtained test item viability results were 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.

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. The test is considered to be not irritating to the skin and is therefore not classified (UN GHS No Category).

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:
weight of evidence
Study period:
August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
July, 2013
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were stored in HBSS containing 1 % (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: Yes
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
The test item was tested as a 20 % suspension (w/v) in saline using sonication for 10 minutes. Prior to treatment of the corneae the pH-value of the test item solution or suspension was determined (pH 7.65).

TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: The test item was tested as a 20 % suspension (w/v) in saline using sonication for 10 minutes.

VEHICLE
- Amount applied: 0.75 mL
- Concentration: Saline (0.9 % NaCl in deionised water)
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3 corneae per group (test item, negative control, positive control)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).

QUALITY CHECK OF THE ISOLATED CORNEAS
The basal opacity of all corneae was recorded. Each corneae with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

NUMBER OF REPLICATES
3 corneae per group (test item, negative control, positive control)

SOLVENT CONTROL USED
Saline (0.9 % NaCl in deionised water)

POSITIVE CONTROL USED
10 % (w/v) benzalkonium chloride in 0.9 % (w/v) NaCl (saline)

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL, 240 min

TREATMENT METHOD:
closed chamber

POST-INCUBATION PERIOD:
no

REMOVAL OF TEST SUBSTANCE
the test item or the control items, respectively, were each rinsed off from the according application sides with saline.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: via opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France))
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

DECISION CRITERIA
IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)


Irritation parameter:
in vitro irritation score
Remarks:
mean
Run / experiment:
1-3
Value:
1.7
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Table 1: Results after 240 Minutes Treatment Time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposedin vitroIrritancy Score

Standard Deviation of in vitro Score

 

 

Mean

 

Mean

 

 

 

 

Negative Control

0

0.00

0.054

0.057

0.81

0.86

Not categorized

0.04

0

0.059

0.89

0

0.058

0.87

Positive Control

121.00*

0.019*

121.29

119.53

Category 1

2.91

116.00*

0.011*

116.17

121.00*

0.009*

121.14

Test Item

2.00*

-0.004*

1.94

1.70

Not categorized

0.57

1.00*

0.003*

1.05

2.00*

0.007*

2.11

*corrected values

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro eye irritation assay (BCOP) according to OECD 437, the test item did not show eye irritating properties.
Executive summary:

The eye irritating properties of the test item were assessed in a BCOP assay using fresh bovine corneae, according to OECD guideline 437.

After an initial opacity measurement of the fresh bovine corneae (t0), the 20 % (w/v) suspension in saline of the test item, the positive, and the negative controls were applied to the different corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed off from the corneae and opacity was measured again (t240). After the opacity measurements, permeability of the corneae was determined by measuring the transfer of sodium fluorescein spectrophotometrically after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

For the negative control (saline, 0.9 % NaCl) neither an increase of opacity nor permeability of the corneae was observed (mean IVIS =0.86). The positive control (10 % (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =119.53) corresponding to a classification as serious eye damaging (UNGHS Category 1). All acceptability criteria were fulfilled.

The test item was tested as suspension. Relative to the negative control, the test item did not cause a relevant increase of the corneal opacity. The calculated mean IVIS was 1.70 (threshold for serious eye damage: IVIS > 55). According to the OECD guideline 437, the test item is not categorized (UN GHS No Category).

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

Additional information

Skin:

An in vitro skin irritation assay (EpiSkin SM) has been performed according to OECD 439 to predict the skin irritation potential of the test item.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material 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 (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The test item has an intrinsic colour (bluish black), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

The test item is a possible MTT-reducer and has an intrinsic colour (bluish black). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

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.

In this in vitro skin irritation test using the EPISKIN model, the test item did not significantly reduce cell viability in comparison to the negative control (mean viability: 100 %). All obtained test item viability results were 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.

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. The test is considered to be not irritating to the skin and is therefore not classified (UN GHS No Category).

Eye:

The objective of the present studies was to determine a possible eye irritating/eye damaging potential of the test substance. A single in vitro assay may not always be sufficient, using the currently available methods, to cover the full range of eye irritating potential. Consequently, in accordance with the IATA for the assessment of eye irritation/eye damage two in vitro assays were performed: The isolated chicken eye test (ICE) according to OECD guideline 438 and the bovine corneal opacity/permeability test (BCOP) according to OECD guideline 437.

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes according to OECD guideline 438. The test compound or imidazole (positive control) was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes (triplicates). One negative control eye was treated with 30 µL saline solution (9 g/L saline). After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. Adherence of the test item and the positive control Imidazole was observed on the cornea surfaces at 240 min after the post-treatment rinse.

Tested corneas were evaluated before treatment and 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. Positive and negative controls showed the expected results. The experiment was considered to be valid.

Test item treatment induced a corneal swelling of 6 % (after 75 min) or 7 % (after 240 min), respectively (ICE class II). A cornea opacity score of 1.0 was determined (ICE class II). A fluorescin retention score of 2.0 was determined (ICE class III).

According to the guideline OECD 438, the overall in vitro classification of the test item is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, the test item has been categorized as “No prediction can be made”.

Since the ICE assay did not lead to a final conclusion, the eye irritating potential of the test item was further assessed in a BCOP assay using fresh bovine corneae, according to OECD guideline 437.

After an initial opacity measurement of the fresh bovine corneae (t0), the 20 % (w/v) suspension in saline of the test item, the positive, and the negative controls were applied to the different corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed off from the corneae and opacity was measured again (t240). After the opacity measurements, permeability of the corneae was determined by measuring the transfer of sodium fluorescein spectrophotometrically after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

For the negative control (saline, 0.9 % NaCl) neither an increase of opacity nor permeability of the corneae was observed (mean IVIS =0.86). The positive control (10 % (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 119.53) corresponding to a classification as serious eye damaging (UNGHS Category 1). All acceptability criteria were fulfilled.

The test item was tested as suspension. Relative to the negative control, the test item did not cause a relevant increase of the corneal opacity. The calculated mean IVIS was 1.70 (threshold for serious eye damage: IVIS > 55). According to the OECD guideline 437, the test item is not categorized (UN GHS No Category).

In conclusion, the test item should be calssified as non- irritation (UN GHS No Category) according to the IATA on eye irritation.

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 and eye irritation, the test item is not classified for skin or eye irritation according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelth time in Regulation (EU) No 2019/521.