Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation/corrosion:

In a study according OECD TG 431 using the Epidermal Model - Epiderm™ (EPI-200-SCT) the test item is considered as non-corrosive in accordance with UN GHS, as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control. However, this test system does not allow discrimination between category of skin irritation and no classification according to CLP.

In a study according OECD TG 439, the test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), as the mean percentage tissue viability was less than 50% of the negative control.

Based on this testing strategy using the results obtained in the OECD TG 431 and OECD TG 439 studies, the test item is identified as skin irritant Cat.2.

Eye irritation:

A study according OECD TG 438 using the Isolated Chicken Eye Test (ICET) was performed. Based on the percentage of corneal swelling, corneal opacity score, fluorescein retention score and morphological effects obtained and on the basis of overall combination of ICE categories obtained for all three end points the test item is characterized as “No Category”.

In a study according OECD TG 492 using the EpiOcular™ model, the test item is identified as requiring classification and labeling according to UN GHS (UN GHS Category 1 or Category 2) as the mean percentage tissue viability is less than 60% of the negative control.

Based on this testing strategy using the results obtained in the OECD TG 438 and 492 studies, the test item is identified as eye irritant Cat 2.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
4 April 2018 - 8 April 2018
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:
6th July 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SOP: In Vitro EpiDerm™ Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SIT).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
The Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SIT) was used as test system (MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic).
Justification for test system used:
As recommended in OECD Guideline No. 439, Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SIT) has been selected as test system for in vitro skin irritation. The RhE test system uses human derived non-transformed keratinocytes as cell source to reconstruct an epidermal model with representative histology and cytoarchitecture.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SIT), MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic
- Tissue batch number: 25891
- Date of initiation of testing: 4th April 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 min at 37 °C, 25 min at room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were rinsed with sterile DPBS by filling and emptying the tissue insert for 15 times to remove any residual test item. The constant stream of DPBS was applied from the nearest distance from the tissue surface. After the 15th rinse with washing bottle, the inserts were completely submerged 3 times in approximately 50 mL of DPBS and shaken to remove all traces of test item/NC/PC.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: plate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS
- Viability: 1.577
- Barrier function: ET50= 5.34 hours
- Morphology: proven presence of a functional stratum corneum, a viable basal cell layer and intermediate spinous and granular layers
- Contamination: sterile


NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
None - The test substance did not directly reduce MTT.

DECISION CRITERIA
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) If the tissue viability after exposure and post-treatment incubation is ≤ 50%.
- The test item is considered as non-irritant to skin in accordance with UN GHS No Category, if the tissue viability after exposure and post-treatment incubation is > 50%.

ACCEPTANCE CRITERIA
1. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 0.8 and ≤ 2.8.
2. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues should be ≤ 20%. If the mean viability of PC tissues is not within the ≤ 20% and the SD of the three tissues replicates above 18%, the assay will be repeated.
3. The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is < 18%.
Note: Test items that provide tissue viabilities in a range of 30 to 70% may provide high SD. If the high SD (above acceptance limits) is typical for the test item and the classification of the test item is consistent in all independent runs, it is recommended to accept this result, although the Assay Acceptance Criterion 3 is not met.


Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 mg

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5%
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
39 hours and 45 minutes
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st run
Value:
9.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
3.3%
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: 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

Table 1: Summary of optical density (OD) and viability (%)

Treatment

 

OD

Viability (%)

Classification

Negative Control

(DPBS)

Mean

1.766

100

NI

±SD

0.011

0.62

n

3

3

Positive Control

(5% Sodium Dodecyl Sulphate)

Mean

0.059

3.3

I

±SD

0.005

0.26

n

3

3

Test Item

Mean

0.165

9.4

I

±SD

0.001

0.03

n

3

3

NI = Non Irritant; I = Irritant; n = No. of tissues; SD = Standard Deviation

Interpretation of results:
other: study cannot be used to decide on classification alone, further information necessary
Conclusions:
Based on the results obtained under the laboratory testing conditions, the test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), as the mean percentage tissue viability was less than 50% of the negative control. Since the test guideline cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion will be required to decide on its final classification.
Executive summary:

A study was conducted to assess the skin irritation potential of the test item according to OECD Guideline No. 439 using Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SIT).

Tissues were topically exposed to 30 µL of DPBS (negative control: NC), 30 µL of 5% aq. SDS solution (positive control: PC) or 25 mg of test item. All the treatments were maintained in triplicates. After 60 minutes of exposure the tissues were washed using DPBS. Later, the tissue inserts were blotted and transferred to fresh medium and incubated in CO2 incubator at 37°C for 22 hours and 57 minutes. After incubation period (Day 1), tissue inserts were shifted from upper wells to lower wells of 6-well plates prefilled with 0.9 mL of assay medium. After the media change, tissues were incubated for an additional 16 hours and 48 minutes in CO2 incubator at 37°C. After the post-incubation period, the bottom of the tissue inserts was blotted and transferred into an MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, was extracted for 2 hours using extraction solvent and the OD was measured at 570 nm.

The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

Percentage viability of negative control, positive control and test item was 100±0.62, 3.3±0.26 and 9.4±0.03 respectively. As the percentage viability of the test item was not greater than 50% of the negative control, the test item is considered as “irritant”. The percentage of viability in the positive control (PC) was less than 50%, which shows the irritative potential of the positive control and the suitability of the test method. As the test method does not allow to distinguish between Category 1 and 2, further testing is required to exclude or confirm a corrosive property.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
5 April 2018 - 6 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29th July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142
Version / remarks:
May 31st, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
As recommended in OECD Guideline No. 431, Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SCT) has been selected as test system for in vitro skin corrosion.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SCT), MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05, Bratislava II, Slovak Republic
- Tissue batch number(s): 25892
- Date of initiation of testing: 5 April 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C in humidified incubator

REMOVAL OF TEST MATERIAL AND CONTROLS
- rinsed with sterile PBS (fill and empty insert 20 times in a constant soft stream of 1xPBS) to remove any residual test item/NC/PC.
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: plate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD (540-570nm) = 1.625
- Barrier function: ET-50 = 4.87 hours
- Morphology: presence of functional stratum corneum, viable basal cell layer, intermediate spinous and granular layers
- Contamination: no

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No additional controls used since no interference observed.

DECISION CRITERIA
A test item is considered to be “corrosive” to skin in accordance with UN GHS Category 1, if:
The tissue viability after 3 minutes exposure is <50%.
The tissue viability after 3 minutes exposure is ≥50% and <15% after 1 hour exposure.
The test item is considered as non-corrosive to skin in accordance with UN GHS, if the tissue viability after 3 minutes exposure is ≥50% and ≥15% after 1 hour exposure.
A test item identified as being corrosive is further subcategorized in accordance with UN GHS based on the following:
The tissue viability after 3 minutes exposure is <25%: optional subcategory 1A
The tissue viability after 3 minutes exposure is ≥25 %: a combination of optional Sub-Categories 1B-and-1C

ACCEPTANCE CRITERIA
The assay is accepted if all the following criteria are met:
The mean OD570 of the NC tissue viability is OD ≥ 0.8 and ≤2.8 for every exposure time.
The mean viability of the tissue replicates exposed for 1 hr with the positive control (8N KOH), expressed as % of the negative control should be PC tissues expressed as % of the negative control should be < 15.
In the range 20% - 100% viability the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
Duration of treatment / exposure:
1 hour (1st experiment); 3 min (2nd experiment)
Duration of post-treatment incubation (if applicable):
None
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st experiment (3 minutes exposure)
Value:
85.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
0.74%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2nd experiment (1 hour exposure)
Value:
61.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
5.4%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes
The technical proficiency of the test method was established by using proficiency chemicals according to OECD Test Guideline No. 431.

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

TABLE 1: SUMMARY of Optical Density (OD) and viability (%)

 

3 Minutes Exposure                                                                            

Treatment

 

OD

Viability (%)

CV (%)

Classification

Negative Control

(Sterile distilled water)

Mean

1.512

100.0

1.06

NC

±SD

0.023

2.1

n

2

2

2

Positive Control

(Glacial acetic acid)

Mean

0.103

6.8

0.74

C (Category 1A)

±SD

0.016

1.5

n

2

2

2

Test Item

Mean

1.288

85.2

0.24

NC

±SD

0.005

0.5

n

2

2

2

                                           

 

1 Hour Exposure

Treatment

 

OD

Viability (%)

CV (%)

Classification

Negative Control

(Sterile distilled water)

Mean

1.447

100.00

0.71

NC

±SD

0.015

1.4

n

2

2

2

Positive Control

(Glacial acetic acid)

Mean

0.078

5.4

0.66

C (Category 1A)

±SD

0.013

1.3

n

2

2

2

Test Item

Mean

0.891

61.5

1.43

NC

±SD

0.029

2.9

n

2

2

2

NC = Non Corrosive; C = Corrosive; n = No. of tissues; SD = Standard Deviation,               CV: Co-efficient of variations

Interpretation of results:
other: non-corrosive
Conclusions:
Based on the results obtained under the conditions of this study, the test item is considered as non-corrosive in accordance with UN GHS, as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control. However, this test system does not allow discrimination between category of skin irritation and no classification according to CLP.
Executive summary:

The in vitro skin corrosion potential of the test item was evaluated in a study according OECD TG 431 by measurement of tissue viability on in the Epidermal Model - Epiderm™ (EPI-200-SCT).

The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

After receipt of the tissues, visual inspection was done to verify the defects.There were no tissue defects, air bubble or excess moisture observed. Tissue inserts were transferred to upper row of 6 well plates prefilled with 0.9 mL of assay medium and incubated in CO2 incubator for 60 minutes.

Exposure with the test item was performed for 1 hour or 3 minutes separately. All the treatments were maintained in duplicates. For 3 minutes treatment, 50 µL of sterile distilled water (NC) was dispensed into the first insert atop the tissue. After 60 seconds the procedure was repeated with second tissue and continued for all other tissues. Similar procedure was followed in the same manner until all the tissues were treated. Tissues were treated with 25 mg test item + 25 µL distilled water and 50 µL of positive control (glacial acetic acid).

For 1 hour treatment, 25 mg test item + 25 µL distilled water, 50 µL of negative control or 50 µL of positive control were dispensed directly atop Epiderm™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 1 hour.

At the end of treatment time tissue inserts were rinsed with sterile PBS (fill and empty insert 20 times in a constant soft stream of 1xPBS) to remove any residual test item. Post rinsing procedure, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and incubated for 3 hours at 37±1°C and 5±1% CO2. Post incubation, the tissue inserts were removed and blotted onto the tissue paper and transferred to a prelabeled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plates were placed on an orbital plate shaker and shaken for 5 hours and 18 minutes (for 1 hour exposure) and 4 hours and 26 minutes (for 3 minutes exposure) at room temperature. At the end of the extraction period, the tissue was pierced with an injection needle and the extract was allowed to run into the well from which the insert was taken. The punctured inserts were discarded and solution was placed on mixer for 15 minutes until it became homogenous. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated.

After 3 minutes exposure, the percentage viability of the negative control, positive control and test item was 100±2.1, 6.8±1.5 and 85.2±0.5 respectively. The percentage viability of the test item was thus greater than 50% of the negative control. The percentage viability of the positive control (PC) is less than 50% of the negative control and thus clearly represents the irritation potential of the positive control.

After 1 hour exposure, the percentage viability of negative control, positive control and test item was 100±1.4, 5.4±1.3 and 61.5±2.9 respectively. The percentage viability of the test item was greater than 15% of the negative control. The percentage viability of the positive control (PC) is was less than 50% of negative control and thus clearly represents the irritation potential of the positive control. Positive and negative controls showed the expected results. The experiments were considered to be valid.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10 April 2018 - 10 May 2018
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:
2017
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:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: local slaughterhouse
- Storage, temperature and transport conditions of ocular tissue: The intact heads are transported from the slaughterhouse at ambient temperature (typically between 18°C and 25°C) in plastic boxes humidified with tissues moistened with isotonic saline. The procedure involving the collection of chicken heads and placing the eyes in the superfusion chamber following enucleation will be completed within two hours to minimize deterioration and/or bacterial contamination.
- Time interval prior to initiating testing: maximally 2 hours
- indication of any existing defects or lesions in ocular tissue samples: eyes applied in the test were unremarkable
- Indication of any antibiotics used: no
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 μL
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
30, 75, 120, 180 and 240 min
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES :
Upon receipt of the chicken heads to the laboratory, first the eyelids were carefully excised, taking care not to damage the cornea. Corneal intergrity was asssessed with a drop of 2% (w/v) sodium fluorescein applied to the corneal surface for few seconds, and then rinsed with isotonic saline. Fluorescein-treated eyes were then examined with a slit lamp microscope to ensure that the cornea is undamaged (i.e., fluorescein retention and corneal opacity scores ≤ 0.5).
Only undamaged eyes were 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. All necessary precautions were taken to aviod any corneal damage due to excessive pressure (i.e, compression artifacts). The visible portion of the optic nerve was left attached to the eye when it was removed from the orbit. Immediately after removing the eye from the orbit, the eye was placed on an absorbent pad and the nictitaing membrane and other connective tissue was removed. 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 appratus. The clamps were positioned in the superfusion appratus in such a way that the entire cornea was supplied with the isotonic saline drip (3 to 4 drops per minute or 0.1 to 0.15 mL/min). The temperature of the chambers of the superfusion appratus was maintained at 32 ± 1.5 °C.
After being placed in the superfusion appratus, the eyes were again examined with a slip-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. Out of the eyes that were not rejected based on the beforementioned criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes of this batch were rejected. During corneal thickness measurements, slit width of slit-lamp microscope was set at 0.095 mm.

EQUILIBRATION AND BASELINE RECORDINGS : Immediately after examination and approval of all eyes, they were incubated for approximately 45 - 60 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 baseline (i.e., time=0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED : physilogical saline, Sodium Chloride injection IP, 0.9 % w/v

POSITIVE CONTROL USED : Imidazole (30 mg)

APPLICATION DOSE AND EXPOSURE TIME : 30 μL for 10 seconds

OBSERVATION PERIOD : approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse (+/- 5 min)

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: isotonic saline (approximately 20 mL) at ambient temperature

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: evaluated by using the area of the cornea that was most densely opacified for scoring
- Damage to epithelium based on fluorescein retention: Fluorescein retention was evaluated at the 30 minutes observation time point
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: 0.095 mm
- Macroscopic morphological damage to the surface: none

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment
- morphological effects (e.g., pitting or loosening of the epithelium)

DECISION CRITERIA: as indicated in OECD TG 438
Irritation parameter:
percent corneal swelling
Run / experiment:
1st trial - at up to 75 min
Value:
11.26
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
1 st trial - at up to 240 min
Value:
15.72
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
2nd trial - at up to 75 min
Value:
11.79
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
2nd trial - at up to 240 min
Value:
14.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
1 st trial - at up to 240 min
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
2nd trial - at up to 240 min
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
1 st trial
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
2nd trial
Value:
0
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: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. 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.
- Acceptance criteria met for positive control: yes. Based on the overall ICE Class the positive control Imidazole was classified as corrosive/severely irritating, UN GHS Classification: Category 1.

Table 1: Data of corneal swelling/thickness and isolated chicken eye (ICE) classification

Treatment

Eye No. and Sw%

Corneal Thickness (µm) at t (minutes)

ICE Class

-50

0

30

75

120

180

240

Negative Control

1

291

264

266

267

263

269

264

I

Sw%

NA

NA

0.76

1.14

-0.38

1.89

0.00

Positive control

2

291

269

486

533

493

570

546

IV

Sw%

NA

NA

80.67

98.14

83.27

111.90

102.97

3

267

261

457

477

459

566

523

Sw%

NA

NA

75.10

82.76

75.86

116.86

100.38

4

268

266

474

510

474

559

507

Sw%

NA

NA

78.20

91.73

78.20

110.15

90.60

Mean sw%

±SD

NA

NA

77.99

2.79

90.88

7.73

79.11

3.79

112.97

3.48

97.99

6.53

Test Item

(Trial 1)

5

271

270

296

302

322

320

329

II

 

 

Sw%

NA

NA

9.63

11.85

19.26

18.52

21.85

6

273

271

310

298

304

306

310

Sw%

NA

NA

14.39

9.96

12.18

12.92

14.39

7

280

284

314

318

314

317

315

Sw%

NA

NA

10.56

11.97

10.56

12.92

14.39

 

Mean Sw%

±SD

NA

NA

11.53

 2.52

11.26

1.13

14.00

4.63

14.35

3.67

15.72

5.59

Test Item

(Trial 2)

5

258

273

296

309

316

319

309

II

Sw%

NA

NA

8.42

13.19

15.75

16.85

13.19

6

266

276

302

309

314

318

317

Sw%

NA

NA

9.42

11.96

13.77

15.22

14.86

7

271

274

303

302

307

312

318

Sw%

NA

NA

10.58

10.22

12.04

13.87

16.06

Mean Sw%

±SD

NA

NA

9.48

1.08

11.79

1.49

13.85

1.86

15.31

1.49

14.70

1.44

Sw%: Corneal Swelling percentage, SD: Standard deviation, t: time.

Table 2: Data of Corneal opacity scores and isolated chicken eye (ICE) classification

Treatment

Eye No.

Corneal Opacity Scores at t (Minutes)

ICE Class

-50

0

30

75

120

180

240

Negative Control

1

0

0

0

0

0

0

0

I

Positive Control

2

0

0

3

3

4

4

4

IV

3

0

0

3

3

4

4

4

4

0

0

2

3

4

4

4

Mean

0

0.0

2.7

3.0

4.0

4.0

4.0

Test Item (Trial 1)

5

0

0

0

0

0

0

0

I

6

0

0

0

0

0

0

0

7

0

0

0

0

0

0

0

Mean

0

0.0

0

0.0

0

0.0

0

Test Item (Trial 2)

1

0

0

0

0

0

0

0

I

2

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

Mean

0

0.0

0

0.0

0

0.0

0

Corneal Opacity Scores

Scores

Observation

0

No opacity

0.5

Very faint opacity

1

Scattered or diffuse areas, details of the iris are 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

Table 3: Data of fluorescein retention, morphological effects and isolated chicken eye (ICE) classification

Treatment

Eye No.

Fluorescein retention at t = (Minutes)

Morphological effects

ICE Class

-50

0

30

Negative Control

 1

0

0

0

No morphological effects observed

I

Positive Control

 2

0

0

3

Loosening of epithelium

IV

 3

0

0

2

Loosening of epithelium

4

0

0

3

Loosening of epithelium

Mean

0

0.0

2.7

NA

Test Item (Trial 1)

5

0

0

0

No morphological effects observed

I

6

0

0

0

No morphological effects observed

7

0

0

0

No morphological effects observed

Mean

0

0.0

0.0

NA

Test Item (Trial 2)

1

0

0

0

No morphological effects observed

I

2

0

0

0

No morphological effects observed

3

0

0

0

No morphological effects observed

 

Mean

0

0.0

0.0

NA

Score

Observation

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

 
Interpretation of results:
other: no catagory
Conclusions:
Based on the percentage of corneal swelling, corneal opacity score, fluorescein retention score and morphologial effects obtained under the laboratory testing conditions and on the basis of overall combination of ICE categories obtained for all three end points the test item is characterized as “No Category”.
Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes according to OECD TG 438. The test compound was applied in a single dose (~30 µL /eye) onto the cornea of isolated chicken eyes. The control and test eyes were examined for corneal thickness and corneal opacity at 0, 30, 75, 120, 180 and 240 min after treatment and results were recorded according to a fixed scoring system. Fluorescein retention by damaged epithelial cells was scored at 30 min post-treatment. Additionally, the eyes treated with negative (0.9% NaCl), positive control (30 mg Imidazole) and test item were evaluated for morphological effects. All examinations were carried out with a slit-lamp microscope and Pachymeter. Three test item treated eyes and three positive control eyes and one negative control eye were used in this study.

For the test item the combination of ICE categories obtained for corneal swelling, corneal opacity, and fluorescein retention was 2 x I, 1 x II ( ICE class of I observed for fluorescein retention and opacity and ICE class II observed for corneal swelling).  Positive and negative controls showed the expected results. The experiments were considered to be valid.

Based on the percentage of corneal swelling, corneal opacity score, fluorescein retention score and morphologial effects obtained under the laboratory testing conditions and on the basis of overall combination of ICE categories obtained for all three end points the test item is characterized as “No Category”.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
5 April 2018 - 7 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
09 October, 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model
Version / remarks:
10 February 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. It was concluded that the EpiOcular™ EIT is able to correctly identify substances and mixtures not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg
Duration of treatment / exposure:
5 h and 45 min
Duration of post- treatment incubation (in vitro):
25 min post-soak and 17 hours and 55 min post-incubation
Number of animals or in vitro replicates:
2
Details on study design:
- RhCE tissue construct used, including batch number : EpiOcular™ human cell construct (OCL-200-EIT) procured from MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava - Slovakia, +421-2-3260-7401, www.mattek.com.; Lot no.: 27030

- Doses of test chemical and control substances used
Test item: 50 mg
Neg. Control: 50 µL
Pos. Control: 50 µL

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
Exposure: 5h and 45 min at 37 °C
Post-exposure immersion: 25 min at room temperature
post-exposure incubation: 17 h and 55 min at 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals :
The test item was considered as non MTT reducer hence no further additional controls were maintained.

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength used for quantifying MTT formazan: 570 nm, 96-well plate reader

- Description of the method used to quantify MTT formazan : Optical density (OD) measurement of the MTT extracts.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :
A test item is labeled as “non-irritant”, if the test item treated percent tissue viability determined by MTT assay is >60% relative to the negative control treated tissue viability. A test item is labeled as “irritant”, if the percent tissue viability determined by MTT assay is <60% relative to the negative control treated tissue viability.

- Complete supporting information for the specific RhCE tissue construct used
Lot number: 27030
Tissue viability: OD (540-570)= 1.677
Barrier function: ET-50= 20.96 min
Sterility: sterile

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals :
Prior to routine use the technical proficiency of the test method was established by using proficiency chemicals according to OECD Test Guideline No. 492.

- Acceptande criteria:
The assay meets the acceptance criterion if the mean OD570 of the NC tissues is >0.8 and < 2.5.
The assay meets the acceptance criterion if the mean relative viability of Positive Control tissues is <50% of negative control viability.
The assay meets the acceptance criterion if the difference of viability between the two relating tissues of a single chemical is < 20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.
Irritation parameter:
other: %viability
Run / experiment:
1st run
Value:
2.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
2.5%
Remarks on result:
positive indication of irritation
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, the assay met the acceptance criterion as the mean OD570 of the NC tissues is 1.976 which is in between >0.8 and ≤2.5.
- Acceptance criteria met for positive control: Yes, the assay met the acceptance criterion if the mean relative viability of PC tissues is 2.5% which is below 60% of negative control viability.

Table 1: Summary of Optical density (OD) and  Viability (%)

Treatment

 

OD

Viability (%)

Viability difference between tissues

Classification

Negative Control

(DBPS)

Mean

1.976

100.00

2.16

No Category

±SD

0.085

4.33

n

2

2

2

Positive Control

(Methyl acetate)

Mean

0.049

2.5

0.08

Cat 2 or Cat 1

±SD

0.003

0.15

n

2

2

2

Test Item

(LYAF (Dried)

Mean

0.053

2.7

0.03

Cat 2 or Cat 1

±SD

0.001

0.05

n

2

2

2

n = No. of tissues

Interpretation of results:
other: UN GHS Category 1 or Category 2
Conclusions:
Based on the results obtained and under the laboratory testing conditions using the EpiOcular™ model , the test item is identified as requiring classification and labeling according to UN GHS (UN GHS Category 1 or Category 2) as the mean percentage tissue viability is less than 60% of the negative control.
Executive summary:

The in vitro eye irritation potential of LYAF (Dried) was evaluated using EpiOcular™ model (OCL-200-EIT) according to OECD TG 492.

Tissues were incubated at standard culture conditions for 32 minutes. Afterwards, 50 mg each of test item, 50 µL of negative control and positive control were dispensed directly atop EpiOcular™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 5 hours and 45 min. All the treatments were maintained in duplicates.

At the end of treatment time with test item, negative control and positive control, tissue inserts were dipped into the first tubes of DPBS, swirled in a circular motion in the liquid for 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first tube. The cultures were then rinsed in the second and third tubes of DPBS.

After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed assay medium in a 12-well plate for 25 minutes immersion incubation (Post-Soak) at room temperature which was intended to remove any test item absorbed. At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted onto the absorbent material, and transferred to the appropriate well of a prelabeled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 17 hours and 55 minutes in CO2 incubator at 37±1°C and 5±1% CO2 (Post-treatment Incubation).

Post 17 hours and 55 minutes of incubation with the assay medium, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and incubated for 2 hours and 55 minutes at 37±1°C and 5±1% CO2. Post incubation, the tissue inserts were removed and blotted onto the tissue paper and transferred to a 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface. The plates were placed on an orbital plate shaker and shaken for 2 hours at room temperature. At the end of the extraction period, the tissue was pierced with an injection needle and the liquid within each insert was decanted into the well from which it was taken. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated.

The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. So no additional controls were maintained.

Mean percentage viability of negative control, positive control and test item were 100±4.33, 2.5±0.15 and 2.7±0.05 respectively. As the mean percentage viability of the test item was less than 60% of the negative control it is identified as requiring classification and labeling according to UN GHS (Cat 2 or Cat 1). Similarly the percentage viability of positive control is less than 60% of negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Based on the results obtained and under the laboratory testing conditions using the EpiOcular™ model, the test item is identified as requiring classification and labeling according to UN GHS (UN GHS Category 1 or Category 2) as the mean percentage tissue viability is less than 60% of the negative control.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion:

In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (OECD 439)

A study was conducted to assess the skin irritation potential of the test item according to OECD Guideline No. 439 using Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SIT).

Tissues were topically exposed to 30 µL of DPBS (negative control: NC), 30 µL of 5% aq. SDS solution (positive control: PC) or 25 mg of test item. All the treatments were maintained in triplicates. After 60 minutes of exposure the tissues were washed using DPBS. Later, the tissue inserts were blotted and transferred to fresh medium and incubated in CO2 incubator at 37°C for 22 hours and 57 minutes. After incubation period (Day 1), tissue inserts were shifted from upper wells to lower wells of 6-well plates prefilled with 0.9 mL of assay medium. After the media change, tissues were incubated for an additional 16 hours and 48 minutes in CO2 incubator at 37°C. After the post-incubation period, the bottom of the tissue inserts was blotted and transferred into an MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, was extracted for 2 hours using extraction solvent and the OD was measured at 570 nm.

The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

Percentage viability of negative control, positive control and test item was 100±0.62, 3.3±0.26 and 9.4±0.03 respectively. As the percentage viability of the test item was not greater than 50% of the negative control, the test item is considered as “irritant”. The percentage of viability in the positive control (PC) was less than 50%, which shows the irritative potential of the positive control and the suitability of the test method. As the test method does not allow to distinguish between Category 1 and 2, further testing is required to exclude or confirm a corrosive property.

 

In vitro skin corrosion: reconstructed human epidermis (RHE) test method (OECD 431)

The in vitro skin corrosion potential of the test item was evaluated in a study according OECD TG 431 by measurement of tissue viability in the Epidermal Model - Epiderm™ (EPI-200-SCT).

The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

After receipt of the tissues, visual inspection was done to verify the defects. There were no tissue defects, air bubble or excess moisture observed. Tissue inserts were transferred to upper row of 6 well plates prefilled with 0.9 mL of assay medium and incubated in CO2 incubator for 60 minutes.

Exposure with the test item was performed for 1 hour or 3 minutes separately. All the treatments were maintained in duplicates. For 3 minutes treatment, 50 µL of sterile distilled water (NC) was dispensed into the first insert atop the tissue. After 60 seconds the procedure was repeated with second tissue and continued for all other tissues. Similar procedure was followed in the same manner until all the tissues were treated. Tissues were treated with 25 mg test item + 25 µL distilled water and 50 µL of positive control (glacial acetic acid).

For 1 hour treatment, 25 mg test item + 25 µL distilled water, 50 µL of negative control or 50 µL of positive control were dispensed directly atop Epiderm™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 1 hour.

At the end of treatment time tissue inserts were rinsed with sterile PBS (fill and empty insert 20 times in a constant soft stream of 1xPBS) to remove any residual test item. Post rinsing procedure, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and incubated for 3 hours at 37±1°C and 5±1% CO2. Post incubation, the tissue inserts were removed and blotted onto the tissue paper and transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plates were placed on an orbital plate shaker and shaken for 5 hours and 18 minutes (for 1 hour exposure) and 4 hours and 26 minutes (for 3 minutes exposure) at room temperature. At the end of the extraction period, the tissue was pierced with an injection needle and the extract was allowed to run into the well from which the insert was taken. The punctured inserts were discarded and solution was placed on mixer for 15 minutes until it became homogenous. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated.

After 3 minutes exposure, the percentage viability of the negative control, positive control and test item was 100±2.1, 6.8±1.5 and 85.2±0.5 respectively. The percentage viability of the test item was thus greater than 50% of the negative control. The percentage viability of the positive control (PC) is less than 50% of the negative control and thus clearly represents the irritation potential of the positive control.

After 1 hour exposure, the percentage viability of negative control, positive control and test item was 100±1.4, 5.4±1.3 and 61.5±2.9 respectively. The percentage viability of the test item was greater than 15% of the negative control. The percentage viability of the positive control (PC) is was less than 50% of negative control and thus clearly represents the irritation potential of the positive control. Positive and negative controls showed the expected results. The experiments were considered to be valid.

Conclusion:

In a study according OECD TG 439, the test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), as the mean percentage tissue viability was less than 50% of the negative control.

In a study according OECD TG 431 using the Epidermal Model - Epiderm™ (EPI-200-SCT) the test item is considered as non-corrosive in accordance with UN GHS, as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control. However, this test system does not allow discrimination between category of skin irritation and no classification according to CLP.

Based on this testing strategy using the results obtained in the OECD TG 431 and OECD TG 439 studies, the test item is identified as skin irritant Cat.2.

Eye Irritation:

Reconstructed human Cornea-like Epithelium (RhCE) test method (OECD 492)

The in vitro eye irritation potential of LYAF (Dried) was evaluated using EpiOcular™ model (OCL-200-EIT) according to OECD TG 492.

Tissues were incubated at standard culture conditions for 32 minutes. Afterwards, 50 mg each of test item, 50 µL of negative control and positive control were dispensed directly atop EpiOcular™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 5 hours and 45 min. All the treatments were maintained in duplicates.

At the end of treatment time with test item, negative control and positive control, tissue inserts were dipped into the first tubes of DPBS, swirled in a circular motion in the liquid for 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first tube. The cultures were then rinsed in the second and third tubes of DPBS.

After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed assay medium in a 12-well plate for 25 minutes immersion incubation (Post-Soak) at room temperature which was intended to remove any test item absorbed. At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted onto the absorbent material, and transferred to the appropriate well of a prelabeled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 17 hours and 55 minutes in CO2 incubator at 37±1°C and 5±1% CO2 (Post-treatment Incubation).

Post 17 hours and 55 minutes of incubation with the assay medium, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and incubated for 2 hours and 55 minutes at 37±1°C and 5±1% CO2. Post incubation, the tissue inserts were removed and blotted onto the tissue paper and transferred to a 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface. The plates were placed on an orbital plate shaker and shaken for 2 hours at room temperature. At the end of the extraction period, the tissue was pierced with an injection needle and the liquid within each insert was decanted into the well from which it was taken. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated.

The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. So no additional controls were maintained.

Mean percentage viability of negative control, positive control and test item were 100±4.33, 2.5±0.15 and 2.7±0.05 respectively. As the mean percentage viability of the test item was less than 60% of the negative control it is identified as requiring classification and labeling according to UN GHS (Cat 2 or Cat 1). Similarly the percentage viability of positive control is less than 60% of negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Based on the results obtained and under the laboratory testing conditions using the EpiOcular™ model, the test item is identified as requiring classification and labeling according to UN GHS (UN GHS Category 1 or Category 2) as the mean percentage tissue viability is less than 60% of the negative control.

Isolated Chicken Eye Test (ICET, OECD 438)

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes according to OECD TG 438. The test compound was applied in a single dose (~30 µL /eye) onto the cornea of isolated chicken eyes. The control and test eyes were examined for corneal thickness and corneal opacity at 0, 30, 75, 120, 180 and 240 min after treatment and results were recorded according to a fixed scoring system. Fluorescein retention by damaged epithelial cells was scored at 30 min post-treatment. Additionally, the eyes treated with negative (0.9% NaCl), positive control (30 mg Imidazole) and test item were evaluated for morphological effects. All examinations were carried out with a slit-lamp microscope and Pachymeter. Three test item treated eyes and three positive control eyes and one negative control eye were used in this study.

For the test item the combination of ICE categories obtained for corneal swelling, corneal opacity, and fluorescein retention was 2 x I, 1 x II ( ICE class of I observed for fluorescein retention and opacity and ICE class II observed for corneal swelling).  Positive and negative controls showed the expected results. The experiments were considered to be valid.

Based on the percentage of corneal swelling, corneal opacity score, fluorescein retention score and morphologial effects obtained under the laboratory testing conditions and on the basis of overall combination of ICE categories obtained for all three end points the test item is characterized as “No Category”.

Conclusion:

In a study according OECD TG 492 using the EpiOcular™ model, the test item is identified as requiring classification and labeling according to UN GHS (UN GHS Category 1 or Category 2) as the mean percentage tissue viability is less than 60% of the negative control.

A study according OECD TG 438 using the Isolated Chicken Eye Test (ICET) was performed. Based on the percentage of corneal swelling, corneal opacity score, fluorescein retention score and morphological effects obtained and on the basis of overall combination of ICE categories obtained for all three end points the test item is characterized as “No Category”.

Based on this testing strategy using the results obtained in the OECD TG 438 and 492 studies, the test item is identified as eye irritant Cat 2.

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 corrosive / irritant properties, the test item is classified and labelled as skin irritant category 2 (H315: "Causes skin irritation") and eye irritant category 2 (H319: "Causes serious eye irritation") according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.