Registration Dossier

Administrative data

Description of key information

Skin:

Based on the results of an in vitro skin irritation assay according to OECD 439, the test substance is considered to possess either skin irritating potential or skin corrosive potential (UN GHS Category 1 or 2 ) (reference 7.3.1-1).

Based on the results of an in vitro skin corrosion assay according to OECD 431, the test substance did not show skin corrosion properties (reference 7.3.1-2).



 

Eye:

Based on the results of an in vitro eye irritation assay according to OECD 437, the test item is not considered to have eye damaging potential (UN GHS: Category 1). No prediction can be made for eye damaging potential UN GHS Category 2 or no classification for eye damaging potential (reference 7.3.2-1).

Based on the results of an in vitro eye irritation assay according to OECD 492, the test substance is considered to possess eye damaging potential (UN GHS: Category 1 or 2) (reference 7.3.2-2).

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:
26 September 2016 - 21 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.
Vehicle:
unchanged (no vehicle)
Details on test system:
SkinEthic™ RHE-model RHE/S/17
- Batch no.: 16-RHE-109
- Expiration date: 24 October 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with minimum 25 mL DPBS; excess DPBS removed by shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Observable damage in the tissue due to washing: no data
- Modifications to validated SOP: none

DYE BINDING METHOD
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin category 2 if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.



Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Application volume: 16 ± 5 µL

NEGATIVE CONTROL
- Application volume: 16 ± 5 µL

POSITIVE CONTROL
- Application volume: 16 ± 5 µL
Duration of treatment / exposure:
42 minutes (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
The test item as well as the positive and negative control were tested in batch-triplicates.
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 1
Run / experiment:
1
Value:
45.73
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 2
Run / experiment:
1
Value:
42.07
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 3
Run / experiment:
1
Value:
44.96
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
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

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 Optical density and Tissue viability

Group

Tissue 1

Tissue 2

Tissue 3

Mean

SD

 

 

OD

viability

OD

viability

OD

viability

OD

viability

viability

Negative Control

1.882

104.27%

1.789

99.12%

1.744

96.61%

1.805

100.00%

3.91%

Positive Control

0.036

2.01%

0.032

1.79%

0.033

1.81%

0.034

1.87%

6.57%

Art. W387800

0.825

45.73%

0.759

42.07%

0.850

47.09%

0.812

44.96%

5.78%

Interpretation of results:
other: Category 1 (corrosive) or Category 2 (irritant) based on GHS criteria
Conclusions:
The results obtained from this in vitro skin irritation test indicated that the test item reveals potential for either skin corrosion (UN GHS Cat 1 ) or skin irritation (UN GHS Cat 2).
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the test item, negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. After treatment with the negative control (DPBS-buffer) the mean OD was 1.805 (study acceptance criterion: > 1.433). Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.87% (study acceptance criterion: <3.14%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 44.96% and, thus, lower than 50%. The results obtained from this in vitro skin irritation test indicated that the test item reveals potential for either skin corrosion (UN GHS Cat 1 ) or skin irritation (UN GHS Cat 2).

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
05 December 2016 - 27 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EU B.40.bis. In vitro skin corrosion: human skin model test
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: INVITTOX Protocol SkinEthicTM Skin Corrosivity Test
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number: 17-RHE-009

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ambient temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing step: using minimum volume of 20 mL DPBS, Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Observable damage in the tissue due to washing: no data
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

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 after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Application volume: 40 ± 3 µL per tissue

NEGATIVE CONTROL
- Application volume: 40 ± 3 µL per tissue

POSITIVE CONTROL
- Application volume: 40 ± 3 µL per tissue
Duration of treatment / exposure:
3 minutes or 1 hour
Number of replicates:
The test item as well as the negative control were tested with two replicate tissues per time point (3 min and 1 hour exposure). The positive control was tested with two replicate tissues only for 1 hour.
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 1 / 3 minutes
Run / experiment:
1
Value:
83.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 1 / 1 hour
Run / experiment:
1
Value:
65.75
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 2 / 3 minutes
Run / experiment:
1
Value:
81.84
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 2 / 1 hour
Run / experiment:
1
Value:
64.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no 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

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: Optical density and tissue viability

Group

Tissue 1

Tissue 2

Mean

CV

OD

viability

OD

viability

OD

viability

viability

Negative
Control

3 min

1.696

93.97%

1.913

106.03%

1.805

100.00%

8.52%

1 hour

1.935

94.85%

2.145

105.15%

2.040

100.00%

7.28%

Positive
Control

1 hour

0.008

0.39%

0.012

0.56%

0.010

0.48%

25.38%

Art.
W387800

3 min

1.507

83.50%

1.477

81.84%

1.492

82.67%

1.42%

1 hour

1.341

65.75%

1.318

64.60%

1.329

65.18%

1.25%

Interpretation of results:
other: Not category 1 (corrosive) based on GHS criteria
Conclusions:
Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin corrosion in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin corrosion potential. Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 40 ± 3 µL of either the liquid test item, the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. After treatment with the negative control (deionised water) the mean OD per tissue replicate was 1.696 and 1.913 after 3 minutes exposure and 1.935 and 2.145 after 1 hour exposure (study acceptance criterion: > 0.8 and < 3.0). Treatment with the positive control (potassium hydroxide, 8N) revealed a mean viability of 0.48% after 1 hour (study acceptance criterion: < 15%). Therefore, the study fulfilled the validity criteria. Following treatment with the test item,the tissue viability was > 50% after 3 minutes exposure (mean viability: 82.67%) and > 15% after 1 hour exposure (mean viability: 65.18%), i.e.according to OECD 431 the test item is not considered as corrosive to skin.

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:
17 May 2017 - 06 July 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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Age at study initiation: 16 -28 months
- Corneal diameter: 26 - 27 mm
- Storage, temperature and transport conditions of ocular tissue: cooled on ice
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Volume applied: 750 µL
- Concentration: 20%

VEHICLE
- Volume applied: 750 µL

Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Three corneas were used per group (negative control, positive control and test item group).
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride solution

POSITIVE CONTROL USED: N,N-dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME: 750 µL of a 20% solution were applied for 10 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes, 120 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:at least 3 times with washing medium

- POST-EXPOSURE INCUBATION: in fresh medium

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: baseline opacity was determined with a calibrated opacitometer, the light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values).
- Corneal permeability: corneas were incubated again in an incubator in a horizontal position at 32 ± 1°C for 90 minutes, amount of fluorescein that crossed the cornea was measured spectrophotometrically 490 nm

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula (referring to OECD Guideline 437) was used to determine the In Vitro Irritancy Score (IVIS) of the negative control:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The following formula was used to determine the In Vitro Irritancy Score (IVIS) of the positive control and the test item:
IVIS = corrected opacity value + (15 x corrected permeability OD490 value)
- The In Vitro Irritancy Score (IVIS) was calculated for each individual treatment and positive control cornea. The mean In Vitro Irritancy Score (IVIS) value of each treated group was calculated from the individual In Vitro Irritancy Score (IVIS) values.

DECISION CRITERIA:
IVIS ≤ 3 No Category (according to GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Serious eye damage, Category 1 (according to GHS)

ACCEPTANCE CRITERIA:
A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean(IVIS positive control: 75.3 - 120.2).
The negative control responses should result in an IVIS that falls within three standard deviations of the current historical mean (IVIS negative control: -1.2 - 5.3).
A single test run with three corneas should be sufficient for a test item when the resulting classification is unequivocal. In cases of the following borderline results in the first testing run, a second test run should be considered.
- 2 of the 3 corneas give discordant predictions from the mean of all 3 corneas or
- 1 of the 3 corneas give discordant predictions from the mean of all 3 corneas, and the discordant result is >10 IVIS units from the cut-off threshold of 55.


Irritation parameter:
in vitro irritation score
Remarks:
replicate 1
Run / experiment:
1
Value:
21.37
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
replicate 2
Run / experiment:
1
Value:
20.265
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
replicate 3
Run / experiment:
1
Value:
26.5
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no
No observations (e.g.tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
The resulting classification of the test item in this study is unequivocal and no borderline results were obtained. Therefore, a single testing run composed of three corneas per group was considered sufficient.

Table 1 Opacity, permeability and IVIS results

 

Opacity

Permeability

IVIS

per cornea

per group
(mean value)

Standard
deviation

Negative
control

0.9%sodium
chloride solution

0.5

-0.001

0.485

1.7

1.5

1.1

0.003

1.145

3.4

-0.002

3.370

Positive
control

N,N-dimethyl-
formamide

102.4

0.627

111.805

103.3

9.1

83.8

0.665

93.775

91.2

0.875

104.325

Testitem

Test item

11.2

0.678

21.370

22.7

3.3

11.1

0.611

20.265

8.8

1.180

26.500

Interpretation of results:
other: not Category 1 (irreversible effects on the eye) based on GHS
Conclusions:
Based on the results of the present study, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (no Category).
Executive summary:

The objective of the present study was to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an acceptedin vitromodel for ocular hazard assessment.

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item. As negative control 0.9% sodium chloride solution and as positive control N,N-dimethylformamide was used.

Three corneas were used per group (negative control, positive control or test item group).

After a first opacity measurement of the untreated bovine corneas, 750 µLof the test item, positive or negative control were applied on the corneas and incubated for 10 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again.

After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically.

The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was1.7 (study acceptance criteria range:-1.2 - 5.3). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 103.3 (study acceptance criteria range:75.3 - 120.2). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with the test item was 22.7 and, thus higher than 3 and lower than 55, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (no Category).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10 July 2017 - 18 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: reconstructed human cornea-like epithelium
Details on test animals or tissues and environmental conditions:
Justification of the test method and considerations regarding applicability:
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

Characterisation of the test system:
- Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
- Lot No.: 27001
- Keratinocyte strain: 4F1188
- Supplier: MatTek In Vitro Life Science Laboratories
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Volume applied: 50 µL/tissue

NEGATIVE CONTROL
- Volume applied: 50 µL/tissue

Duration of treatment / exposure:
30 ± 2 minutes
Duration of post- treatment incubation (in vitro):
12 ± 2 minutes at room temperature, 120 ± 15 minutes at 37°C and 5% CO2
Number of animals or in vitro replicates:
The test item as well as the positive and negative control were tested in batch-duplicates.
Details on study design:
- Details of the test procedure used:

Preparation:
On day of receipt, the tissues were equilibrated in their 24-well shipping container to room temperature for about 15 minutes. Afterwards the tissues were removed from the shipping container using sterile forceps and transferred to 6-well plates containing 1 mL pre-warmed (37°C) assay medium. Any agarose adhering to the inserts was removed by gentle blotting on gauze or paper towel. Afterwards, the tissues were incubated at 37°C and 5% CO2 overnight (16 - 24 hours) without medium exchange.

Pre-Treatment:
After the overnight incubation, the tissues were pre-wetted with 20 µL DPBS and incubated at 37°C and 5% CO2 for 30 minutes (± 2 minutes).

Treatment:
After the 30 minute DPBS pre-treatment, the liquid test item, the negative and the positive control were tested by applying 50 µL topically on the EpiOcular™ tissues. The tissues were placed back into the culture medium after dosing and incubated at 37°C and 5% CO2 for 30 ± 2 minutes.
At the end of the 30 ± 2 minutes treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (room temperature) DPBS. Three clean beakers, containing a minimum of 100 mL each of DPBS were used per group. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the two tissues per group were rinsed simultaneously by holding the replicate inserts together by their collars using forceps. The test item or control articles were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 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 at least two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS at least three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material.
After rinsing, the tissues were immediately transferred in 5 mL of pre-warmed (room temperature) assay medium in a 12-well plate for 12 ± 2 minutes at room temperature.

Post-treatment incubation:
After the 12 ± 2 minutes incubation, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert were blotted on absorbent material and transferred in 6-well plates filled with 1 mL of pre-warmed (37°C) assay medium for 120 ± 15 minutes at 37°C and 5% CO2.

- RhCE tissue construct used, including batch number:
Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 27001
Keratinocyte strain: 4F1188
Supplier: MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used: 50 µL

- Duration and temperature of exposure, post-exposure: exposure: 30 ± 2 min at 37 °C; post-exposure: 12 ± 2 min at room temperature + 120 ± 15 min at 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No. The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties.

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

- Wavelength used for quantifying MTT formazan: 570 nm

- Description of the method used to quantify MTT formazan:
MTT assay:
After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37°C and 5% CO2.
The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 24-well plate containing 2 mL isopropanol in each well so that isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. At the end of the extraction period, the tissues were pierced and the liquid within each insert is decanted into the well from which it was taken.
The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate. The results of the functional test and the printout of the measurement were included in the raw data of the study.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category). If the test item-treated tissue viability is <60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).

- Acceptance Criteria:
The results are acceptable if:
1. The negative control OD >0.8 and <2.5,
2. The mean relative viability of the positive control is:
a) 30 minute exposure: below 50% of control viability
b) 6 hour exposure: below 50% of control viability
3. Acceptable variability between tissue replicates: < 20 %
Irritation parameter:
other: tissue viability %
Remarks:
tissue 1
Run / experiment:
1
Value:
20.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
other: tissue viability %
Remarks:
tissue 2
Run / experiment:
1
Value:
23.4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e.the test item is not a direct MTT reducer and the test item has no colorant properties.

Table 1 Results viability

Group

Tissue 1

Tissue 2

Mean

SD

Difference
between tissue
replicates

OD

Viability

OD

Viability

OD

Viability

Viability

Negative
Control

1.614

100.1%

1.611

99.9%

1.613

100.0%

0.14

0.2%

Positive
Control

0.119

7.4%

0.126

7.8%

0.123

7.6%

0.28

0.4%

Test item

0.330

20.5%

0.378

23.4%

0.354

22.0%

2.05

2.9%

Acceptability of the test:

The negative control OD is >0.8 and <2.5 (1.611 and 1.614).

The mean relative viability of the positive control is below 50% of the negative control viability (7.6%).

The difference of viability between the two relating tissues of a single chemical is <20% (values between 0.2% to 2.9%) in the same run(for positive and negative control tissues and tissues of single chemicals).

The study met all acceptance criteria.

Interpretation of results:
other: Category 1 (irreversible effects on the eye) or Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Under the conditions of the present study, the test item did show an eye hazard potential. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or Category 2).
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model.

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 µL of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 1.613 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 7.6% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 22.0% and, thus, lower than 60%, i.e.according to OECD 492 the test item is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or Category 2).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

 

OECD 439

The objective of the present study (reference 7.3.1-1) was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the test item, negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. After treatment with the negative control (DPBS-buffer) the mean OD was 1.805 (study acceptance criterion: > 1.433). Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.87% (study acceptance criterion: <3.14%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 44.96% and, thus, lower than 50%. The results obtained from this in vitro skin irritation test indicated that the test item reveals potential for either skin corrosion or skin irritation (UN GHS Cat 1 or 2).

OECD 431

The objective of the present study (reference 7.3.1-2) was to investigate the potential of the test item to induce skin corrosion in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin corrosion potential. Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour.Duplicates with the positive control were only treated for 1 hour. 40 ± 3 µL of either the liquid test item, the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. After treatment with the negative control (deionised water) the mean OD per tissue replicate was 1.696 and 1.913 after 3 minutes exposure and 1.935 and 2.145 after 1 hour exposure (study acceptance criterion: > 0.8 and < 3.0). Treatment with the positive control (potassium hydroxide, 8N) revealed a mean viability of 0.48% after 1 hour (study acceptance criterion: < 15%). Therefore, the study fulfilled the validity criteria.

Following treatment with the test item, the tissue viability was > 50% after 3 minutes exposure (mean viability: 82.67%) and > 15% after 1 hour exposure (mean viability: 65.18%), i.e. according to OECD 431 the test item is not considered as corrosive to skin.

 

 

Conclusion: Since one in vitro test alone cannot predict reliably the skin irritating/corrosive potential of the test item, an integrated test strategy, consisting of the tests according to OECD Guideline 439 (skin irritation, reference 7.3.1-1) and OECD Guideline 431 (skin corrosion, reference 7.3.1-2) was performed. According to the results of the performed tests (skin irritating, not skin corrosive) the test item is considered to be skin irritating (UN GHS: Category 2, H315).

 

Eye irritation

 

OECD 437

The objective of the present study (reference 7.3.2-1) was to examine the potential of the test item to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item. As negative control 0.9% sodium chloride solution and as positive control N,N-dimethylformamide was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the test item, positive or negative control were applied on the corneas and incubated for 10 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS). After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.7 (study acceptance criteria range: 1.2 - 5.3).Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 103.3 (study acceptance criteria range: 75.3 - 120.2). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 22.7 and, thus higher than 3 and lower than 55, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (no Category).

 

OECD 492

The objective of the present study (reference 7.3.2-2) was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 µL of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. After treatment with the negative control (sterile deionized water) the mean OD was 1.613 (study acceptance criterion: > 0.8 and < 2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 7.6% (study acceptance criterion: < 50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 22.0% and, thus, lower than 60%, i.e.according to OECD 492 the test item is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or Category 2).

 

Conclusion: Since one in vitro test alone cannot predict reliably the eye irritating/damaging potential of the test item, an integrated test strategy, consisting of the tests according to OECD Guideline 437 (eye damage, reference 7.3.2-1) and OECD Guideline 492 (eye irritation, 7.3.2-2) was performed. According to the results of the performed tests the test item is considered to be eye irritating (UN GHS Category 2, H319).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is considered to be classified for skin irritation (UN GHS: Category 2, H315) and eye irritation (UN GHS: Category 2, H319) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.