Registration Dossier

Administrative data

Description of key information

Skin irritation / corrosion

In a study according OECD TG 439, the test item is not identified as requiring classification and labelling according to UN GHS, as the mean percentage tissue viability was higher than 50% of the negative control (reference 7.3.1-1).

Eye irritation

In a study according OECD TG 492 using the EpiOcular™ model, the tissue viability was 1.1% and, thus, lower than 60%,i.e.according to OECD TG 492 no prediction could be made regarding the eye hazard potential and further testing was necessary (reference 7.3.2 -1). In a study according OECD TG 437 the IVIS obtained after treatment with the test item was 1.1 and, thus, i.e.according to OECD TG 437 lower than 3, the test item is not requiring classification for eye irritation or serious eye damage (reference 7.3.2 -2).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 20, 2018 -June 22,2018
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:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM, Performance Standards for In-Vitro Skin Irritation Test Methods based on Reconstructed Human Epidermis (RHE)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
other: SkinEthic Skin Irritation Test-42bis Standard Operating Procedure (SOP): Using the Reconstructed Human Epidermis (RHE) model, INVITTOX Version 2.1
Version / remarks:
2009
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:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17 obtained from Episkin/SkinEthic Laboratories, Lyon, France
- Tissue batch number: 18-RHE-068

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C and 5% CO2.

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

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours (± 5 minutes) at 37°C and 5% CO2 protected from light
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD =1.2 (CV= 3.1 %)
- Barrier function: 4.5 h
- Morphology: well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum, absence of significant histological abnormalities
- Contamination: none

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
A test item is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is ≥ 50%.
A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is ≤ 50%. Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.

ACCEPTABILITY CRITERIA
The OD values for the negative control shall be in the range of ≥ 0.8 and ≤ 3.0 as given in OECD Guideline 439.
Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:
The negative control data meet the acceptance criteria if the mean OD value of the 3 tissues is ≥ 1.2 at 570 nm. The standard deviation value is considered valid if ≤ 18% of group mean-value.
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is < 40%. The standard deviation value is considered valid if ≤ 18% of group mean-value.
Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:
The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundary at 570 nm. The boundary is two standard deviations below the current historical mean (1.450).
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is three standard deviations above the current historical mean (2.87%).
Test Item Data Acceptance Criteria:
The standard deviation of the three tissues treated with the test item should be ≤ 18%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied (volume or weight with unit): 16 mg ± 2 mg per tissue
Before adding 16 mg of the test item, 10 µL of deionised water were spread to the epidermis surface to improve further contact between the test item and the epidermis.

NEGATIVE CONTROL
- Concentration: 16 µL ± 0.5 µL per tissue

POSITIVE CONTROL
- Concentration: 16 µL ± 0.5 µL per tissue
Duration of treatment / exposure:
42 minutes (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st experiment
Value:
107.5
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: no
- Colour interference with MTT: 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.

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: Results

Group

Tissue 1

 

Tissue 2

 

Tissue 3

Mean

SD

 

 

OD

viability

OD

viability

OD

viability

OD

viability

viability

Negative
Control

1.794

107.7%

1.607

96.5%

1.594

95.7%

1.665

100.00

6.7%

Positive
Control

0.025

1.5%

0.021

1.3%

0.021

1.3%

0.022

1.4%

7.1%

Test item

1.993

119.7%

1.682

101.0%

1.693

101.7%

1.789

107.5%

9.9%

Table 2: Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

 

Acceptance Criterion

Result

Negative control OD

> 0.8 and < 3.0

1.594 to 1.794

Table 3: Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories

 

Acceptance Criterion

Result

Mean OD negative control

≥1.2

1.665

Mean viability positive control

< 40%

1.4%

SD of group-mean value

 

 

 

≤ 18%

7.1% (positive control)

 

6.7% (negative control)

 

Table 4: Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory

 

Acceptance Criterion

Result

Mean OD negative control

≥ 1.450

1.665

Mean viability positive control

≤ 2.87%

1.4%

Table 5: Test Item Data Acceptance Criteria:

 

Acceptance Criterion

Result

SD of group-mean value

≤ 18%

9.9%

 

 

Table 6: Historical Data

The mean of negative control and positive control of all performed experiments in the testing laboratory is given in the following table.

Positive Control

Negative Control

Mean Viability [%]

142

Mean Absorption [OD570]

1.987

Standard Deviation

0.48

Standard Deviation

0.268

Based on the historical data, acceptability ranges for the positive and negative control were stated.

 

Interpretation of results:
GHS criteria not met
Conclusions:
Following treatment with the test item, the tissue viability was 107.5% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin irritation in anin vitro human skin model according to OECD TG 439. 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 minutes1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met.

Following treatment with the test item, the tissue viability was 107.5% and, thus, higher than 50%,i.e.according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April 24, 2018 - April 26, 2018
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:
2017
Deviations:
no
Qualifier:
according to
Guideline:
other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular™ EIT
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; For use with MatTek Corporation's Reconstructed Human EpiOcular Model
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
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.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 mg


Duration of treatment / exposure:
6 hours (± 15 minutes)
Duration of post- treatment incubation (in vitro):
18 hours (± 15 minutes)
Number of animals or in vitro replicates:
The test item as well as the positive and negative control were tested in batch-duplicates. Therefore, a total number of six tissues was used in this study.
Details on study design:
- RhCE tissue construct used, including batch number
The EpiOcular™ Tissues (OCL-200, OCL-212) (Lot No: 27036) was obtained from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia.

- Doses of test chemical and control substances used
Solid test item: 50 mg per tissue
Negative control: 50 µL per tissue
Positive control: 50 µL per tissue

- Duration and temperature of exposure
incubated at 37°C and 5% CO2 for 6 hours (± 15 minutes)
At the end of the 6 hours treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (room temperature) DPBS. After rinsing, the tissues were immediately transferred in 5 mL of pre-warmed (room temperature) assay medium in a 12-well plate for 25 minutes (± 2 minutes) at room temperature. After the 25 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 18 hours (± 15 minutes) at 37°C and 5% CO2.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
The non-colored test item was tested for its ability to become colorant after contact with water or isopropanol. For this purpose, 50 mg of the test item were added to 1.0 mL of water in a 6-well plate and the mixture was incubated for 1 hour at 37 ± 1°C and 5% CO2 protected from light. Furthermore, 50 mg were added to 2 mL isopropanol, the same amount as used for MTT extraction, and was incubated in 6-well plates for 2 to 3 hours at room temperature.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
The test item as well as the positive and negative control were tested in batch-duplicates. Therefore, a total number of six tissues was used in this study.

- Description of the method used to quantify MTT formazan
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 6-well plate containing 2 ml isopropanol so that no 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. The corresponding negative and positive controls were treated identically.
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.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test item is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability is more than 60%. In this case no further testing in other test methods is required. If the mean percent tissue viability is less than or equal 60%, no prediction can be made. In this case, further testing with other test methods will be required because RhCE test methods show a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.

- Acceptable 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. 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:
1.1
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: 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1: Results

Group

Tissue 1

Tissue 2

Mean

SD

Difference
between tissue
replicates

 

 

OD

Viability

OD

Viability

OD

Viability

Viability

 

 

Negative
Control

1.992

97.9%

2.076

102.1%

2.034

100.0%

2.97

4.2%

Positive Control

0.515

25.3%

0.690

33.9%

0.603

29.6%

6.08

8.6%

Test item

0.021

1.0%

0.022

1.1%

0.022

1.1%

0.07

0.1%

Interpretation of results:
other: no prediction can be made; further testing with other test methods is required because RhCE test methods cannot resolve between UN GHS Categories 1 and 2
Conclusions:
Following treatment with the test item, the tissue viability was 1.1% and, thus, lower than 60%, i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.
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 according to OECD TG 492. 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 mg 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 2.034 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 29.6% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 1.1% and, thus, lower than 60%,i.e.according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted and further testing is necessary.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
November 2, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
2017
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
- Characteristics of donor animals: 12-41 months old cattle
- Storage, temperature and transport conditions of ocular tissue: The eyes were kept and transported in transport medium 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: no
- Indication of any antibiotics used: Streptomycin and Penicillin (5 mL/500 mL HBSS) in transport medium
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: According to OECD Guideline 437 surfactant substances are tested at a concentration of 10% (w/v). Therefore, the test item was prepared with 0.9% sodium chloride solution to a concentration of 10% (w/v).

Duration of treatment / exposure:
10 min
Duration of post- treatment incubation (in vitro):
120 min
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded.
Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.
For equilibration, the corneas in the holder were incubated (BSS 160, Grumbach Brutgerâte GmbH, Asslar, Germany) in a vertical position at 32 ± 1°C for about one hour. At the end of the incubation period, the incubation medium was replaced by fresh pre-warmed (32 ± 1 °C) incubation medium in both compartments.

QUALITY CHECK OF THE ISOLATED CORNEAS
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). Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded. Three corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control group.

NUMBER OF REPLICATES
three

NEGATIVE CONTROL USED
0.9% sodium chloride solution

SOLVENT CONTROL USED
0.9% sodium chloride solution

POSITIVE CONTROL USED
N,N-dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME
Fresh incubation medium was filled into the posterior compartment, while the surface of the cornea in the anterior compartment was treated with 750 µL of either the test item preparation, negative or positive control. The test item preparation, the negative and positive control preparations were introduced with the closed-chamber method through the dosing holes of the chamber. After application, the corneas were incubated in an incubator in a horizontal position at 32 ± 1°C for 10 minutes.

TREATMENT METHOD
closed chamber

POST-INCUBATION PERIOD: yes
After the incubation period, the test item, the negative and positive control were removed from the anterior chamber without opening the chamber. The corneal surface was washed at least three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. Fresh incubation medium was replaced in both compartments and the corneas were incubated for additional 120 minutes at 32 ± 1°C. Afterwards, the incubation medium was replaced in both compartments prior to reading the opacity value after treatment.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others: pertinent visual observations

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Decision criteria as indicated in the TG was used.
IVIS ≤3 (UN GHS No Category)
IVIS >3; ≤55 (No prediction can be made)
IVIS >55 (UN GHS Category 1)
Irritation parameter:
in vitro irritation score
Run / experiment:
1st run
Value:
1.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Tween 20 and Trichloroacetic acid were tested as reference substances. Under the conditions of thist study, the eye hazard classifications of the test items Tween 20 and Trichloroacetic acid were correctly identified as given in OECD Guideline 437 (see Table 3).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1: Results

 

 

Opacity

 

 

Permeability

 

 

IVIS

per cornea

per group
(mean value)

Standard
deviation

Negative
control

 

 

 

0.9% sodium
chloride solution

 

 

 

1.0

0.002

1.030

1.2

 

 

 

0.8

 

 

 

2.1

0.002

2.130

0.5

0.001

0.515

Positive
control

 

 

 

N,N-dimethyl-
formamide

 

 

 

82.7

0.704

93.260

96.8

 

 

 

10.0

 

 

 

71.1

1.197

89.055

86.9

1.416

108.140

Test item

 

 

 

 

 

 

0.2

0.012

0.380

1.1

 

 

 

1.2

 

 

 

0.3

0.011

0.465

2.2

0.024

2.560

Table 2: Historical Data

Positive Control

Negative Control

Mean IVIS

98.05

Mean IVIS

2.03

Standard Deviation

10.20

Standard Deviation

1.13

 

Table 3: Results from study using Tween 20 and Trichloroacetic acid

 

 

Opacity

 

 

Permeability

 

 

IVIS

per cornea

per group
(mean value)

Standard
deviation

Negative
control

 

 

 

0.9% sodium
chloride solution

 

 

 

1.3

0.000

1.300

0.9

 

 

 

1.0

 

 

 

1.6

0.003

1.645

-0.3

0.002

-0.270

Positive
control

 

 

 

N,N-

dimethylformamide

 

 

 

82.3

0.402

88.330

92.0

 

 

 

13.5

 

 

 

72.2

0.576

80.840

90.8

1.078

106.970

Test item 1

 

 

 

Tween 20

 

 

 

-0.4

0.000

-0.400

-0.4

 

 

 

0.4

 

 

 

0.0

0.000

0.000

-0.8

0.000

-0.800

Test item 2

 

 

 

 

 

Trichloroacetic acid

 

 

 

209.2

-0.002

209.170

213.6

 

 

 

4.0

 

 

 

216.8

-0.003

216.755

215.0

-0.004

214.940

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is not requiring classification for eye irritation or serious eye damage (UN GHS: 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 according to OECD TG 437. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment.

To determine the eye hazard potential of the test item the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item. As is a surfactant, a 10% (w/v) suspension in a 0.9% sodium chloride solution was tested as required by OECD 437. 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 preparation, positive or negative control were applied on the corneas and incubated for 10 minutes. After this exposure time fresh incubation medium was replaced in both compartments and the corneas were incubated for additional 120 minutes at 32 ± 1°C. Afterwards, the incubation medium was replaced in both compartments prior to reading the opacity value after treatment. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution. 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 1.2 (study acceptance criteria range: -1.4 - 5.4). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 96.8 (study acceptance criteria range: 77.7 - 118.4). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 1.1 and, thus, lower than 3,i.e.according to OECD 437 the test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

The potential of the test item to induce skin irritation was investigated in an in vitro human skin model according to OECD TG 439. 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 negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met. Following treatment with the test item, the tissue viability was 107.5% and, thus, higher than 50%, i.e.according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category) (reference 7.3.1 -1).

Eye irritation

The potential of the test item to induce eye irritation was investigated in an in vitro human cornea model according to OECD TG 492. 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 mg 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 2.034 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 29.6% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 1.1% and, thus, lower than 60%,i.e.according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item. Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted and further testing is necessary (reference 7.3.2 -1).

In a further study the potential of the test item to induce serious eye damage was investigated in the BCOP assay according to OECD TG 437. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment. To determine the eye hazard potential of the test item the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item. As the test item is a surfactant, a 10% (w/v) suspension in a 0.9% sodium chloride solution was tested as required by OECD 437. 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 preparation, positive or negative control were applied on the corneas and incubated for 10 minutes. After this exposure time fresh incubation medium was replaced in both compartments and the corneas were incubated for additional 120 minutes at 32 ± 1°C. Afterwards, the incubation medium was replaced in both compartments prior to reading the opacity value after treatment. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically.The opacity and permeability assessments were combined to determine anIn Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.2 (study acceptance criteria range: -1.4 - 5.4). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 96.8 (study acceptance criteria range: 77.7 - 118.4). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 1.1 and, thus, lower than 3, i.e.according to OECD 437 the test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category) (reference 7.3.2 -2).

Justification for classification or non-classification

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

The available data for skin irritation/corrosion and eye irritation/corrosion are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is neither considered to be classified for skin irritation/corrosion nor for eye irritation/corrosion (UN GHS: no category) under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.