Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion:

In conclusion, it can be stated that under the experimental conditions reported, the test item is non-irritant to skin according to UN GHS and EU CLP regulation.

Eye irritation:

In conclusion, according to the performd study the test item is not an eye irritant according to UN GHS and CLP regulation.

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:
2019-03-07 to 2019-04-17
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
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: reconstruceted human skin model
Justification for test system used:
The applied test system is recommended by the guidelines followed.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Kit
- Tissue batch number: 28690

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsing with PBS for at least 15 times. After rinsing, the inserts were submerged in PBS at least three times. Afterwards the inserts were once again rinsed with sterile PBS from the inside and the outside. Excess PBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: yes
- Barrier function: yes
- Morphology: yes
- Contamination: none
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
not applicable since the test item did not direclty interfere with MTT or changed color in the presence of water.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if mean tissue viability after 1 h exposure is equal to or less than 50 % of the control
- The test substance is considered to be non-irritating to skin if mean tissue viability after 1 h exposure is greater than 50 % of the control
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL

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):
24 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
94.51
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
- 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 results after 60 min treatment with the test item

Treatment group

Mean OD of 3 tissues

SD

Mean Rel Viability [%]

Negative control

1.840

4.9

100.0

Positive control

0.059

0.1

3.20

Test Item

1.739

1.5

94.51

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is non-irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not change colour when mixed with deionised water (pre-test for colour interference). Also, its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5 % SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system.

After treatment with the test item the mean relative viability value decreased to 94.51 % compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50 %. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, is non-irritant to skin.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-18 to 2019-04-26
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)
Deviations:
yes
Remarks:
The positive control is 2-Ethoxyethanol (purity: 99%) since the laboratory historical control data are established with this chemical.
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Freshly isolated bovine cornea
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Number of animals:
- Characteristics of donor animals: 14 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were stored in HBSS containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used: none
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL test item, positiv or negativ control
Duration of treatment / exposure:
10 minutes
Number of animals or in vitro replicates:
3 replicates per test group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of 14 month old donor cattle were collected from the abattoir. All eyes were carefully examined macroscopically for defects. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each isolated cornea was mounted in a specially designed cornea holder, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium.

QUALITY CHECK OF THE ISOLATED CORNEAS
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement (t0). At the end of the incubation period, the basal opacity was determined (t0). Only corneae with a value of the basal opacity < 7 were used. Sets of three corneae were used for treatment with the test item and for the negative and positive controls.

NUMBER OF REPLICATES : three

NEGATIVE CONTROL USED : Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED : 2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME
The anterior compartment received the test item or the negative or positive controls at a volume of 0.75 mL on the surface of the corneae, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted ten minutes (± 30 seconds).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 4 times - 3 times with EMEM containing phenol red and 1 time with cMEM without phenol red
- POST-EXPOSURE INCUBATION: The corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130). In the second step of the assay, permeability of the corneae was determined.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France); The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value.
- Corneal permeability: the optical density at 490 nm (OD490) was measured with a microplate reader (Versamax® Molecular Devices)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Depending on the score obtained, the test item was classified according to OECD guideline 437.
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
test item group
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Results after 10 Minutes Treatment Time

Test Group

Opacity value = Difference (t130-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposed in vitro Irritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

1

0.33

0.094

0.078

2.41

1.51

No category

0

0.072

1.08

0

0.069

1.04

Positive Control

87.67*

1.417*

108.92

99.81

Category 1

75.67*

1.059*

91.55

82.67*

1.087*

98.97

Test Item

-0.33*

-0.032*

0.00#

0.00

No category

-0.33*

-0.030*

0.00#

-0.33*

-0.024*

0.00#

 

With the negative control (saline), neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.51).

The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 99.81) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

The test item was tested undiluted. Relative to the negative control, the test item did not cause any increase of the corneal opacity or permeability. The calculated mean IVIS was 0.00 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not classified.

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, according to the current study the test item is not an eye irritant.
Executive summary:

To assess the corneal damage potential of the test item, an in vitro study according to OECD TG 437 was conducted. After a first opacity measurement of the fresh bovine cornea (t0), the undiluted test item, the positive control, and the negative control were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 °C. After the incubation phase the test item, the positive control, and the negative controls were each rinsed from the cornea. Further, the corneae were incubated for another 120 min at 32 °C in a vertical position. Afterwards, opacity was measured a second time (t130). Permeability of the cornea was also determined after opacity measurements.

The acceptance criteria for the negative and positive control were met. With the negative control (saline), neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.51). The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 99.81) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

In comparison to the negative control, the test item did not cause any increase of the corneal opacity or permeability. The calculated mean IVIS was 0.00 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not classified as serious eye damaging or as eye irritating.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD 439:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not change colour when mixed with deionised water (pre-test for colour interference). Also, its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5 % SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system.

After treatment with the test item the mean relative viability value decreased to 94.51 % compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50 %. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, is non-irritant to skin.

OECD 437:

To assess the corneal damage potential of the test item, an in vitro study according to OECD TG 437 was conducted. After a first opacity measurement of the fresh bovine cornea (t0), the undiluted test item, the positive control, and the negative control were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 °C. After the incubation phase the test item, the positive control, and the negative controls were each rinsed from the cornea. Further, the corneae were incubated for another 120 min at 32 °C in a vertical position. Afterwards, opacity was measured a second time (t130). Permeability of the cornea was also determined after opacity measurements.

The acceptance criteria for the negative and positive control were met. With the negative control (saline), neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.51). The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 99.81) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

In comparison to the negative control, the test item did not cause any increase of the corneal opacity or permeability. The calculated mean IVIS was 0.00 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not classified as serious eye damaging or as eye irritating.

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. As a result the substance is not considered to be classified for skin corrosion/irritation and eye damage/irritation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.