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Diss Factsheets

Administrative data

Description of key information

Skin:

In an in vitro skin irritation assay according to OECD Guideline 439 (RhE), the test substance did not show skin irritating properties (UN GHS: No Category) (reference 7.3.1-1).

Eye:

In an in vitro eye irritation assay according to OECD Guideline 437 (BCOP), the test substance did not show an eye hazard potential (UN GHS: No Category) (reference 7.3.2-1).

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:
10 May 2017 - 17 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SkinEthic Skin Irritation Test42bis Standard Operating Procedure
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
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 (Episkin/SkinEthic Laboratories, Lyon, France)
- Tissue batch number: 17-RHE-071
- Date of initiation of testing: On day of receipt the pre-incubation phase of the tissues started

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the test item, negative and positive control were removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. 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

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: ELx800 (BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- 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 item is considered to be non-corrosive to skin if the tissue viability after exposure and post-treatment incubation is > 50%.
- The test item is considered to be irritant to the skin Category 2 if the viability is less than or equal to 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 16 ± 2 mg

NEGATIVE CONTROL
- Amount applied: 16 ± 0.5 µL

POSITIVE CONTROL
- Amount applied: 16 ± 0.5 µL
Duration of treatment / exposure:
42 ± 1 min
Duration of post-treatment incubation (if applicable):
42 ± 1 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1, Tissue 1
Value:
101.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1, Tissue 2
Value:
101.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1, Tissue 3
Value:
105.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1, Mean
Value:
102.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
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

Group

Tissue 1

Tissue 2

Tissue 3

Mean

SD

OD

Viability (%)

OD

Viability (%)

OD

Viability (%)

OD

Viability (%)

Viability (%)

Negative Control

1.874

103.2

1.707

94.0

1.866

102.8

1.816

100.0

5.2

Positive Control

0.024

1.3

0.025

1.4

0.025

1.4

0.025

1.4

7.1

Test Item

1.836

101.1

1.839

101.3

1.913

105.3

1.863

102.6

2.3

Table 1: Optical Density and Tissue Viability

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin irritation assay according to OECD Guideline 439 (RhE), the test substance did not show skin irritating properties.
Executive summary:

In an in vitro skin irritation assay according to OECD Guideline 439 (RhE), the skin irritating properties of the test item were determined. 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 and the positive control were met.

Following treatment with the test item, the mean tissue viability was 102.6% 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
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 June 2017 - 14 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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
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. QA statement)
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: 19 - 28 months
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. 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: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20% (w/v)

VEHICLE CONTROL
- Amount applied: 750 µL
- Concentration: 0.9% (w/v)

POSITIVE CONTROL
- Amount applied: 750 µL
- Concentration: 20% (w/v)
Duration of treatment / exposure:
4 h
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
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.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. The baseline opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). 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.

NUMBER OF REPLICATES
3

SOLVENT CONTROL USED
Yes

POSITIVE CONTROL USED
Yes

APPLICATION DOSE AND EXPOSURE TIME
750 µL, 20%, 4 h

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The corneal surface was washed 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 prior to reading the opacity value after treatment.

- POST-EXPOSURE INCUBATION: No

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany).
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany) at OD490.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Opacity: The opacity value of each individual cornea was corrected for background opacity by subtracting the initial baseline opacity reading from the post treatment opacity reading. In addition, the opacity values of both the treatment and positive control groups were corrected for the mean negative control opacity values. From the individual corrected opacity values, a mean corrected opacity value was calculated for each group.
Permeability: For each cornea either treated with the positive control or the test item, an individual corrected OD490 value was calculated by subtracting the average negative control permeability value from each individual permeability reading. From the individual corrected permeability values, a mean corrected permeability value was calculated for each group.

In Vitro Irritancy Score (IVIS) Calculation: 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: The following IVIS cut-off values for identifying test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) were applied. IVIS score below or equal to 3: UN GHS No Category
IVIS score above 3 and less or equal to 55: No prediction can be made
IVIS score above 55: UN GHS Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
1, Tissue 1
Value:
0.145
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
1, Tissue 2
Value:
0.6
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
1, Tissue 3
Value:
2.6
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
1, Mean
Value:
1.1
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle 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: Results

 

Opacity

Permeability

IVIS

Per Cornea

Mean

SD

Solvent Control

0.0

0.002

0.030

0.2

0.4

0.0

0.003

0.045

0.6

0.004

0.660

Positive Control

71.0

2.506

108.590

106.4

2.3

71.2

2.352

106.480

72.4

2.112

104.080

Test Substance

0.1

0.003

0.145

1.1

1.3

0.6

0.002

0.630

2.6

0.000

2.600

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

In an in vitro irritation assay according to OECD Guideline 437 (BCOP) the eye hazard potential of the test item was determined. The induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) solution in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test substance group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the dissolved test item, positive or negative control were applied on the corneas and incubated for 240 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 the calculated IVIS was 0.2 (study acceptance criteria range: -1.4 - 3.2). Treatment with the positive control revealed an IVIS of 106.4 (study acceptance criteria range: 80.5 - 133.0). Therefore, the study fulfilled the acceptance criteria. The mean IVIS obtained after treatment with the test substance was 1.1 and, thus, lower than 3, i.e. according to OECD 437 the test substance 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)

Additional information

Skin (reference 7.3.1-1):

In an in vitro skin irritation assay according to OECD Guideline 439 (RhE) the skin irritating properties of the test item were determined. 16 µg of the test item were 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 (1 mg/mL) 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 and the positive control were met. Following treatment with the test substance, the mean tissue viability was 102.6% 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).

Eye (reference 7.3.2-1):

In an in vitro irritation assay according to OECD Guideline 437 (BCOP) the eye hazard potential of the test item was determined. The induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) solution in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole 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 dissolved test item, positive or negative control were applied on the corneas and incubated for 240 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 the calculated IVIS was 0.2 (study acceptance criteria range: -1.4 - 3.2). Treatment with the positive control revealed an IVIS of 106.4 (study acceptance criteria range: 80.5 - 133.0). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test substance was 1.1 and, thus, lower than 3, i.e. according to OECD 437 the test substance is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Justification for classification or non-classification

Classification, Labeling, 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 test item is not considered to be classified for skin or eye irritation/corrosion under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

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