Registration Dossier

Administrative data

Description of key information

Skin:

In an in vitro skin irritation assay (RhE) according to OECD Guideline 439, a cell viability of 76.7 % was determined. Thus the test substance is not considered as skin-irritant (UNG GHS: no Category) (reference 7.3.1-1).

Eye:

Based on the results of the OECD 437 study, the test substance induced serious eye damage (UN GHS: Category 1) (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:
25 April 2017 - 04 October 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)
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: The SkinEthic™ RHE-model RHE/S/17, Episkin/SkinEthic Laboratories, Lyon, France.
- Tissue batch number: 17-RHE-086

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: 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. The inserts were placed in 6-well plates with 2 mL fresh pre-warmed (room temperature) growth medium.
- 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: Spectrophotometer (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: not specified
- N. of replicates : 3
- Method of calculation used: see "Any other information on materials and methods"

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritating to skin if the mean tissue viability is > 50 % after incubation period.
- The test substance is considered to be irritating to skin if the mean tissue viability is ≤ 50 % after incubation period.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 16 µL ± 0.5 µL per tissue

NEGATIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue

POSITIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue
Duration of treatment / exposure:
42 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
76.7
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: Yes
- 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

 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

1.930 to 2.127

 

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

 

Acceptance Criterion

Result

Mean OD negative control

≥ 1.2

2.019

Mean viability positive control

< 40 %

1.0 %

SD of group-mean value

18 %

10.0 % (positive control) 4.9 % (negative control)

 

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

 

Acceptance Criterion

Result

Mean OD negative control

≥ 1.463

2.019

Mean viability positive control

2.98 %

1.0 %

 

Test Item Data Acceptance Criteria:

 

Acceptance Criterion

Result

SD of group-mean value

18 %

7.3 %

 

The study met all acceptance criteria.

The means of the negative controls and the positive control of all performed experiments in the testing laboratory are given in the following table:

Positive Control

Negative Control

Mean Viability [%]

1.45

Mean Absorption [OD570]

2.015

Standard Deviation

0.51

Standard Deviation

0.276

The results obtained after treatment of the reconstructed human epidermis model with the test substance are given in the following table:

Group

Tissue 1

Tissue 2

Tissue 3

Mean

SD

OD

Viability (%)

OD

Viability (%)

OD

Viability (%)

OD

Viability (%)

Viability (%)

Negative Control

2.127

105.3

1.930

95.6

2.001

99.1

2.019

100.0

4.9

Positive Control

0.021

1.0

0.019

0.9

0.022

1.1

0.021

1.0

10.0

Test Substance

1.510*

71.0

1.587*

82.2

1.540*

77.0

1.546

76.7

7.3

* corrected optical density after true metabolic conversion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro skin irritation assay (RhE) according to OECD Guideline 439, a cell viaability of 76.7 % was determined. Thus the test substance is not considered as skin irritant.
Executive summary:

In an in vitro skin irritation assay (RhE) according to OECD Guideline 439, the skin irritating potential of the test item was investigated. 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 test item, the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. The test item has the ability to directly reduce MTT. To evaluate the extent of non-specific interaction, three killed tissues were treated with the test item and three killed untreated tissues were used as negative control. The treatment and MTT assay of the killed tissues was similar to the handling of the living tissues. The obtained OD for a non-specific reduction was subtracted from OD-values obtained after treatment of living tissues with the test item to calculate the cell viability. 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 substance, the tissue viability was 76.7 % 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). Under the conditions of the present study, the test substance is not considered to possess an irritant potential 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:
18 July 2017 - 22 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:
other: isolated bovine cornea
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Number of animals: not specified
- Characteristics of donor animals: 14 - 196 months old
- 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: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin was added for the transport (5 mL/500 mL HBSS).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3 corneas per group
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 and scratches were discarded. At the end of the incubation period, the basal 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.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes

POST-INCUBATION PERIOD: 120 minutes

REMOVAL OF TEST SUBSTANCE:
After the incubation 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The light transmission through the corneas was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany).
- Corneal permeability: The amount of fluorescein that crossed the cornea was measured spectrophotometrically with a microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS ≤ 3 No Category (according to GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Serious eye damage according, Category 1 (according to GHS)
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
156.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
2
Value:
146
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Value:
132.4
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: none

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

Study Acceptance Criteria

After treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 1.2 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.1 - 5.3). After treatment with the positive control (N,N-dimethylformamide) the calculated IVIS was 95.2 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS 76.1 - 120.0). Therefore, the study fulfilled the acceptance criteria. 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.

Results

 

Opacity

Permeability

IVIS

per cornea

per group (mean value)

Standard Deviation

Negative Control

1.0

0.009

1.135

1.2

0.6

1.08

0.005

1.875

0.6

0.007

0.705

Positive control

80.5

0.236

84.040

95.2

11.0

86.1

0.615

95.325

87.2

1.259

106.085

Test Substance

156.7

0.000

156.700

145.0

12.2

145.6

0.027

146.005

132.3

0.008

132.420

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Based on the results of the OECD 437 study, the test item induced serious eye damage (UN GHS: Category 1).
Executive summary:

The objective of the present study was to examine the potential of the test substance to induce serious eye damage in the BCOP assay. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item according to OECD Guideline 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, 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 1.2 (study acceptance criteria range: -1.1 - 5.3). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 95.2 (study acceptance criteria range: 76.1 - 120.0). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with the test item was 145.0 and, thus, higher than 55, i.e. according to OECD 437 the test item induced serious eye damage (UN GHS: Category 1).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation study:

In an in vitro skin irritation assay (RhE) according to OECD Guideline 439, the skin irritating potential of the test item was investigated (reference 7.3.1-1). 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 test item, the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. The test item has the ability to directly reduce MTT. To evaluate the extent of non-specific interaction, three killed tissues were treated with the test item and three killed untreated tissues were used as negative control. The treatment and MTT assay of the killed tissues was similar to the handling of the living tissues. The obtained OD for a non-specific reduction was subtracted from OD-values obtained after treatment of living tissues with the test item to calculate the cell viability. 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 substance, the tissue viability was 76.7 % 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 irritation study:

The objective of the study according to OECD guideline 437 was to examine the potential of the test item to induce serious eye damage in the BCOP assay (7.3.2-1). 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 1.2 (study acceptance criteria range: -1.1 - 5.3). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 95.2 (study acceptance criteria range: 76.1 - 120.0). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with the test item was 145.0 and, thus, higher than 55, i.e. according to OECD 437 the test item induced serious eye damage (UN GHS: Category 1).

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 considered to be classified for serious eye damage (UN GHS: Category 1, H318) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.

Furthermore, the test substance is not considered to be classified as skin-irritant under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.