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Description of key information

Skin irritation

Based on the results of an OECD 431 study, the test item is determined not to be corrosive to skin (not UN GHS Cat. 1).

Based on the results of an OECD 439 study, the test item is determined to be irritant or corrosive to skin (UN GHS Cat. 1 or 2).

As overall result of the performed in vitro studies, the test item is determined to be irritant to skin (UN GHS Cat. 2).

 

Eye irritation

Based on the results of an OECD 437 study, the test item is determined to have an eye damaging potential (UN GHS Cat. 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
23 October 2017 - 07 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Methode B.40 bis (In vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Reconstructed Human Epidermis (RHE)
Justification for test system used:
The use of the EpiDerm Kit is state of the art.
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-212-SCT) from MatTek In Vitro Life Science Laboratories, Bratislava
- Tissue batch number(s): 25850
- Delivery date: 24 Oct. 2017
- Date of initiation of testing: 26 Oct. 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: thoroughly rinsed with DPBS
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Anthos Reader 2010 Flexi (Anthos Microsysteme GmbH)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied to the tissues: ca. 25 mg

NEGATIVE CONTROL
- Amount applied: 50 µL water

POSITIVE CONTROL
- Amount applied: 50 µL potassium hydroxide
- Concentration: 8 M
Duration of treatment / exposure:
3 and 60 minutes
Number of replicates:
2 tissues per exposure period, 3 replicates each
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
exposure period 3 minutes
Value:
72.8
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
exposure period 60 minutes
Value:
19.3
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
- OTHER EFFECTS:
- 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
Interpretation of results:
other: not Category 1 (corrosive) based on GHS criteria
Conclusions:
The test item is considered to be non-corrosive to skin.
Executive summary:

The test substance was tested for skin corrosion in vitro using the EpiDermTM test kit according to the OECD guideline 431 under GLP conditions. In brief, reconstructed human epidermis was exposed to either water (negative control), 8 N potassium hydroxide (positive control), or the test material (25 mg with 25 µL distilled water) for 3 and for 60 minutes. Three replicates were run per dose and exposure period. Thereafter, the tissues were incubated with MTT medium. MTT is reduced to a blue formazan dye which is extracted and measured at 570 nm as a parameter for cell viability.

Following 3 minutes treatment with the test item, the mean value of relative tissue viability was reduced to 72.8 %.This value is above the threshold for corrosion potential (50%). After 1 hour treatment, mean value of relative tissue viability was reduced to 19.3 %.This value, too, is above the threshold for corrosion potential (15%). Therefore, the test item is considered to be non-corrosive to skin in the Reconstructed Human Epidermis (RhE) Test Method.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 November 2017 - 23 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human skin model EpiDermTM (MatTek In Vitro Life Science Laboratories, Bratislava)
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT) from MatTek In Vitro Life Science Laboratories, Bratislava
- Tissue batch number(s): 25864
- Delivery date: 12 December 2017

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
- Washing steps: rinsed immediately after 1 hour incubation
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Anthos Reader 2010 Flexi (Anthos Microsysteme GmbH)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the viability after exposure is less than or equal 50%.
- The test substance is considered not to be corrosive or irritant to skin if the viability after exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied to the tissues: ca. 25 mg

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 85%
Duration of treatment / exposure:
1 h
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3 tissues, 2 measurements each
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 1
Value:
4
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 2
Value:
4.3
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 3
Value:
4
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
OTHER EFFECTS
- 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
Interpretation of results:
other: Category 1 (corrosive) or Category 2 (irritant) based on GHS criteria
Conclusions:
The test item is considered to be irritant or corrosive to skin.
Executive summary:

An in vitro skin irritation study according to OECD guideline 439 was performed to assess the skin irritation potential of the test item. Three tissues of the human skin model EpiDermTM were treated with the test item for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance values was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.5. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.2% (required: ≤ 20%). The variation within the tissue replicates of negative, control, positive control and test item was ≤ 18% as required. After the treatment with the test item, the mean value of relative tissue viability was reduced to 4.1%. This value is well below the threshold for skin irritation potential (50%). Test items that induce values below the threshold of 50% are considered irritant or corrosive to skin. Therefore, the test item is considered to be irritant or corrosive to skin in the reconstructed human epidermis test method.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
27 November 2017 - 23 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
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)
Test material information:
Composition 1
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany
- Characteristics of donor animals: 12 - 60 months old
- Storage, temperature and transport conditions of ocular tissue: transported in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container
- indication of any existing defects or lesions in ocular tissue samples: corneas were examined and only corneas which were free from damages were used
Vehicle:
Hank's balanced salt solution
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL of the dissolved test item (150 mg/750 µL 20% HBSS)
- Concentration: 20 %

VEHICLE
- Amount applied: 750 µL
- Concentration: 20 %
- Lot/batch no.: 20171207
Duration of treatment / exposure:
4 h
Number of animals or in vitro replicates:
3 replicates per group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the arrival of the corneas, they were examined and only corneas which were free from damages were used.

NUMBER OF REPLICATES: 3 replicates per group

SOLVENT CONTROL USED: Balanced Salt Solution (HBSS)

POSITIVE CONTROL USED: Imidazole solution (20%)

APPLICATION DOSE AND EXPOSURE TIME:
- application dose: 750 µL of the dissolved test item (150 mg/750 µL 20% HBSS)
- exposure time: 4 h

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: decision criteria as indicated in OECD guideline 437
Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1 / Replicate 1
Value:
94.27
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1 / Replicate 2
Value:
94.74
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1 / Replicate 3
Value:
94.9
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: Yes
- Acceptance criteria met for positive control: Yes
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test item was determined to be have an eye damaging potential (UN GHS Category 1).
Executive summary:

The eye damaging potential of the test item was assessed in a Bovine Corneal Opacity and Permeability (BCOP) test according to OECD guideline 437. Bovine corneas were collected from slaughtered cattle that were between 12 and 60 months old. The test item solution (20% in HBSS) was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In Vitro Irritancy Score) was 1.81. 20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS was 94.64. Under the conditions of this study, the test item induced serious eye damage on the cornea of the bovine eye. The calculated IVIS was 99.44. According to OECD guideline 437, a substance with an IVIS > 55 induces serious eye damage and should be classified as UN GHS Category 1. Therefore, the test item was determined to have an eye damaging potential and is classified as 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

 

OECD 431

The test substance was tested for skin corrosion in vitro using the EpiDermTM test kit according to the OECD guideline 431 under GLP conditions. In brief, reconstructed human epidermis was exposed to either water (negative control), 8 N potassium hydroxide (positive control), or the test material (25 mg with 25 µL distilled water) for 3 and for 60 minutes. Three replicates were run per dose and exposure period. Thereafter, the tissues were incubated with MTT medium. MTT is reduced to a blue formazan dye which is extracted and measured at 570 nm as a parameter for cell viability. Following 3 minutes treatment with the test item, the mean value of relative tissue viability was reduced to 72.8 %.This value is above the threshold for corrosion potential (50%). After 1 hour treatment, mean value of relative tissue viability was reduced to 19.3 %.This value, too, is above the threshold for corrosion potential (15%). Therefore, the test item is considered to be non-corrosive to skin.

OECD 439

An in vitro skin irritation study according to OECD guideline 439 was performed to assess the skin irritation potential of the test item. Three tissues of the human skin model EpiDermTM were treated with the test itemfor 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. After treatment with the negative control, the mean absorbance values was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.5. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.2% (required: ≤ 20%). The variation within the tissue replicates of negative, control, positive control and test item was ≤ 18% as required. After the treatment with the test item, the mean value of relative tissue viability was reduced to 4.1%. This value is well below the threshold for skin irritation potential (50%). Test items that induce values below the threshold of 50% are considered irritant or corrosive to skin. Therefore, the test item is considered to be irritant or corrosive to skin in the reconstructed human epidermis test method.

Eye irritation

 

OECD 437

The eye damaging potential of the test item was assessed in a Bovine Corneal Opacity and Permeability (BCOP) test according to OECD guideline 437. Bovine corneas were collected from slaughtered cattle that were between 12 and 60 months old. The test item solution (20% in HBSS) was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In Vitro Irritancy Score) was 1.81. 20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS was 94.64. Under the conditions of this study, the test item induced serious eye damage on the cornea of the bovine eye. The calculated IVIS was 99.44. According to OECD guideline 437, a substance with an IVIS > 55 induces serious eye damage and should be classified as UN GHS Category 1. Therefore, the test item was determined to have an eye damaging potential and is classified as UN GHS Category 1.

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 1272/2008. Based on this data, the substance is considered to be classified for skin irritation (UNG GHS Cat. 2, H315) and eye damaging potential (UN GHS Cat. 1, H318) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.