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Description of key information

Based on the results of the OECD 431 study and the OECD 439 study, the test item is considered to be corrosive to skin (CLP/GHS).

Based on the results of the OECD 437 study, the test item is serious eye damaging (CLP/EPA/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:
key study
Study period:
11 April 2017 - 21 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: B.40 bis (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2008
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: EpiDerm TM Kit (human-derived epidermal keratinocytes)
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Epi-200 kits and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia). EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
Justification for test system used:
The EpiDerm TM is an in vitro system recommended by the corresponding guideline.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 Kit
- Tissue batch number: 25811
- Date of initiation of testing: 2017-05-10

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C for 3 minutes; room temperature for 60 minutes

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 20 times
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax Molecular Devices, Softmax Pro
- 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 to skin if:
- < 50% after 3 minutes exposure
- ≥ 50% after 3 minutes exposure AND < 15% after 60 minutes exposure
The test substance is considered to be non-corrosive to skin if:
- ≥ 50% after 3 minutes exposure AND ≥ 15% after 60 minutes exposure
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL
Duration of treatment / exposure:
3 min and 60 min
Number of replicates:
3
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
1_Exposure: 3 min
Value:
97.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
2_exposure: 3 min
Value:
94.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
Corrected Mean_Exposure: 3 min
Value:
91.5
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
1_Exposure: 1 hour
Value:
21.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
2_Exposure: 1 hour
Value:
24.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (%)
Run / experiment:
Corrected Mean_Exposure: 1 hour
Value:
6
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: yes, an additional test with freeze-killed tissues was performed to determine a correction factor for calculating the true viability in the main experiment
- Colour interference with MTT: none

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:
Category 1 (corrosive) based on GHS criteria
Conclusions:
The test item is considered to be corrosive to skin according to EU CLP and UN GHS.
Executive summary:

An in vitro study was performed to assess the corrosive potential of the test substance by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item passed the colour interference pre-test. Since it proved to be a MTT reducer in the MTT interference pre-test, an additional test with freeze-killed tissues was necessary.

Independent duplicate tissues of EpiDermTM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for 17.6 hours at room temperature.

After exposure to the test item the corrected relative absorbance value decreased to 91.5% after 3 minutes exposure. After 1 hour exposure the corrected relative absorbance value was reduced to 6.0%. The 1-hour-value fell below the threshold for corrosivity which is defined to be 15%.

The required acceptability criteria were met.

The test item is considered to be corrosive.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 February 2017 - 11 July 2017
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:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Epi-200 SIT kits and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
Justification for test system used:
The EpiDerm TM is an in vitro system recommended by the corresponding guideline.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Kit
- Tissue batch number: 23399
- Date of initiation of testing: 2017-03-14

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C for 35 minutes; room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with DPBS at least 15 times
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax Molecular Devices, Softmax Pro
- 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 irritant to skin (Category 2) if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control
- The test substance is considered to be non-irritant to skin if the mean relative tissue viability of three individual tissues is reduced > 50% of the negative control
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
25 h + 18 h
Number of replicates:
3
Irritation / corrosion parameter:
other: relative absorbance (% of negative control)
Run / experiment:
Test Item - Tissue 1
Value:
3.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (% of negative control)
Run / experiment:
Test Item - Tissue 2
Value:
4.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (% of negative control)
Run / experiment:
Test Item - Tissue 3
Value:
4.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: relative absorbance (% of negative control)
Run / experiment:
Corrected Mean
Value:
1.4
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: no
- Direct-MTT reduction: yes (additional test with freeze-killed tissues)
- 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 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 passed the colour interference pre-test. Due to its MTT reducing property, an additional test with freeze-killed tissues was performed.

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SLS) for 60 minutes.

The required acceptability criteria were met.

After treatment with the test item the corrected mean relative absorbance value decreased to 1.4% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

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:
17 February 2017 - 15 March 2017
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)
Version / remarks:
July 2013
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:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: isolated bovine cornea
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 months old
- Storage temperature: ambient temperature
- Transport conditions of ocular tissue: The isolated eyes were transported to the laboratory in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin).
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and were directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
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):
2 hours
Number of animals or in vitro replicates:
3 corneas per group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
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 according to the description given in OECD guideline 437. The endothelial side of the cornea was positioned against the sealing ring (Oring) 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. The posterior compartment was filled first. Care was taken to assure no air bubbles were present within the compartments. The corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

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 (t0). Each corneae with a value of the basal opacity > 7 was discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 min

POST-INCUBATION PERIOD: yes (2 hours)

REMOVAL OF TEST SUBSTANCE
- POST-EXPOSURE INCUBATION: 2 hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: OP_KIT opacitometer (changes in the light transmission passing through the cornae)
- Corneal permeability: Microtiter plate reader (OD490, passage of sodium fluorescein dye)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS ≤ 3 No Category (UN GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Category 1 (UN GHS)
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
87.07
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
2
Value:
96.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Value:
75.48
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
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 is serious eye damaging (CLP/EPA/GHS (Cat 1)).
Executive summary:

To assess the corneal damage potential of the test item, an in vitro study according to OECD TG 437 was performed.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive control (2-Ethoxyethanol), and the negative control (saline) were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured a second time (t130).

After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

The test item was tested undiluted. Relative to the negative control, the test item caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 86.42 (threshold for serious eye damage: IVIS ≥ 55).

The required acceptability criteria were met.

The test item is considered to be classified as serious eye damaging.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation study:

An 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 passed the colour interference pre-test. Due to its MTT reducing property, an additional test with freeze-killed tissues was performed.

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SLS) for 60 minutes.

The required acceptability criteria were met.

After treatment with the test item the corrected mean relative absorbance value decreased to 1.4% 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 considered to possess an irritant potential.

Skin corrosion study:

An in vitro study was performed to assess the corrosive potential of the test substance by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item passed the colour interference pre-test. Since it proved to be a MTT reducer in the MTT interference pre-test, an additional test with freeze-killed tissues was necessary.

Independent duplicate tissues of EpiDermTM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for 17.6 hours at room temperature.

After exposure to the test item the corrected relative absorbance value decreased to 91.5% after 3 minutes exposure. After 1 hour exposure the corrected relative absorbance value was reduced to 6.0%. The 1-hour-value fell below the threshold for corrosivity which is defined to be 15%.

The required acceptability criteria were met.

The test item is considered to be corrosive.

 

Eye irritation study:

To assess the corneal damage potential of the test item, an in vitro study according to OECD TG 437 was performed.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive control (2-Ethoxyethanol), and the negative control (saline) were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured a second time (t130).

After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

The test item was tested undiluted. Relative to the negative control, the test item caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 86.42 (threshold for serious eye damage: IVIS ≥ 55).

The required acceptability criteria were met.

The test item is considered to be classified as serious eye damaging.

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 1272/2008. Based on this data, the substance is considered to be skin corrosive and eye irritant (Cat. 1, H314, H318) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.