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Administrative data

Description of key information

Based on the results of an OECD 439 study the test item is not requiring classification for skin irritation (UN GHS: No Category) (reference 7.3.1 -1).

Based on the results of an OECD 437 study the test item is not requiring classification for eye irritation or serious eye damage (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:
26 September 2016 - 21 November 2016
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:
adopted July 28, 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation 440/2008, ATP 640/2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: reconstituted human epidermis
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
- Tissue batch number(s): 16-RHE-109
- 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: rinsed with minimum 25 mL DPBS; excess DPBS removed by shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Observable damage in the tissue due to washing: no data
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader ELx800, BioTek Instruments 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 irritant to skin category 2 if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 16 mg
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 triplicate tissues (3 aliquots per tissue)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 1
Value:
91.62
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 2
Value:
87.19
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 3
Value:
81.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
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:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).
Executive summary:

An in vitro study in accordance with the OECD Guideline OECD 439 was performed to assess the skin irritation potential of the test item.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes. 16 µL of either the negative control (DPBS) or the positive control (5% SLS) 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.

The test item did not reduce MTT, and it did not indicate colour interference. All of the acceptability criteria were met. The absorbance values of the negative control were between 1.7 and 1.9. The positive control induced a sufficient decrease of the relative absorbance as compared to the negative control. The relative standard deviations of three replicates were below 18%.

As a result, the mean relative absorbance was determined to be 86.8% compared to the relative absorbance of the negative control after treatment with the test item. Therefore, the test item is not considered to possess an irritant potential to skin under the experimental conditions chosen (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:
03 January 2017 - 17 February 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:
Commission Regulation (EU) No. 1152/2010
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
- Characteristics of donor animals: 18 - 24 months old
- Storage, temperature and transport conditions of ocular tissue: 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.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL of the dissolved test item (i.e. 0.15 g/0.75 mL 0.9% sodium chloride solution)

VEHICLE
- Amount applied: 0.75 mL
- Concentration: 0.9%
- Lot/batch no.: 15464012 (B. Braun Melsungen AG, Germany)
Duration of treatment / exposure:
240 min
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: No, vehicle control used

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL of the dissolved test item (i.e. 0.15 g/0.75 mL 0.9% sodium chloride solution) and 240 min

POST-INCUBATION PERIOD: no

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)
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:
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)

ACCEPTANCE CRITERIA:
A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean(IVIS positive control: 79.9 - 134.6). The negative control responses should result in an IVIS that falls within three standard deviations of the current historical mean (IVIS negative control: -1.4 - 3.4).
A single test run with three corneas should be sufficient for a test item when the resulting classification is unequivocal. In cases of the following borderline results in the first testing run, a second test run should be considered.
- 2 of the 3 corneas give discordant predictions from the mean of all 3 corneas or
- 1 of the 3 corneas give discordant predictions from the mean of all 3 corneas, and the discordant result is >10 IVIS units from the cut-off threshold of 55.
Irritation parameter:
in vitro irritation score
Remarks:
replicate 1
Run / experiment:
1
Value:
0.585
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
replicate 2
Run / experiment:
1
Value:
0.72
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
replicate 3
Run / experiment:
1
Value:
1.115
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: Yes
- Acceptance criteria met for positive control: Yes
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the OECD 437 study the test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).
Executive summary:

To assess the corneal damage potential of the test item, an in vitro study according to OECD Guideline 437 was performed. 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 in an incubator in horizontal position for 240 minutes at 32°C. 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 at 32°C. 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 vehicle control (0.9% sodium chloride solution) the calculated IVIS was 1.2 (study acceptance criteria range: -1.4 - 3.4). Treatment with the positive control (20% Imidazole) revealed an IVIS of 102.3 (study acceptance criteria range: 79.9 - 134.6). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with the test item was 0.8 and, thus, lower than 3, i.e. according to OECD 437 the test item 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)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation study

An in vitro study in accordance with the OECD Guideline OECD 439 was performed to assess the skin irritation potential of the test item.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes. 16 µL of either the negative control (DPBS) or the positive control (5% SLS) 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.

The test item did not reduce MTT, and it did not indicate colour interference. All of the acceptability criteria were met. The absorbance values of the negative control were between 1.7 and 1.9. The positive control induced a sufficient decrease of the relative absorbance as compared to the negative control. The relative standard deviations of three replicates were below 18%.

As a result, the mean relative absorbance was determined to be 86.8% compared to the relative absorbance of the negative control after treatment with the test item. Therefore, the test item is not considered to possess an irritant potential to skin under the experimental conditions chosen (UN GHS: No Category).

Eye irritation/corrosion study

To assess the corneal damage potential of the test item, an in vitro study according to OECD Guideline 437 was performed. 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 in an incubator in horizontal position for 240 minutes at 32°C. 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 at 32°C. 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 vehicle control (0.9% sodium chloride solution) the calculated IVIS was 1.2 (study acceptance criteria range: -1.4 - 3.4). Treatment with the positive control (20% Imidazole) revealed an IVIS of 102.3 (study acceptance criteria range: 79.9 - 134.6). Therefore, the study fulfilled the acceptance criteria.

The IVIS obtained after treatment with the test item was 0.8 and, thus, lower than 3, i.e. according to OECD 437 the test item 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 1272/2008. Based on this data, the substance is not considered to be classified for skin irritation and eye irritation under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.