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Description of key information

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:
From August 07, 2017 to August 11, 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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No.: WD0066442; Purity: 100 %; Appearance: homogeneous amber liquid
Test system:
human skin model
Remarks:
EpiDermTM tissues
Source species:
other: human-derived epidermal keratinocytes
Cell type:
non-transformed keratinocytes
Cell source:
other:
Vehicle:
unchanged (no vehicle)
Details on test system:
The test system is a commercially available EpiDermTM-Kit, procured by MatTek. The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.

Origin:
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
One plate was used as negative control; each tissue was treated with 30 μL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate was used as positive control; each tissue was treated with 30 μL 5% SDS solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.

One plate was used for treatment with the test substance:
For each plate, 3 tissues were used. The tissues were wetted with 25 μL DPBS buffer before applying the test substance and spreading it to match the tissue size.
The following amounts were applied to the tissues:
Replicate 1: 26.6 mg
Replicate 2: 26.2 mg
Replicate 3: 26.7 mg

Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2. 1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals. After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). Then, the tissues were set in the incubator for 23 hours 30 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.

Medium Renewal
After post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-wellplate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 hours 5 minutes for post-incubation at 37 ± 1 °C and 5.0 ± 0.5% CO2.
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
23 hours and 30 minutes
Number of replicates:
3 replicates
Irritation / corrosion parameter:
% tissue viability
Value:
72.4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Results:
% Tissue viability (tissue 1): 71.5 %
% Tissue viability (tissue 2): 73.1 %
% Tissue viability (tissue 3): 72.6 %
% Tissue viability (mean) 72.4 %

Tissue Viability

The photometric absorbance of the negative controls is considered as 100%. For each replicate of test substance and positive control, tissue viability is calculated as % photometric absorbance compared with the mean of the negative controls:
% Tissue Viability = (OD replicate test substance resp. positive control x 100%) / OD negative controls

Acceptance of the results:

- The test substance is considered as non-irritant to skin.
- After the treatment, the relative absorbance values were reduced to 99.4%. This value is above the threshold for skin irritation (50%).
- The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8.
- The positive control has met the acceptance criterion too, demonstrating the validity of the test system.
- Variation within replicates was within the accepted range for negative control, positive control and test substance (required: ≤ 18%).

Validity:
- OD of negative control: 1.6 (Required: ≥ 0.8 and ≤ 2.8)
- % Tissue viability of positive control SDS: 2.2% (Required: ≤ 20 % of negative control)
- SD of mean viability of the tissue replicates (≤18 %):

9.7% (negative control)
0.2% (positive control)
0.8% (test substance)
Interpretation of results:
GHS criteria not met
Remarks:
non-irritant
Conclusions:
Under the study conditions, after the treatment with the test substance, the mean value of relative tissue viability was reduced to 72.4%. This value is above the threshold for skin irritation potential (50%). Therefore, the test substance is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) test method.
Executive summary:

A study was conducted to determine the skin irritation potential of the test substance using the Reconstructed Human Epidermis (RHE) Test Method according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. One valid experiment was performed. Three tissues of the human skin model EpiDermTM were treated with the test substance for 60 minutes. The test substance 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 absorbance values with a mean OD of 1.6, were within the required acceptability criterion of 0.8≤ mean OD ≤2.8. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.2%. The variation within the tissue replicates of negative control, positive control and test substance was acceptable (required: ≤ 18%). Under the study conditions, after the treatment with the test substance, the mean value of relative tissue viability was reduced to 72.4%. This value is above the threshold for skin irritation potential (50%). Therefore, the test substance is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) test method (Andres, 2017).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 11, 2017 to September 28, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No.: WD0066442; Purity: 100 % (UVCB); Appearance: amber, homogeneous liquid
Species:
other: EpiOcularTM tissue
Details on test animals or tissues and environmental conditions:
Specification:
Commercially available EpiOcularTM kit.
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

Origin:
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Batch no.: 27006
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Tissue 1: 51.1 mg
Tissue 2: 51.6 mg
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
Pre-Tests:
Assessment of direct reduction of MTT by the test substance:
50.9 mg of the test substance was added to 1 mL of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 and 80 – 100 % relative humidity for 3 hours. 1 mL of MTT solution plus 50 μL of H2O demin was used as negative control. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.

Assessment of coloured or staining test substances:
51.9 mg of the test substance was added to 2 mL isopropanol, incubated in 6-well plates on an orbital shaker for 3 hours at room temperature. Then, two 200 μL aliquots of the resulting solution and two 200 μL aliquots of neat isopropanol were transferred into a 96-well plate and measured with a plate reader at 570 nm. After subtraction of OD for isopropanol, the OD of the test substance solution was 0.00 (≤ 0.08). Therefore, the main test was performed without colourant controls.


Main Test:
On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use.
The assay medium was warmed in the water bath to 37 ± 1 °C. 6-well-plates were labelled with test substance, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour.
After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours.


Exposition and Post-Treatment:
After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes. After that, 50 μL of the controls and a defined amount of the test substance were applied in duplicate in 1- min- intervals. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature.

After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. After the post-treatment incubation, the MTT Assay was performed.


MTT Assay and Extraction:
A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 3 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.

Measurement:
The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate. Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm

Irritation parameter:
other: tissue viability (%)
Run / experiment:
6 h
Value:
2.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Validity criteria:
OD of negative control: >0.8 and <2.5, result: 1.6
% mean relative viability of positive control: < 50 % of negative control, result: 42.2 %
Variation within replicates: < 20 %,
result:
0.2 % (negative control)
3.5 % (positive control)
0.4 % (test substance)
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the study conditions, the test substance is considered either eye irritant or inducing serious eye damage in the EpiOcularTM eye irritation test.
Executive summary:

A study was conducted to determine an eye irritation potential of the tests substance according to OECD Guideline 492, in compliance with GLP. The test substance was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 h. The jellylike test substance was applied to two tissue replicates (51.1 and 51.6 mg). After treatment, the respective substance was rinsed from the tissue and cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. After treatment with the negative control, the mean absorbance values was 1.6, being within the required acceptability criterion of mean OD >0.8 and <2.5. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 42.2% (<50%). Variation within tissue replicates was acceptable (<20%). After treatment with the test substance, the mean value of relative tissue viability was reduced to 2.4%. This value is below the threshold for eye irritation potential (≤60%). According to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required. Under the study conditions, the test substance is considered either eye irritant or inducing serious eye damage in the EpiOcularTM eye irritation test (Andres, 2017).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 17, 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)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No.: WD0066442; Purity: 100 %; Appearance: homogeneous amber liquid
Species:
cattle
Strain:
other: Bos primigenius Taurus
Details on test animals or tissues and environmental conditions:
Specification:
Species Bos primigenius Taurus (fresh bovine corneas)

Origin:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1 % Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Neat test substance tested in three replicates:
Replicate 1: 1563.6 mg
Replicate 2: 1516.2 mg
Replicate 3: 1531.5 mg
Duration of treatment / exposure:
10 minutes at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
2 hours at 32 ± 1 °C
Details on study design:
Preparations:
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C. On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate. After the arrival of the corneas, they were examined and only corneas which were free from damages were used. 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. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

Method description:
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage, therefore, all corneas were used. For each treatment group (negative control solution, test substance and positive control solution), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative control solution, test substance or positive control solution were applied to each replicate.

Open Chamber Method:
The “open chamber-method” is used for solid substances. In order to apply the test substance, the nut was unscrewed to remove the glass disc. The test substance could be applied directly on the cornea now. The following amounts of the test substance were tested neat and applied directly on the cornea using a weighing funnel: 1563.6 mg, 1516.2 mg, 1531.5 mg.

The test substance was applied on the epithelium in such a manner that as much as possible of the cornea was covered with test substance. Exposure time on the corneas was 10 minutes at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the corneas were stored for additional 2 hours at 32 ± 1 °C (post-incubation). Then, the final illuminance value of each cornea was recorded.

Permeability Test:
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas. For the open chamber method, a sodium fluorescein solution with a concentration of 4 mg/mL was used. The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured as optical density of the liquid with a microtiter plate photometer at 492 nm.


Irritation parameter:
in vitro irritation score
Value:
19.21
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
SD: 92.67%
Other effects / acceptance of results:
According to the guideline, the test is considered valid if the positive control causes an IVIS that falls within the two standard deviations of the current historical mean value. The negative control has to show an IVIS ≤ 3.
IVIS of negative control: 0.44 (criterion: ≤ 3)
IVIS of positive control: 72.28 (criterion: 41.63 – 148.12)
Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.
Interpretation of results:
study cannot be used for classification
Conclusions:
Under the study conditions, the test substance showed effects on the cornea of the bovine eye. The calculated IVIS was established at 19.21. According to OECD Guideline 437, a substance with an IVIS >3 and ≤55 induces effects on the cornea and cannot be classified in an UN GHS Category for eye damage based on this study design only.
Executive summary:

A study was conducted to determine the corneal damage potential by the test substance according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. One valid experiment was performed. Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old. The test substance was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1°C for 1 h and whose opacity had been measured. The test substance was incubated on the cornea for 10 minutes at 32 ± 1°C. After removal of the test substance and 2 h post-incubation, opacity and permeability values were measured. HBSS-solution was used as negative control and undiluted Dimethylformamide (DMF) was used as positive control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) was 0.44. 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 72.28. Under the study conditions, the test substance showed effects on the cornea of the bovine eye. The calculated IVIS was established at 19.21. According to OECD Guideline 437, a substance with an IVIS >3 and ≤55 induces effects on the cornea and cannot be classified in an UN GHS Category for eye damage based on this study design only (Anders, 2017).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

A study was conducted to determine the skin irritation potential of the test substance using the Reconstructed Human Epidermis (RHE) Test Method according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. One valid experiment was performed. Three tissues of the human skin model EpiDermTM were treated with the test substance for 60 minutes. The test substance 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 absorbance values with a mean OD of 1.6, were within the required acceptability criterion of 0.8≤ mean OD ≤2.8. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.2%. The variation within the tissue replicates of negative control, positive control and test substance was acceptable (required: ≤ 18%). Under the study conditions, after the treatment with the test substance, the mean value of relative tissue viability was reduced to 72.4%. This value is above the threshold for skin irritation potential (50%). Therefore, the test substance is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) test method (Andres, 2017).

Eye irritation

Study 1:

A study was conducted to determine the corneal damage potential by the test substance according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. One valid experiment was performed. Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old. The test substance was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1°C for 1 h and whose opacity had been measured. The test substance was incubated on the cornea for 10 minutes at 32 ± 1°C. After removal of the test substance and 2 h post-incubation, opacity and permeability values were measured. HBSS-solution was used as negative control and undiluted Dimethylformamide (DMF) was used as positive control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) was 0.44. 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 72.28. Under the study conditions, the test substance showed effects on the cornea of the bovine eye. The calculated IVIS was established at 19.21. According to OECD Guideline 437, a substance with an IVIS >3 and ≤55 induces effects on the cornea and cannot be classified in an UN GHS Category for eye damage based on this study design only (Anders, 2017).

Study 2:

A study was conducted to determine an eye irritation potential of the tests substance according to OECD Guideline 492, in compliance with GLP. The test substance was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 h. The jellylike test substance was applied to two tissue replicates (51.1 and 51.6 mg). After treatment, the respective substance was rinsed from the tissue and cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. After treatment with the negative control, the mean absorbance values was 1.6, being within the required acceptability criterion of mean OD >0.8 and <2.5. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 42.2% (<50%). Variation within tissue replicates was acceptable (<20%). After treatment with the test substance, the mean value of relative tissue viability was reduced to 2.4%. This value is below the threshold for eye irritation potential (≤60%). According to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required. Under the study conditions, the test substance is considered either eye irritant or inducing serious eye damage in the EpiOcularTM eye irritation test (Andres, 2017).

Justification for classification or non-classification

The available OECD 492 study indicates that the test substance is ‘’eye irritating’’ or ‘’induces serious eye damage’’. However, the result of the OECD 437 study do not trigger the Category 1 classficiation. Based on both studies, a classification as Eye Damage 2 - H319: (causes serious eye irritation) is proposed according to EU CLP (EC 1272/2008) criteria.