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Administrative data

Description of key information

- not skin irritating [OECD 439, GLP]

- not eye irritating [OECD 492, GLP]

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:
2019-01-14 - 2019-01-18 (experimental phase)
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)
Version / remarks:
OECD Guideline for the Testing of Chemicals, Version 439, adopted 28. July 2015, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EU) No. 640/2012 amending Regulation (EC) No. 761/2009, Annex III, EU method B.46 “IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST”, adopted 06. Jul. 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage: Fridge (2 – 8 °C); keep away from light; keep away from humidity
- Stability: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: multiple donors, not specified
Source strain:
other: not applicable
Details on animal used as source of test system:
Source: 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.

Test System

Specification
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
Day of delivery: 15. Jan. 2018
Batch no.: 28679
Justification for test system used:
as stipulated under REACH and the respective Guidelines
Vehicle:
unchanged (no vehicle)
Remarks:
The tissues were wetted with 25 µL DPBS buffer before applying the test item.
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava, EPI-200-SIT
- Tissue batch number(s): 28679
- Delivery date: 2018-01-15

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
- Observable damage in the tissue due to washing: none stated
- Modifications to validated SOP: none stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h
- Spectrophotometer: plate spectrophotometer: 96-well-plate photometer, Anthos Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 replicates for each test item, positive and negative control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment, 3 replicates

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if the viability after 1h exposure is greater than or equal to 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
One plate was used for treatment with the test item:
The tissues were wetted with 25 µL DPBS buffer before applying the test item and spreading it to match the tissue size.
The following amounts were applied to the tissues:
Tissue Amount
1 26.6 mg
2 23.9 mg
3 25.6 mg

NEGATIVE CONTROL
One plate (3 tissues) 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.

POSITIVE CONTROL
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.
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
23 hours and 30 minutes
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of three tissues
Value:
74.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
tissue 1
Value:
84.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
tissue 2
Value:
78.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
tissue 3
Value:
61.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- 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: EU-GHS criteria not met
Conclusions:
- mean value of relative tissue viability was reduced to 74.6 %
- test item is considered non-irritant to skin [since obtained value is above the threshold for skin irritation potential (50%)]
Executive summary:

An OECD 439 (in vitro skin irritation) study under GLP was performed in order to evaluate the potential of 2-hydroxy-1-[4-(4-(2-hydroxy-2-methylpropionyl)phenoxy)phenyl]-2-methylpropan-1-one to evoke skin irritation in a reconstructed human epidermis (RhE) test method.

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 value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.6. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.6 % (required: ≤20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%).

After the treatment with the test item, the mean value of relative tissue viability was reduced to 74.6 %. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin.

Therefore, the test item 2-hydroxy-1-[4-(4-(2-hydroxy-2-methylpropionyl)phenoxy)phenyl]-2-methylpropan-1-one is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

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:
2018-12-10 - 2018-12-13 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP
Qualifier:
according to guideline
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)
Version / remarks:
OECD Guideline for the Testing of Chemicals, Part 492, adopted 25. Jun. 2018, “Re-constructed 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. QA statement)
Remarks:
Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Specific details on test material used for the study:
The test item was stored in the test facility in a closed vessel in the refrigerator (2 – 8 °C); protected from light and humidity.
Species:
human
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The EpiOcularTM Eye Irritation Test (EIT) predicts the acute eye hazard potential of chemi-cals by measurement of tissue damage caused by cytotoxic effects in the reconstructed human cornea-like tissue model. Within a testing strategy, the EpiOcularTM EIT can be used as a replacement of the in vivo Draize Eye Irritation Test.
It is utilized for the classification and labelling of chemicals concerning their eye hazard potential. The EpiOcular™ EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. A limitation of this guideline is that it neither allows discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B). For these purposes, further testing with other suitable test methods is required.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
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, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 11. Dec. 2018
Batch no.: 27085
The cells used to produce EpiOcular tissues are screened for potential biological contaminants. None of the following potential contaminants were detected:
HIV-1 virus (Oligonucleotide-directed amplification)
Hepatitis B virus (Oligonucleotide-directed amplification)
Hepatitis C virus (Oligonucleotide-directed amplification)
Bacteria, yeast, other fungi (long-term antibiotic, antimycotic free culture)
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 52.6 and 50.8 mg / tissue
- Concentration (if solution): undiluted

VEHICLE
none
Duration of treatment / exposure:
6 hours
Observation period (in vivo):
n/a
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
- RhCE tissue construct used, including batch number
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.
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-212-EIT
Day of delivery: 11. Dec. 2018
Batch no.: 27085
- Doses of test chemical and control substances used
Test chemical: 52.6 and 50.8 mg/tissue
Controls: 50 µL of the controls
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
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 ≥ 95% relative humidity for 29 minutes. After that, 50 µL of the controls and a defined amount of the test item (see table 7.2-a) were applied in duplicate in one- minute- 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 ≥ 95% 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 17 hours and 45 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
After the post-treatment incubation, the MTT assay was performed.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
Spectrophotometer, 570 nm
- Description of the method used to quantify MTT formazan
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 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% 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 was 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-well-plate. Eight wells with 200 µL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
OD value; > 60% of control
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
Parameter Optical Density Negative Control Relative Tissue Viability Positive Control
Demineralised H2O Methyl acetate
Exposure time 6 hours
Mean 1.631 34.8%
Standard deviation 0.271 7.3%
Range 1.047 - 2.340 21.1 - 53.9%
Current study 1.725 42.7%

- Complete supporting information for the specific RhCE tissue construct used
- Reference to historical data of the RhCE tissue construct
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
The validity of the EpiOcularTM test at the laboratory was demonstrated in a proficiency study. For this purpose 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized. Therefore, the proficiency of the EpiOcularTM test was demonstrated.

- Positive and negative control means and acceptance ranges based on historical data
Yes
- Acceptable variability between tissue replicates for positive and negative controls
< 20%
- Acceptable variability between tissue replicates for the test chemical
< 20%
Irritation parameter:
other: % tissue viability
Run / experiment:
mean test item
Value:
77.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: % tissue viability
Run / experiment:
mean positive control
Value:
42.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not examined
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none stated

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not different
Interpretation of results:
other: EU-GHS criteria not met
Conclusions:
The study was conducted under GLP according to OECD guideline 492 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritation / corrosion potential of the test substance to the eye in vitro.
After treatment with the test item, the mean value of relative tissue viability was 77.4 %. This value is well above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant.
According to the Guideline, the EpiOcular™ EIT, SkinEthic™ HCE EIT and LabCyte CORNEA-MODEL24 EIT are recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) (1) without further testing, within a testing strategy such as the Bottom-Up/Top-Down approach.
So the test item does not need to be classified as eye irritant according to Regulation 1272/2008 and amendments, and further testing is not required.
Executive summary:

One valid experiment was performed according to OECD TG 492 under GLP.

The test item 2-hydroxy-1-[4-(4-(2-hydroxy-2-methylpropionyl)phenoxy)phenyl]-2-methylpropan-1-one was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours.

 

After treatment, the respective substance was rinsed from the tissue; then, 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.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.7. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 42.7 % (< 50%).

The variation within tissue replicates of the controls and the test item was acceptable (< 20%).

 

After treatment with the test item, the mean value of relative tissue viability was 77.4 %.

This value is well above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant.

 

Under the conditions of the test, 2-hydroxy-1-[4-(4-(2-hydroxy-2-methylpropionyl)phenoxy)phenyl]-2-methylpropan-1-one is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A reliability score of 1 was assigned for the eye and skin irritation study, in accordance with the criteria for assigning data quality in line with the principles described by Klimisch (1997).

Justification for classification or non-classification

Based on reliable in vitro data, the substance 2-Hydroxy-1-[4-(4-(2-hydroxy-2-methylpropionyl)phenoxy)phenyl]-2-methylpropan-1-one is considered to be non-irritant to eye/skin and does not require classification according to Regulation (EC) No. 1272/2008.