2-hydroxy-1-[4-[4-(2-hydroxy-2-methylpropanoyl)phenoxy]phenyl]-2-methylpropan-1-one

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

472-110-0

EC Name:

-

Cas Number:

71868-15-0

Molecular formula:

C20H22O5

IUPAC Name:

2-hydroxy-1-{4-[4-(2-hydroxy-2-methylpropanoyl)phenoxy]phenyl}-2-methylpropan-1-one

Test material form:

solid: particulate/powder

Remarks:

off white to pale yellow powder

Details on test material:

Storage: Fridge (2 – 8 °C); keep away from light; keep away from humidity

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

In vitro test system

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

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Applicant's summary and conclusion

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:

A study was performed to assess the potential of the test substance to cause skin irritation in a reconstructed human epidermis (RhE) method according to OECD Guideline 439, in compliance with GLP. Three EpiDermTM human skin tissues were exposed 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 as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability range of 0.8 ≤ mean OD ≤ 2.8. The positive control showed clear irritating effects. Mean relative tissue viability was reduced to 2.6% (required: ≤20%). The variation within the tissue replicates of negative control, positive control and test substance was acceptable (required: ≤ 18%). After treatment with the test substance, mean relative tissue viability was reduced to 74.6%. This is above the threshold for skin irritation (50%). Substances that induce values above the threshold are considered non-irritant. Therefore, under the study conditions, the test substance was concluded to be non-irritating to skin (Andres, 2019).