3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 29. Jul. 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU-Method B.40 BIS. “IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST“

Version / remarks:

30. May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

EC Number:

204-729-7

EC Name:

3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

Cas Number:

125-20-2

Molecular formula:

C28H30O4

IUPAC Name:

3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

Details on test material:

- Storage conditions: Room Temperature (20 ± 5 °C)
- Expiry date: 16. Dec. 2019

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 arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures 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 arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.

Origin
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Day of delivery: 17. Jul. 2018
Batch no.: 28634

Justification for test system used:

As stipulated under REACH and the guideline

Vehicle:

unchanged (no vehicle)

Remarks:

test item were applied together with 25 µL demineralised water

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): 28634
- Delivery date: 2018-07-17

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: After the respective incubation time (“3 minutes” and “1 hour”) at 37 ± 1°C and 5.0 ±0.5% CO2, the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with DPBS, blotted with sterile cellulose tissue and set into the respec-tive holding plate, using the wells containing assay medium.
- 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 (choose relevant statement)
- 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
25 mg of the test item were applied together with 25 µL demineralised water.
The following amounts were applied to the tissues:
Tissue Amount
1 25.7 mg
2 25.0 mg

NEGATIVE CONTROL
Demineralised water

POSITIVE CONTROL
KOH, CAS No. 1310-58-3, solution in demineralised water (8 M)

Duration of treatment / exposure:

1 hour

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value

Value:

94.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

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:

GHS criteria not met

Remarks:

not corrosive

Conclusions:

The study was conducted under GLP according to OECD guideline 431 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 corrosive potential of the test substance to the skin in vitro. The viability of the cells treated with the test item was ≥ 50% (3 min treatment) and ≥ 15% (60 min) of negative control. Hence, the test item was considered to be non-corrosive to the skin.
In a tiered in vitro testing strategy, this result (non-corrosive) may however only give an indication of the skin-damaging potential, but does not allow a definitive classification according to Regulation 1272/2008. Hence, an additional skin irritation test according to OECD 439 was conducted.

Executive summary:

The skin corrosion potential of the test item has been determined in the Reconstructed Human Epidermis (RHE) Test Method following OECD Guideline 431 and EU Method B.40-BIS (GLP).

One valid experiment was performed. Two tissues of the human skin model EpiDermTM were treated with the test item for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size.

Demineralised water was used as negative control, 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.7 (3 minutes experiment) and 1.8 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 5.4% for the 1 hour treatment.

After 3 minutes treatment with the test item, the mean value of relative tissue viability was reduced to 93.9%. This value is above the threshold for corrosion potential (50%). After 1  hour treatment, mean value of relative tissue viability was decreased to 94.3%. This value, too, is above the threshold for corrosion potential (15%).

Therefore, the test item is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method and will be further tested in an in vitro skin irritation test.

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