Cerium gadolinium oxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 APR 2021 to 10 MAY 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cerium gadolinium oxide

EC Number:

953-470-2

Molecular formula:

Gd2CeO5

IUPAC Name:

Cerium gadolinium oxide

Test material form:

solid: nanoform

Specific details on test material used for the study:

The test item was a (white) powder, which is an appropriate form to test solids in this type of test.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

not specified

Justification for test system used:

The in vitro test system of reconstructed human epidermis closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. This model used in this study was a three-dimensional human epidermis model, consisting of adult human-derived epidermal keratinocytes which had been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which resulted in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model (EPISKIN-SM, 0.38 cm2) - Source: SkinEthic Laboratories, Lyon, France.

- Tissue batch number: 21-EKIN-018.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature.
- Temperature of post-treatment incubation: 37°C.

PREPARATION OF THE TEST SYSTEM:
- On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 23 hours at 37°C.

ENVIRONMENTAL CONDITIONS FOR INCUBATIONS:
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 67 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

REMOVAL OF TEST MATERIAL AND CONTROLS
- The tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS (phosphate bufferred saline).
- Incubation time: 3 hours at 37°C.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.

NUMBER OF REPLICATE TISSUES: 3.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1.

PREDICTION MODEL / DECISION CRITERIA :
- A test item is considered irritant in the skin irritation test if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is < 50% of the mean viability of the negative controls.
- A test item is considered non-irritant in the in vitro skin irritation test if the relative mean tissue viability of three individual tissues alter 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: the skin was moistened with 5µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item (19.1 to 37.2 mg) was added into 12-well plates on top of the skin tissues.

NEGATIVE CONTROL
- Three tissues were treated with 25 µL PBS (negative control).

POSITIVE CONTROL
- Three tissues were treated with 25 µL 5% SDS (positive control). The positive control was re-spread after 7 minutes contact time.

Duration of treatment / exposure:

The exposure period was 15 +/- 0.5 minutes.

Duration of post-treatment incubation (if applicable):

The post treatment incubation duration was 42 +/- 1 hours.

Number of replicates:

The test was performed on a total of 3 tissues per test item together with negative and positive controls.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

One experiment lasting 15 minutes +/- 0.5 minutes.

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The absolute mean OD 570 of the negative control tissues was within the laboratory historical control data range.

Positive controls validity:

valid

Remarks:

The positive control had a mean cell viability of 33% after 15 ± 0.5 minutes exposure .

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- Test of color interference: the test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted alter subtraction of the blank in an OD of -0.0007 and 0.0053, respectively. Therefore it was concluded that the test item did not induce color interference.
- Test of MTT reduction: in addition, because no color change was observed in the presence of MTT, it was concluded that the test item did not interact with the MTT endpoint (no direct reduction of MTT).
- The standard deviation value of the percentage viability of three tissues treated identically was <14%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 101%. Since the mean relative tissue viability for the test item was above 50%, the test item was considered to be non-irritant in the in vitro skin irritation test under the experimental conditions described for this study and should not be classified according to the GHS and CLP criteria.

Executive summary:

The objective of this study was to evaluate the test substance for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes.

The study was carried out according to the OECD guideline 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method, (corrected 26 June 2020).

Skin tissue was moistened with 5µL of Milli-Q water and at least 10 mg of the test item was applied as such directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 ± 1 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 101%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment, the test item was considered to be non-irritant.

The positive control had a mean cell viability of 33% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 14%, indicating that the test system functioned properly.

In conclusion, the test item was non-irritant in the in vitro skin irritation test under the experimental conditions of this study and should not be classified according to GHS and CLP criteria.