Lutetium 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 2016-09-21 to 2017-07-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

28 July 2015

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:

6 July 2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiSkin(TM) SOP, ECVAM Skin Irritation Validation Study: Validation of the EpiSkin(TM) test method 15 min - 42 h for the prediction of acute skin irritation of chemicals

Version / remarks:

February 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Lutetium oxide

EC Number:

234-764-3

EC Name:

Lutetium oxide

Cas Number:

12032-20-1

Molecular formula:

Lu2O3

IUPAC Name:

Lutetium oxide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): dilutetium trioxide
- Physical state/Appearance: white solid
- Further information confidential.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was applied as supplied, no formulation was required.

OTHER:
- Correction factor: No correction for purity of the test item was applied.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified (adult)

Source strain:

other: not applicable

Justification for test system used:

The EPISKIN TM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439). Therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM (SM) (Manufacturer: SkinEthic, France)
- Tissue batch number(s): 16-EKIN-038 (expiry date: 26 September 2016) used in Experiment I and 16-EKIN-040 (expiry date: 10 October 2016) used in Experiment II
- Date of initiation of testing: 21 September (Experiment I) and 5 October 2016 (Experiment II)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 23.2-25.9°C in Experiment I, 23.3-24.8°C in Experiment II
- Temperature of post-treatment incubation (if applicable): 37°C ± 1.0°C
- All incubations were carried out in a humid atmosphere (> 95%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C. Temperature and humidity were continuously 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. Any variation to these conditions were evaluated and maintained in the raw data.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the 15 minutes incubation time, the EPISKIN (SM) units were removed and rinsed thoroughly with phosphate buffered saline (PBS) to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: yes, plate reader, not further specified
- Wavelength: 570 nm
- Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). As the test item had an intrinsic colour, further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of the test item to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non-specific OD evaluation. This tissue followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

NUMBER OF REPLICATE TISSUES: 5 for the test item, 3 for negative controls and 3 for positive controls

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

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered to be non-irritant to skin if the mean relative viability of three individual tissues after 15 minutes exposure to the test item and 42 hours post incubation is more than 50% of the mean variability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

NEGATIVE CONTROL (Phosphate Buffered Saline)
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL (Sodium Dodecyl Sulphate solution)
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 h)

Number of replicates:

5 replicates for the test item, 3 negative controls and 3 positive controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 replicates, experiment I

Value:

93

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: As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation (experiment II). The mean optical density (measured at 570 nm) of these two tissues was 0.005. Non Specific Colour % was calculated to be 0.6%. This value was below 5%, therefore additional data calculation was not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD value of the three negative control tissues was in the recommended range (0.854 in Experiment I and 0.764 in Experiment II). Standard deviation of the viability results for negative control samples was 14.0 in Experiment I and 6.8 in Experiment II.
- Acceptance criteria met for positive control: Yes. The three positive control treated tissues showed 3.8% (Experiment I) and 5.3% (Experiment II) viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.2 in Experiment I and Experiment II.
- Acceptance criteria met for variability between replicate measurements: Yes. The standard deviation of viability values of the five test item-treated tissue samples in the MTT assay was 10.7.
- The mean OD value of the blank samples (acidified isopropanol) was 0.048 in Experiment I and 0.045 in Experiment II.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Any other information on results incl. tables

Optical Density (OD) and the calculated relative viability % of the samples (Experiment 1)

	Optical Density (OD)			Viability
Substance		Measured	Blank Corrected	% RV
Negative Control:	1	0.884	0.835	97.8
Phosphate Buffered saline	2	0.794	0.745	87.2
	3	1.031	0.982	115.0
	Mean	----	0.854	100.0
Positive Control:	1	0.070	0.021	2.5
5% (w/v) SDS solution	2	0.087	0.039	4.5
	3	0.087	0.038	4.5
	Mean	----	0.033	3.8
Test Item:	1	0.709	0.660	77.3
Dilutetium trioxide	2	0.837	0.788	92.3
	3	0.966	0.917	107.4
	4	0.840	0.791	92.6
	5	0.863	0.815	95.4
	Mean	----	0.794	93.0

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study (in vitro EPISKIN model test according to OECD guideline 439), the test item was determined to be non-irritant to skin. Based on these results, the test item is considered not classified according to the CLP Regulation.