Yttrium chloride hexahydrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 August 2016 - 17 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

28 July 2015

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:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Yttrium chloride hexahydrate

Cas Number:

10025-94-2

Molecular formula:

YCl3.6H2O

IUPAC Name:

Yttrium chloride hexahydrate

Test material form:

solid: particulate/powder

Details on test material:

- Name of the test material (as cited in the report): yttrium trichloride hexahydrate
- Physical state: solid
- Appearance: white to yellowish crystalline powder
- Further information on test material confidential.

Specific details on test material used for the study:

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 (SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431). 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) (0.38 cm²), SkinEthic, France
- Tissue batch number(s): 16-EKIN-032
- Expiry date: August 15, 2016
- Date of initiation of testing: August 11, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 24.2-25.3°C
- Temperature of post-treatment incubation (if applicable): 37.0°C
- All incubations were carried out in a humid atmosphere (80-100%) 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 incubation time, the test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with phosphate buffered saline (PBS) to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly. The rest of the PBS was removed from the epidermal surface using 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

NUMBER OF REPLICATE TISSUES: 2

ADDITIONAL CONTROL TISSUES
- No additional control tissues were used for direct interference with MTT as there was no indication of direct interference during a preliminary experiment.
- As the test item had an intrinsic colour, two additional test item-treated living tissues were used for the non-specific OD evaluation. These tissues 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 readings were conducted following the same conditions as for the other tissues.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
For 2 disks:
If both disks have mean viability of ≥ 35% = Non Corrosive
If both disks have mean viability of < 35% = Corrosive (at the corresponding incubation period)

Otherwise:
If the mean value is ≥ 35% and the variability is less than 50% = Non Corrosive
If the mean value is < 35% and the variability is less than 50% = Corrosive

VALIDITY OF THE TEST
- The mean OD value of the two negative control tissues should be ≥ 0.6 and ≤ 1.5 and negative control OD values should not be below historically established boundaries.
- The acceptable mean viability % range for positive control is ≤ 20%.
- The difference of viability between the two tissue replicates should not exceed 30%.
- The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.

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 (100 μL physiological saline was added to the test item to ensure good contact with the epidermis)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 0.9% (w/v)
- Lot/batch no. (if required): 52532Y05-2
- Expiry date: 31 May 2018

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): no data
- Lot/batch no. (if required): 15A160011
- Expiry date: 30 November 2017

Duration of treatment / exposure:

4 hours (± 10 min)

Number of replicates:

2 per group (test item, positive control, negative control)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 2 replicates

Value:

86.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range: 85.4 to 88.0%

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 living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined to be 0.003 and Non Specific Colour % (NSCliving%) was calculated as 0.4%. This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions in each case.
The mean OD value of the two negative control tissues was in the recommended range (0.917).
The two positive control treated tissues showed 0.6% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 3.0%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

RESULTS
Following exposure with yttrium trichloride hexahydrate, the mean cell viability was 86.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

Any other information on results incl. tables

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the additional control tissues

Additional control		OD Measured	OD Blank Corrected	NSC% (living)
Treated with	1	0.054	0.007
Test Item	2	0.047	0.000	0.4
	Mean	-	0.003

Note: Mean blank value was 0.047. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

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

Substance		OD Measured	OD Blank Corrected	Viability (%RV)
Negative Control	1	0.940	0.893	97.4
(0.9% (w/v) NaCl)	2	0.988	0.941	102.6
	mean	-	0.917	100.0
Positive Control	1	0.055	0.008	0.9
(Glacial Acetic Acid)	2	0.050	0.003	0.3
	mean	-	0.005	0.6
Test Item	1	0.854	0.807	88.0
	2	0.830	0.783	85.4
	mean	-	0.795	86.7

Note: Mean blank value was 0.047. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with the test item yttrium trichloride hexahydrate, the mean cell viability was 86.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in the in vitro EPISKIN™(SM) model test, the results indicated that the test item is non-corrosive to skin.