Yttrium

Endpoint summary

Description of key information

Skin irritation/corrosion

The test item was demonstrated to be non-irritant in an in vitro study performed according to OECD guideline 439 (Reconstructed Human Epidermis Test method, EPISKIN) and conform GLP requirements (Kovács, 2018). This study was considered reliable without restrictions (Klimisch 1) and is considered as the key study for endpoint coverage.

Eye irritation

The test item was demonstrated to be non-irritant to eyes in an in vitro study performed according to OECD guideline 492 (Reconstructed Human Cornea-like Epithelium test method, EpiOcular study) and conform GLP requirements (Gerbeix, 2018). This study was considered reliable without restrictions (Klimisch 1) and is considered as the key study for endpoint coverage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-08-10 to 2018-01-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 in its original form, no formulation was required (although it was grounded to fine powder).

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 (SM) (0.38 cm²), SkinEthic, France
- Tissue batch number(s): 17-EKIN-032
- Expiry date: 14 August 2017
- Date of initiation of testing: 10 August 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 23.2-27.6°C
- Temperature of post-treatment incubation (if applicable): 37.0 ± 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 2 x ~25 mL 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). Note that the test item was stuck on the surface of the epidermis and could not be removed completely from the surface ot 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: 3

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

ADDITIONAL CONTROL TISSUES
- For non-specific OD evaluation: 2 additional test item-treated living tissues. The MTT incubation step was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item.
- For MTT-interaction: 2 additional test item-treated killed epidermis and 2 negative control-treated killed epidermis (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues. The same treatment steps were followed as for the living tissues.
- Because the test item was identified as producing both direct MTT reduction and colour interference, a third set of controls was required (to avoid a potential double correction for colour interference). Killed tissue samples were used for this third set of controls (2 replicates).
- Procedure used to prepare the killed tissues (if applicable): Living epidermis units (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-017, Expiry Date: 1 May 2017) were placed in a 12-well plate with 2 mL of distilled water, then incubated at 37°C in an incubator with 5% CO2, in a > 95% humidified atmosphere for 48 hours (± 1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen on 28 April 2017 (the frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Maintenance Medium). Further use of killed tissues was similar to living tissues.

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 viability 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:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 replicates

Value:

101

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
- Colour interference with MTT: Yes. 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 tissues was 0.020, and Non-Specific Colour % (NSCliving%) was calculated to be 2.7%. This value was below 5%, therefore additional data calculation was not necessary.
- Direct-MTT reduction: Yes. As colour change (purple) was observed after three hours of incubation of the test item in MTT working solution, the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Based on the observed mean OD (0.022), the calculated NSMTT (Non-Specific MTT reduction) is 2.9%.
- As the test item was shown to be an MTT-interacting substance and the test item had an intrinsic colour, two additional test item-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.042, and Non-Specific Colour % (NSCkilled%) was calculated as 5.6%. Because the NSCliving% was not used, correction with NSCkilled% 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 (0.751). Standard deviation of the viability results for negative control samples was 3.8%.
- Acceptance criteria met for positive control: Yes. The positive control treated tissues showed 8.0% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 2.3%.
- Acceptance criteria met for variability between replicate measurements: Yes. The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 3.1%.
- The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

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

Additional control	Optical Density (OD)			NSC (living)%
		Measured	Blank corrected
Treated with	1	0.072	0.025
yttrium metal	2	0.062	0.015	2.7
	mean	-	0.020

Optical Density (OD) and the calculated Non Specific MTT Reduction (NSMTT) of the additional control tissues (killed epidermis)

Additional control	Optical Density (OD)			NSMTT	NSMTT %
		Measured	Blank corrected
Treated with yttrium metal	1	0.096	0.049	0.026	2.9
	2	0.088	0.041	0.018
	mean	-	0.045	0.022
Treated with PBS	1	0.078	0.031
	2	0.063	0.016
	mean	-	0.023

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

Additional control	Optical Density (OD)			NSC (killed)%
		Measured	Blanc corrected
Treated with	1	0.084	0.037
yttrium metal	2	0.093	0.046	5.6
	mean	-	0.042

Notes (for all 3 tables above): 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	Optical density (OD)				Viability
		Measured	Blank corrected	TODTT	(% RV)
Negative control:	1	0.819	0.772		102.8
Phosphate Buffered Saline	2	0.765	0.718		95.7
	3	0.809	0.762		101.5
	mean	-	0.751	-	100.0
Positive control:	1	0.111	0.064		8.5
5% (w/v) SDS solution	2	0.122	0.075		10.0
	3	0.088	0.041		5.4
	mean	-	0.060		8.0
Test item:	1	0.852	0.805	0.783	104.3
yttrium metal	2	0.805	0.758	0.736	98.1
	3	0.824	0.777	0.755	100.6
	mean	-	0.780	0.758	101.0

Notes:

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3. TODTT: the measured values were corrected for non-specific MTT reduction (by subtracting the NSMTT value of 0.022)

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-08-29 to 2018-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- No correction factor was applied in this study.
- The test item was used in its original form, as supplied by the Sponsor.

Species:

human

Strain:

other: Reconstructed human cornea-like epithelium (tissues)

Details on test animals or tissues and environmental conditions:

- Source: MatTek, Bratislava, Slovak Republic
- Expiry date: The EpiOcular tissues were used within 72 hours of their production.
- Selection: At receipt, the tissues were inspected for obvious defects as they could have been rejected based on blistering, excess fluid or air bubbles below the tissue insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
- Storage conditions: At receipt, the living EpiOcular tissues were stored on their day of arrival, at 37°C, 5% CO2, in a humidified incubator.
- Description of the cell system used: The EpiOcularTM model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm²), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinised epithelium which models the corneal epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotype 3D cornea-like model. The 3D tissue consists of highly organised cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 51 mg (± 1 mg)

NEGATIVE CONTROL (sterile deionised water)
- Amount(s) applied (volume or weight with unit): 50 μL

POSITIVE CONTROL (99% methyl acetate)
- Amount(s) applied (volume or weight with unit): 50 μL

Duration of treatment / exposure:

6 hours (± 15 minutes)

Duration of post- treatment incubation (in vitro):

18 hours (± 15 minutes)

Number of animals or in vitro replicates:

2

Details on study design:

Details of the test procedure used:
- RhCE tissue construct used, including batch number: EpiOcular tissue, MatTek, Bratislava, Slovak Republic. Batch number documented in a certificate of analysis archived in the study files.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Exposure for 6 hours at 37°C, then (after rinsing with Dulbecco's Phosphate Buffered Saline) soaked in assay medium for 25 minutes at room temperature, blotted, and then incubated for 18 hours at 37°C.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): As the test item was found in the preliminary test not to have any colouring potential and any direct MTT reducing properties, no additional controls were run during the main test.
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan: MTT formazan precipicates are extracted using isopropanol and quantified using spectrophotometry. Formazan extraction was performed during 2 to 3 hours at room temperature by placing the plates on an orbital plate shaker.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: A test substance is predicted as ocular irritant, if the mean relative tissue viability (%) of two tissues exposed to the test item is ≤ 60%.
- Positive and negative control means and acceptance ranges based on historical data: Negative control acceptance criteria: mean cOD between 0.8 and 2.5. Positive control acceptance criteria: relative mean viability of the positive control is < 50% of the relative mean viability of the negative control.
- Acceptable variability between tissue replicates for the test chemical: Acceptable if the difference of viability between the two tissue replicates is < 20%.

Irritation parameter:

other: relative viability in %

Run / experiment:

mean of duplicate tissues

Value:

105

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

ACCEPTANCE OF RESULTS
- Acceptance criteria met for negative control: Yes. Mean cOD for the two replicate tissues was 1.506 and 1.623.
- Acceptance criteria met for positive control: Yes. 25 and 29% viability was observed for the two tissue replicates in the positive control, whereas 96 and 104% viability was observed for the two tissue replicates in the negative control. The relative mean viability in the positive control is hence < 50% of that in the negative control.
- Acceptable variability between replicate tissues treated with test item: Yes. A difference of only 2% was obtained between the replicate tissues.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was tested for its eye irritation potential in a Reconstructed Human Cornea-like Epithelium assay performed according to OECD guideline 492. Since mean viability in the treated tissues after MTT reduction was 105%, which is higher than the cut-off level of 60%, the test results meet the criteria for a non-irritant response. Therefore, the test substance is not to be classified for eye irritation under the CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

One reliable (Klimisch 1) in vitro study is available (Kovács, 2018). This study was performed according to OECD guideline 439 (Reconstructed Human Epidermis Test method, EPISKIN) and conform GLP requirements. The mean % tissue viability in the three tissue replicates exposed to the test item was 101%. A 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 viability of the negative controls. Since the mean viability in the negative controls was 100%, the test item was concluded to be non-irritant under the conditions of the study. Based on these results, the test item is considered not classified according to the CLP Regulation. The study of Kovács (2018) is considered as the key study for endpoint coverage.

Eye irritation

One reliable (Klimisch 1) in vitro study is available. This study (Gerbeix, 2018) was performed according to OECD guideline 492 (Reconstructed Human Cornea-like Epithelium assay) and conform GLP requirements. Since mean viability in the treated tissues after MTT reduction was 105%, which is higher than the cut-off level of 60%, the test results met the criteria for a non-irritant response. Therefore, the test substance could be concluded not to be classified for eye irritation under the CLP Regulation. This study is considered the key study for endpoint coverage.

Justification for classification or non-classification

Skin irritation/corrosion

The test item was demonstrated not to be irritant to skin in an in vitro study performed according to OECD guideline 439 and is therefore not to be classified according to the criteria of the CLP regulation.

Eye irritation

The test item was demonstrated not to be irritant to eyes in an in vitro study performed according to OECD guideline 492 and is therefore not to be classified according to the CLP Regulation.