Erbium tetraoxide vanadium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-07 to 2017-12-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

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:

2015-07-28

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

2015-06-29

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Test material

Test material information

Constituent 1

Reference substance name:

Erbium tetraoxide vanadium

EC Number:

237-037-9

EC Name:

Erbium tetraoxide vanadium

Cas Number:

13596-17-3

Molecular formula:

ErO4V

IUPAC Name:

Erbium tetraoxide vanadium

Test material form:

solid: particulate/powder

Details on test material:

- State of aggregation: pink powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature. Keep container tightly sealed. Protect from humidity and water.

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

JUSTIFICATION OF THE TEST METHODS AND CONSIDERATIONS REGARDING APPLICABILITY:
In a prevalidation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye irritation test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt has turned out to be sufficiently promising predictor for eye irritancy potential.
A limitation of the Test Guideline OECD 492 is that it does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), as defined by UN GHS. For these purposes further testing with other suitable test methods is required.
The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.

RhCE TISSUE CONSTRUCT USED: EpiOcular™ (Lot No.: 27018; source: MatTek Corporation (82105 Bratislava, Slovakia))
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes that have been cultured to form a stratified squamous epithelium similar to that in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the in vivo corneal epithelium.
Please also refer to the field "Attached background material " below.

Test system

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): approx. 50 mg of the test item

Duration of treatment / exposure:

6 hours

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

approx. 18 hours

Number of animals or in vitro replicates:

Number of EpiOcular tissues:
Test item: duplicates
Negative control: duplicates
Positive control: duplicates

Details on study design:

DETAILS ON THE TEST PROCEDURE USED:
- on day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. Medium was aliquoted into the appropriate wells of pre-labeled 6-well plates.
- each shipping container was removed from its plastic bag under sterile conditions and its surface disinfected.
- sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert.
- tissues were removed from the shipping containers and the insert was transferred into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium.
- after one hour, the Assay Medium was replaced by fresh Assay Medium at 37 °C
- EpiOcular™ tissues were incubated at 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH overnight (16 -24 hours).

- after the overnight incubation, the tissues were pre-wetted with Ca2+Mg2+free-DPBS and incubated at 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH for 30 minutes.
- next, the test and control items (negative and positive control) were tested by applying approx. 50 mg (test item) or 50 µL (controls) topically on the EpiOcular™ tissues.
- tissues were incubated at 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH for 6 hours.
- after the 6 hours treatment, the test item was removed by rinsing the tissues with Ca2+Mg2+-free DPBS (brought to room temperature).

- by using several rinsing steps, the test item and controls were removed (decanted) from the tissue surface with Ca2+Mg2+-free DPBS.
- since it was not possible to remove the visible test item completely, this was noted and no further rinsing was done.
- after rinsing, the tissues were immersed in previously-warmed assay medium (room temperature) for a 25 minutes immersion incubation at room temperature in order to remove any test or control item absorbed by the tissue.
- next, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted and transferred to the appropriate well of the pre-labelled 6-well plate containing warm assay medium. The tissues were incubated for approx. 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2.

MTT ASSAY
- at the end of the post-treatment incubation of 18 hours, each insert was removed from the 6-well plate.
- tissues were placed into the 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and were incubated for 180 minutes at standard culture conditions.
- next, the insert was transferred into a 6-well plate containing isopropanol, which were sealed with parafilm or a standard plate sealer, and were immediately extracted (shaken for 2 to 3 hours at room temperature).
- extract solution was mixed and two 200 µL aliquots were transferred into the appropriate wells of a pre-labelled 96-well plates.
- absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

TEST FOR DIRECT MTT-REDUCERS AND COLOURING TEST CHEMICALS
1) Assessment of direct MTT reduction by the test item:
The test item was evaluated for its potential to interfere with the MTT assay. To test if a test item directly reduces MTT, approx. 50 mg of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently. If the MTT solution would turn blue/purple, the test item was presumed to have reduced the MTT and an additional test with freeze-killed tissues would be necessary in order to examine if the test material is binding to the tissues.
Since the MTT solution colour did not turn blue/purple, the test item was not assumed to be a MTT reducer, and an additional test with freeze-killed tissues was not necessary.

2) Assessment of coloured or staining materials:
The test item was checked for its colouring properties, since these properties might interfere with the MTT measurements.
Since the test item was coloured, additional tests had to be performed to assess, if it becomes coloured after contact with water or isopropanol. Therefore, approx. 50 mg each of the test item were added to 1.0 mL of water or 2 mL isopropanol. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for one hour, whereas the isopropanol mixture for 3 hours at room temperature.
Since the test item did not change in water or isopropanol it was not considered to interact with the MTT measurement. Thus, an additional test on viable tissues (with medium instead of MTT addition) did not have to be performed.

DESCRIPTION OF DATA EVALUATION
1) mean optical density (OD) value of the blank control wells (ODBlk) for each experiment was calculated.
2) mean ODBlk from each mean OD value of the same experiment was subtracted (blank corrected values).
3) mean value of the two replicates for each tissue was calculated.
4) mean value of the two relating tissues for each control (negative control (NC) and positive control (PC) and test item (TI) was calculated (ODTI, ODNC, ODPC).
5) mean OD value of the negative control corresponds to 100% viability.
Corrected negative control OD = Negative Control OD - ODBlk = 100% Viability
6) OD of the extraction solvent alone should be sufficiently small, i.e. OD <0.1
7) percent viability of each of the two relating tissues for each control and test item relative to the negative control (= 100%) was calculated.
Viability (%) = 100 x (ODTI/ODPC/ODNC / meanODNC)
8) difference of the viability between duplicate tissues was calculated. If the difference is > 20 %, the test is considered as non-valid.
9) mean viability of the test item (TI viability) was calculated and the test item was classified according to the prediction model.
10) the results of positive and negative controls of the teset method demonstrate reproducibility over time.

PREDICTION MODEL
- if the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is identified as not requiring classification and labelling according to UN GHS (No Category), i.e. is not an eye irritant.
- if the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1), i.e. is an eye irritant.
- according to OECD guideline 492 a single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60 ± 5 %, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

DEMOSTRATING OF PROFICIENCY IN PERFORMING THE TEST METHOD BEFORE ROUTINE USE BY TESTING OF THE PROFICIENCY CHEMICALS
Prior to routine use of EpiOcularTM EIT for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the eye irritation potential of fifteen proficiency chemicals listed in Table 1 of OECD TG 492. The respective proficiency certificate given by MatTek is attached in the field "Attached background material" below.

ACCEPTABILITY OF THE ASSAY
The results are acceptable, if:
1) the negative control OD is > 0.8 and < 2.5,
2) the mean relative viability of the positive control is below 50% of the negative control viability.
3) the difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the viabilities of freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated relative to the viability of the negative control.
4) the results of positive and negative controls of the test method demonstrate reproducibility over time.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability (mean)

Value:

87.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the negative control optical density (OD) is > 0.8 and < 2.5 (1.432 and 1.510).
- Acceptance criteria met for positive control: the mean relative viability of the positive control is below 50% of the negative control viability (10.3%).
- The difference between the viability of two relating tissues of a single item is < 20 % (values between 0.3 % and 3.8 %) in the same run (for positive and negative control tissues and tissues of single test items).
- The OD of the extraction solvent is sufficiently small, i.e. OD < 0.1

The acceptance criteria were met. Regarding the reproducibility of the data, the absorbance of the negative controls and positive controls is within the historical range of absorbance.

Please also refer for information on the results to the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

Table 1:Absorbance of tissues after treatment for 6 hours with erbium tetraoxide vanadium and the controls

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Mean OD of Treatment Group blank corrected	Rel. Viability [%] Tissue 1, 2 *	Absolute Value of the Difference of Rel. Viability  Tissue 1,2[%]	Mean Rel. Viability [%]**
Blank		0.035	0.036	0.0355
Negative Control	1	1.482	1.432	1.457	1.422	1.436	99.0	2.0	100.0
	2	1.510	1.460	1.485	1.450		101.0
Positive Control	1	0.183	0.179	0.181	0.146	0.148	10.1	0.3	10.3
	2	0.188	0.184	0.186	0.150		10.5
Test Item	1	1.279	1.240	1.260	1.224	1.251	85.3	3.8	87.2
	2	1.320	1.308	1.314	1.278		89.1

* Relative viability [rounded values]: (100 x (absorbance test item/positive control/negative control)) / (mean absorbance negative control)

** Mean relative viability [rounded values]: (100 x (mean absorbance test item/positive control/negative control)) / (mean absorbance negative control)

Table 2: Historical data

Positive Control		Negative Control [OD570]
Mean Viability	30.16%	Mean Absorption	1.54
Standard Deviation	0.10	Standard Deviation	0.248
Range of Viabilities	8.10% - 42.54%	Range of Absorbance	1.02 – 2.05
Mean Absorption	0.463
Standard Deviation	0.169
Range of Absorbance	0.078 - 0.776

Data of 25 studies performed from July 2015 until end of November 2017

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the reported experimental conditions of the in vitro eye irritation / human cornea model test according to OECD TG 492, erbium tetraoxide vanadium does not possess an eye irritating potential. Thus, erbium tetraoxide vanadium does not require classification as eye irritant according to Regulation (EC) No 1272/2008 and subsequent adaptations.