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Diss Factsheets

Administrative data

Description of key information

An in vitro skin irritation test according to OECD 439 was performed, and results indicate that erbium tetraoxide vanadium is not irritant to skin.

An in vitro eye corrosion test according to OECD 437 was performed, and results indicate that erbium tetraoxide vanadium is not corrosive to the eye but the hazardous properties with regard to corneal irritation (CLP (Cat 2)) could not be predicted.

A further in vitro eye irritation test according to OECD 492 was performed, and results indicate that erbium tetraoxide vanadium is not irritating to the eye.

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:
2017-05-30 to 2017-06-30
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)
Version / remarks:
2015-07-28
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009-07-23
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200).
Version / remarks:
2015-06-29
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-09-14
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: keep container tightly sealed. Protect from humidity and water
Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: humans
Source strain:
other: not applicable
Details on animal used as source of test system:
not applicable
Justification for test system used:
In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
other: Dulbecco's phosphate buffered saline
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin model (source: MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue lot number: 25825
- Delivery date: 2017-06-27

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37 ± 1.5 °C (23 hours)
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 minutes, room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were rinsed with DPBS for at least 15 times in order to remove any residual test material.

After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with DPBS. The tissues were then transferred into plates with assay medium. Tissues were incubated for nearly 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation, the medium was changed (pre-warmed fresh medium). Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was approx. 41 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/ well)
- Incubation time with MTT: 3 hours
- Extraction of Formazan: after the incubation period, the tissues were rinsed three times with DPBS. The tissues were transferred into new plates containing extractant solution (isopropanol) in each well so that the tissues were covered completely and the plate was sealed to inhibit the evaporation of isopropanol. The formazan salt was extracted for 2.5 hours while shaking at room temperature.
After the extraction was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken and the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution after the 15 minutes exposure were transferred into a 96-well flat bottom microtiter plate. The optical density was read in a microplate reader. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm

TEST FOR COLOUR INTERFERENCE
Before the test started, a functional check for colour interference was performed. 25 ± 2 mg of the test item were added to deionised water. The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 minutes. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated.

TEST FOR DIRECT MTT REDUCTION
The test item was evaluated for its potential to interfere with MTT assay. To test if a test item directly reduces MTT, 25 ± 2 mg of the test item were added to 1 mL of the MTT-solution (1mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1- virus, Hepatitis B virus, and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: relative viability(%) = (mean OD test item or positive control/ mean OD of negative control) x 100
For the test item and the positive control, the mean relative viability ± relative standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model: if the mean relative tissue viability of three individual tissues is less or equal to 50% of the negative control, the test item needs to be classified and labelled for its skin irritation potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 25 mg (~ 39 mg/cm²) of the test item, wetted with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% sodium lauryl sulfate (SLS) solution
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
approx. 41 hours
Number of replicates:
triplicates
Irritation / corrosion parameter:
% tissue viability
Value:
98.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: since the test item did not dye water relevantly (milky), an additional test with viable tissues (but without MTT addition) was not necessary.
- Direct-MTT reduction: the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control, the absorbance values (1.684, 1.738, and 1.588 (mean: 1.670)) were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 3.3 % (acceptability criterion: positive control is ≤ 20 %).
- Acceptance criteria met for variability between replicate measurements: the relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 15 % (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%).
Please refer to the field "Any other information on results incl. tables" below

HISTORICAL DATA

Positive Control

Negative Control [OD570]

Mean Viability

4.37 %

Mean Absorption

1.74

Rel. Standard Deviation

21.60 %

Rel. Standard Deviation

9.40 %

Range of Viabilities

2.20 % - 6.78 %

Range of Absorbance

1.34 – 2.00

Mean Absorption

0.08

 

Rel. Standard Deviation

20.12 %

Range of Absorbance

0.03 - 0.11

Data of 103 studies performed from July 2015 until March 2017

Table 1: Results after treatment with erbium vanadate and the controls

Dose Group

Tissue No.

Absor-bance 570 nm
Well 1

Absor-bance 570 nm
Well 2

Absor-bance 570 nm
Well 3

Mean Absor-bance of 3 Wells

Mean-Absorbance

of three wells blank

corrected

Mean

Absorbance

of 3 tissues

Mean Rel. Absorbance

[% of Negative Control]*

Blank

 

0.038

0.038

0.037

0.037

 

 

Negative Control

1

1.708

1.718

1.740

1.722

1.684

1.670

100.0

2

1.804

1.757

1.767

1.776

1.738

3

1.659

1.606

1.611

1.625

1.588

Positive Control

1

0.090

0.095

0.094

0.093

0.055

0.055

3.3

2

0.093

0.093

0.091

0.092

0.055

3

0.092

0.093

0.093

0.093

0.055

Test Item

1

1.967

1.920

1.925

1.938

1.900

1.639

98.1

2

1.490

1.465

1.460

1.472

1.434

3

1.642

1.611

1.606

1.620

1.582

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

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the in vitro skin irritation test (OECD 439), erbium tetraoxide vanadium is not irritating to the skin. According to Regulation (EC) No 1272/2008 and its subsequent regulations, erbium tetraoxide vanadium does not meet classification criteria neither for skin irritation nor skin corrosion.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

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
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
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
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.
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.
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.
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.

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

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.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-02 to 2017-07-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2013-07-26
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details
Version / remarks:
April 1997
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-09-14
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: keep container tightly sealed. Protect from humidity and water.
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: isolated eyes were stored in Hank’s Buffered Salt Solution (HBSS) containing penicillin / streptomycin in the cooled slaughter-house until transportation on the same morning to the laboratory using a styrofoam box.
- Time interval prior to initiating testing: corneae were isolated and used on the same day after delivery of the eyes
Vehicle:
other: 0.9% (w/v) NaCl in deionised water
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration: 20 % suspension (w/v) in vehicle (using sonication for 10 minutes)
Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
not required
Number of animals or in vitro replicates:
Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- all eyes were examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- cornea was removed from the eye
- each isolated cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with incubation medium cMEM (MEM, supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin and 1 % fetal calf serum, equivalent to EMEM).
- for equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE CORNEAS
- at the end of the equilibration period of the corneae in the holder, the basal opacity was determined (t0).
- each corneae with a value of the basal opacity > 7 was discarded.

APPLICATION DOSE AND EXPOSURE TIME
- the anterior compartment received the test item suspension or negative or positive control at a volume of 0.75 mL each on the surface of the corneae.
- corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath (incubation time: 240 minutes).
- following incubation, the test item or control items were rinsed off with saline.
- fresh incubation medium was added into the anterior compartment and opacity was measured (t240).
- permeability of the corneae was determined.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the opacitometer (OP_KiT opacitometer (Electro Design)) was calibrated and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Evaluation of opacity:
- the change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
- the average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (Versamax® Molecular Devices)(OD490).
- after the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by 0.5 % (w/v) sodium fluorescein solution in HBSS
- corneae were incubated in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C.
- incubation medium from the posterior compartment was removed, mixed and the optical density at 490 nm was determined with the microplate reader .
Evaluation permeability:
- the corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values of each individual treatment and positive control cornea.
Depending on the IVIS score obtained, the test item is classified into the following category according to OECD guideline 437 (please refer to table 1 in the field "Any other information on material and methods incl. tables" below).

ACCEPTANCE CRITERIA:
The test will be acceptable if:
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
Irritation parameter:
in vitro irritation score
Remarks:
(mean)
Run / experiment:
Experiment 1
Value:
4.89
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: due to inconsistent results in the 1st experiment, two independent experiments were performed for the confirmation of the response.
Irritation parameter:
in vitro irritation score
Remarks:
(mean)
Run / experiment:
Experiment 2
Value:
4.72
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
NOTE: due to inconsistent results in the 1st experiment, two independent experiments were performed for the confirmation of the response.

- relative to the negative control, the test item erbium tetraoxide vanadium caused an increase of the corneal opacity.
- in both experiments, exposure of the corneae to the negative control (saline) did not result in an increase of opacity or permeability of the corneae (mean IVIS = 1.37 and 1.18, respectively).
- exposure of the corneae to the positive control (10% (w/v) Benzalkonium chloride in saline) led in both tests to clear opacity and distinctive permeability effects on the corneae (mean IVIS = 119.75 and 98.30, respectively) corresponding to a classification Irreversible effects on the eye / serious eye damage (CLP (Cat 1)).

ACCEPTANCE OF RESULTS (both experiments):
- Acceptance criteria met for negative control: opacity and permeability of the negative control are less than the respective established upper limits for background opacity and permeability.
- Acceptance criteria met for positive control: the positive control falls within two standard deviations of the current historical mean.

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

Table 1: Results of the first experiment after 240 minutes treatment time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposedin vitroIrritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

1

0.33

0.069

0.069

2.04

1.37

Not categorized

0

0.070

1.05

0

0.069

1.04

Positive Control

112.67*

0.330*

117.61

119.75

Category 1

122.67*

0.523*

130.51

105.67*

0.364*

111.12

Erbium tetraoxide vanadium

8.67*

0.029*

9.10

4.89

No prediction can be made

2.67*

0.005*

2.74

2.67*

0.011*

2.83

*corrected values (subtraction of average negative control from measured permeability of each replicate)

Table 2: Results of the second experiment after 240 minutes treatment time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposed in vitro Irritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

0

0.00

0.084

0.078

1.26

1.18

No Category

0

0.070

1.05

0

0.081

1.22

Positive Control

97.00*

0.016*

97.24

98.30

Category 1

102.00*

0.019*

102.28

95.00*

0.025*

95.37

Erbium tetraoxide vanadium

4.00*

0.000*#

4.00

4.72

No prediction can be made

5.00*

0.008*

5.12

5.00*

0.003*

5.04

*corrected values (subtraction of average negative control from measured permeability of each replicate)

# A negative permeability is not possible and thus this value should be set to zero

Table 3: Historical Data

 

Positive Control

Negative Control

Mean IVIS

112.39

1.34

Standard Deviation of IVIS

9.29

0.18

Range of IVIS

98.30 — 127.22

1.01 — 1.64

95 % Control limits of IVISpos

93.81 — 130.98

 

Mean Opacity t130min

110.64

0.21

Standard Deviation of
Opacity t130min

12.46

0.17

Range of Opacity t130min

82.00 — 131.67

0.00 — 0.33

Mean Permeability

0.12

0.07

Standard Deviation of Permeability

0.13

0.01

Range of Permeability

-0.01 — 0.52

0.07 — 0.09

Values of 30 studies with solid test items sharing 14 sets of controls between them, performed from January 2017, calendar week 3, to July 2017, calendar week 28.

Interpretation of results:
other: does not meet classification criteria of Category 1 (irreversible effects on the eye)
Conclusions:
Based on a Bovine Corneal Opacity and Permeability Test (OECD 437), erbium tetraoxide vanadium does not meet classification criteria for serious eye damage (Category 1), but hazardous properties with regard to corneal irritation cannot be predicted.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification

Skin irritation

The substance does not possess a skin irritating potential based on an in vitro OECD 439 (2015) test and does not require classification as skin irritating according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

The substance does not posses an eye irritating potential based on an in vitro study according to OECD 492 (2017) and does not require classification as eye irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.