Aluminium vanadium tetraoxide

Endpoint summary

Description of key information

An in vitro skin irritation test according to OECD 439 was performed, and results indicate that aluminium vanadium tetraoxide may have a potential for skin irritation/corrosion. Based on the in vitro Membrane Barrier Test for Skin Corrosion (OECD 435), aluminium vanadium tetraoxide is not corrosive to the skin.

An in vitro eye corrosion test according to OECD 437 was performed, and results indicate that aluminium vanadium tetraoxide 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 the potential for eye irritation cannot be fully characterised. Since other in vitro test systems do not appear to be appropriate to characterise the eye irritation potential (CLP Category 2) and an in vivo test cannot be performed according to Annex VII of Regulation (EC) No 1907/2006, the eye irritating potentail of aluminium vanadium tetraoxide cannot be fully characterised.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-07 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: store at room temperature. 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 tissues 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.
The test item dyed the water (brown), therefore, an additional test with viable tissues (but without MTT addition) had to be performed to determine a correction factor for calculating the true viability in the main experiment.
To check the tissue-binding of a coloured test item, two viable tissues were exposed to 25 ± 2 mg of the test item. In parallel, two tissues were exposed to DPBS (negative control). All procedures were followed as described for the MTT assay used in the main experiment, except that the tissue were incubated for 3 hours in culture media without MTT (37 ± 1.5°C, 5 ± 0.5% CO2,) instead of incubating in media containing MTT. After the 3 hour incubation, the tissues were rinsed and the tissues were extracted using isopropanol and the optical density (OD) at 570 nm was measured.
Since the tissues treated by the coloured test item (or test item) had a mean OD between 5% and 30% of the negative control tissue (treated with DPBS), the real MTT OD (unaffected by the interference with the coloured test items) was calculated using the following formula:
OD = ODcolored tissue (MTT assay) – ODcolored tissue (no MTT assay)

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 (1 mg/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:

26.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Colour interference with MTT: the test item dyed the water (brown), therefore, an additional test with viable tissues (without MTT addition) had to be performed to determine a correction factor for calculating the true viability in the main experiment.
- Direct-MTT reduction: the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional functional 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 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 5 % (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 aluminium vanadium tetraoxide 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-Absor-bance of three Wells blank corrected	Mean Absor-bance of 3 Tissues	Mean Rel. Ab-sorbance [%]*	Corrected Mean Rel. Absorbance [%]**
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	0.527	0.507	0.505	0.513	0.476	0.445	27.3	26.6
	2	0.482	0.474	0.472	0.476	0.438
	3	0.499	0.486	0.484	0.489	0.452
Negative Controladd. viable	1	0.043	0.043	0.043	0.043	0.005	0.007	0.4
	2	0.047	0.048	0.046	0.047	0.009
Test Itemadd. viable	1	0.047	0.047	0.047	0.047	0.010	0.010	0.6
	2	0.048	0.049	0.048	0.049	0.011

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

**                  Mean relative absorbance [rounded values]: [100 x (mean absorbance test item - mean absorbance test item add viable)]/(mean absorbance negative control)

               

Interpretation of results:

other: may meet classification criteria for skin irritation or corrosion

Conclusions:

Based on the in vitro skin irritation test (OECD 439), aluminium vanadium tetraoxide is irritating to the skin. According to Regulation (EC) No 1272/2008 and its subsequent regulations, aluminium vanadium tetraoxide may meet classification criteria for skin irritation/corrosion.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-13 to 2017-12-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Version / remarks:

2015-07-28

Deviations:

no

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: store at room temperature. Keep container tightly sealed. Protect from humidity and water.

Test system:

artificial membrane barrier model

Source species:

other: not specified

Cell type:

other: synthetic macromolecular bio-barrier

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

not applicable

Justification for test system used:

Corrositex TM is a validated and accepted in vitro method to assess if a test item can produce skin corrosion and to distinguish between GHS corrosivity categories 1A, 1B, and 1C.

Vehicle:

unchanged (no vehicle)

Details on test system:

SOURCE AND COMPOSITION OF MEMBRANE BARRIER USED
- Was the Corrositex® test kit used: yes (lot no. CT120516; supplier: ROMER Labs Deutschland GmbH, 35510 Butzbach, Germany)
- Components: a synthetic macromolecular bio-barrier and a chemical detection system (CDS)
- Apparatus and preparation procedures: one day prior to testing the bio-barrier matrix was prepared. The bio-barrier powder was solved in the bio-barrier diluent and heated for 20 ± 2 minutes at 68 – 70 °C in a water bath under continuous stirring. The temperature did not exceed 70 °C. The mixture was allowed to cool in the turned-off water bath for another 10 minutes. The mixture was then filled into the membrane holders (200 μL per membrane holder). Air bubbles were avoided. The filled membrane holders were sealed with parafilm and were stored at 2 – 8 °C until further use.

WAS THE COMPATIBILITY TEST PERFORMED: yes
In order to test whether the test system is suitable for the test item, approximately 100 mg of the test item were applied into the “Qualify Test Vial”. The vial was shaken until the solution appeared homogenous, and incubated for 1 minute. Afterwards, the colour change was noted.
The test item induced a change in colour in the qualify test after 1 minute incubation. Since a change in colour was visible in the “Qualify Test Vial”, the test item was considered to be suitable for the next step.

WAS THE TIMESCALE CATEGORY TEST PERFORMED: yes
In the categorisation test the observation period of the test item after application to the bio-barrier was determined:
- Category 1: observation period after application is 4 hours
- Category 2: observation period after application is 1 hour
Approximately 100 mg of the test item were applied into the “Category A Vial” as well as into the “Category B Vial”. The vials were shaken until the solution appeared homogenous. After 1 minute the colour change was recorded.
Based on the observed colour change, a test item is assigned to a category. If an intense colour change (similar to the category 1 colour chart) is observed in “Category A Vial” or in “Category B Vial” the test item is assigned to category 1. If a less intense colour change (similar to the category 2 colour chart) is observed in “Category A Vial” or in “Category B Vial” the test item will be assigned to category 2. If a colour change is not observed in either of the vials, a confirmation test is conducted. For the confirmation test two drops of the confirm reagent were added to the “Category B Vial”. The vial was shaken for 5 seconds. The colour of the solution matched one of the colours shown in the accompanying colour chart, confirming that the test item is a category 2 substance.
The test item induced a change in colour in the Category A vial and in the Category B vial after 1 minute of incubation. According to the colour scheme the test item does not appear to be a strong acid or base and was assigned to category II. It was concluded that the required duration of the classification test was 60 minutes.

TEMPERATURE USED DURING TREATMENT: room temperature

METHOD OF DETECTION
- Chemical or electrochemical detection system: chemical detection system

METHOD OF APPLICATION (CLASSIFICATION TEST):
8 vials containing the CDS were pre-warmed to room temperature (test item: 4 vials; negative control: 1 vial; positive control (sulfuric acid): 1 vial; positive control (sodium hydroxide): 1 vial; colour reference for CDS: 1 vial).
The prepared bio-barriers were placed atop the CDS vials (not longer than 2 minutes prior to application) and the test item or the respective controls were applied per bio-barrier for 1 hour, as results of the categorisation test. The time interval of the possible colour change or precipitation in the CDS solution was recorded.

INTERPRETATION OF THE RESULTS:
For each vial the period until observable change in CDS solution was determined. The mean of the quadruplicate measurement was calculated. The test item was classified according to the criteria in table 1 as presented in the field "Any other information on materials and methods incl. tables" below.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 500 mg of the neat test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 500 µL of citric acid (10 % (w/v) solution in deionised water)

POSITIVE CONTROL (two positive controls were used)
- Amount(s) applied (volume or weight):
1) 500 µL of sulfuric acid 95 - 97 %
2) 110 ± 15 mg of sodium hydroxide

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

Test item: quadruplicates
Negative control: single measurement
Positive control: single measurement

Irritation / corrosion parameter:

penetration time (in minutes)

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Colour change was not observed up to 60 minutes (treatment period)

Other effects / acceptance of results:

QUALIFY TEST
The test item induced a change in colour in the qualify test after 1 minute incubation. Since a change in colour was visible in the “Qualify Test Vial”, the test item was considered to be suitable for the next step.

CATEGORISATION TEST:
The test item induced a change in colour in the Category A vial and in the Category B vial after 1 minute of incubation. According to the colour scheme the test item does not appear to be a strong acid or base and was assigned to category II. It was concluded that the required duration of the classification test was 60 minutes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: negative control did not induce a change in the colour of CDS reagent after 60 minutes (acceptability criteria: negative control does not induce a physical change (colour or precipitation) up to 60 minutes in the CDS solution in the classification test).

- Acceptance criteria met for positive control: the positive control sulfuric acid showed a distinct change in the colour of the CDS reagent in the time interval of 0 – 3 minutes. The positive control sodium hydroxide induced a change in the colour of the CDS reagent after 3 – 60 minutes.

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

HISTORICAL DATA:

Mean time until a colour change of positive control Sulfuric acid [minutes]	1.24
Standard deviation [minutes]	0.37
Range of mean times	0.50 - 2.00

Data of 88 studies performed from September 2007 until March 2016.

CLASSIFICATION TEST:

Test Group		Time Interval of Colour Change	UN GHS
Negative Control		Colour change was not observed up to 60 minutes	Non-corrosive
Positive Control Sulfuric acid		1 minute 13 seconds	Corrosive subcategory 1A
Positive Control Sodium Hydroxide		6 minutes 30 seconds	Corrosive subcategory 1B
Test Item	Colour change was not observed up to 60 minutes		Non-corrosive

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the in vitro Membrane Barrier Test for Skin Corrosion (OECD 435), aluminium vanadium tetraoxide is not corrosive to the skin. Thus, according to Regulation (EC) No 1272/2008 and its subsequent adaptations, aluminium vanadium tetraoxide does not meet classification criteria of skin corrosion.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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 test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a 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 which have been cultured to form a stratified squamous epithelium similar to that found 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 normal 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.
- at the end of the 6 hours treatment time, 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 item or control 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 to 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 to the appropriate wells of a pre-labelled 96-well plate(s).
- 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 presumed 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 either to 1.0 mL of water or to 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, the isopropanol mixture for 3 hours at room temperature.
Since the test item became coloured more intensively in water, it had to be considered as possibly interacting with the MTT measurement and an additional test on viable tissues (without MTT addition) had 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) mean relative viablity of the test item and positive control were calculated as follows:
mean relative viability [rounded values]: (100 x (mean absorbance test item/positive control/negative control))/ mean absorbance negative control

Calculation for viability plus colorant control (CC) test
1) Optical density (OD) values of the additional viable tissue experiment (without MTT addition; each two tissues and two replicates) were determined and blank corrected. The mean value of the two replicates for the negative control (NC) and test item (TI) was calculated (test item: ODTI_CC; negative control: ODNC_CC). The mean OD of the two negative control tissues was calculated (mean ODNC_CC).
2) viability of the two relating tissues was calculated according to the following formula:
Test ItemCC viability [5] = 100 x (ODCC/mean ODNC)
3) difference of the viability of the two tissues was calculated. If the difference is >20%, the additional viable tissues (without MTT addition) test is considered as non-qualified.
4) mean TestItemCC Viability for the additional viable tissues (without MTT addition) will be calculated and was subtracted from the test item viability determined above (TI viability) to determine the CC corrected test item viability.
CC corrected test item viability = TI viability – mean TestItemCC Viability
5) test item was classified regarding the additional viable tissues (without MTT addition) corrected viability according to the prediction model.

PREDICTION MODEL
- If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
- If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.

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 of the 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 freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating 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:

49.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Since the viability value of the test item exposed tissues decreased below 60%, the test item is may be considered to possess an eye irritating potential. However, the in vitro eye irritation / human cornea model test according to OECD TG 492 may not be suitable to characterize the potential for eye irritation of the test item, an inorganic powder that could not be rinsed off the tissue after the test.

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 0.0 % and 5.4 %) in the same run (for positive and negative control tissues and tissues of single test items). This applied also to the killed controls (items and negative control) and the additional viable tissues (without MTT addition)s which were calculated as percent values related to the viability of the relating negative control.
- 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 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.

It was not possible to remove the visible test item, a poorly soluble inorganic metal oxide powder, completely. Thus, the test system may not be suitable to characterize the eye irritation poential of inorganic metal oxide powders such as aluminium vanadium tetraoxide.

Table 1: Results after treatment for 6 hours with aluminium vanadium tetraoxide 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	0.805	0.787	0.796	0.760	0.722	53.0	5.4	49.8***
	2	0.724	0.713	0.718	0.683		47.6
Negative Control Viable Tissues	1	0.043	0.041	0.042	0.006	0.006	0.4	0.0	0.4
	2	0.041	0.041	0.041	0.006		0.4
Test Item Viable Tissues	1	0.047	0.041	0.044	0.008	0.007	0.6	0.1	0.5
	2	0.042	0.042	0.042	0.006		0.4

* 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)

*** corrected value

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:

other: cannot be fully characterized

Conclusions:

Under the reported experimental conditions of the in vitro eye irritation / human cornea model test according to OECD TG 492 with its limitations, the eye irritation potential of aluminium vanadium tetraoxide cannot be fully characterized.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-06

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: store at room temperature. 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 % 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 technique 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)

Value:

6.03

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- relative to the negative control, the test item aluminium vanadate caused an increase of the corneal opacity.
- for the negative control (saline) an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.42).
- the positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =108.47) corresponding to the 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 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	1	0.33	0.060	0.072	1.90	1.42	Not categorized
	0		0.079		1.19
	0		0.078		1.17
Positive Control	100.67*		0.036*		101.20	108.47	Category 1
	119.67*		-0.008*		119.54
	104.67*		0.00*		104.66
Aluminium vanadate	4.67*		0.028*		5.08	6.03	No prediction can be made
	5.67*		0.075*		6.79
	5.67*		0.037*		6.22

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

Table 2: Historical Data

	Positive Control	Negative Control
Mean IVIS	119.75	1.30
Standard Deviation IVIS	12.94	0.29
Range of IVIS	98.49 – 167.85	0.73 – 2.04
Mean Opacity t240min	117.92	0.17
Standard Deviation Opacity t240min	15.46	0.22
Range of Opacity t240min	88.67 – 183.00	-0.33 – 0.67
Mean Permeability	0.131	0.139
Standard Deviation Permeability	0.212	0.221
Range of Permeability	-0.003 – 1.438	0.054 – 1.100

Values of 51 studies with solid test items performed from February 2015 (calendar week 7) until January 2017 (calendar week 2)

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), aluminium vanadium tetraoxide 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:

adverse effect observed (irritating)

Additional information

Regarding the characterisation of the eye irritation potential applying validated in vitro test systems, the BCOP test (EU B.47/OECD TG 437) does not consider conjunctival and iridal injuries, it addresses corneal effects. Further, the BCOP test is not recommended for the identification of chemicals that should be classified as irritating to eyes (CLP Category 2). Other validated test methods with regulatory acceptance do also not consider e.g. conjunctival and iridal injuries:

-The ICE test does not consider conjunctival and iridal injuries as evaluated in the rabbit ocular irritancy test, it addresses corneal effects. The Isolated Chicken Eye (ICE) test (EU B.48/OECD TG 438) is not recommended for the identification of chemicals that should be classified as irritating to eyes (i.e., CLP Category 2). 

- The purpose of the Fluorescein leakage (FL) test (OECD TG 460) is to identify ocular corrosives/severe irritants only. The FL test is not recommended for the identification of chemicals which should be classified as mild/moderate irritants or of chemicals which should not be classified for ocular irritation (substances and mixtures) (i.e. CLP Cat. 2, no category). 

-Short Time Exposure In Vitro Test Method (STE) for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (OECD 491) aims to evaluate the eye hazard potential of a test chemical based on its ability to induce cytotoxicity in the Short Time Exposure Test method. The cytotoxic effect of chemicals on corneal epithelial cells is an important mode of action (MOA) leading to corneal epithelium damage and eye irritation.

- The in vitro EpiOcular™ Eye Irritation Test (EIT), i.e. Reconstructed human Cornea-like Epithelium (RhCE) test  (OECD 492), aims to identify chemicals which do not require classification and labelling for eye irritation or serious eye damage according to UN GHS without further testing within a testing strategy.

Based on results of the BCOP test (OECD 437) and the RhCE test (OECD 492), aluminium vanadium tetraoxide is not corrosive to the eye but the hazardous properties with regard to irritation (CLP Category 2) could not be characterised. Since other in vitro test systems do not appear to be appropriate to characterise the eye irritation potential (CLP Category 2) and an in vivo test cannot be performed according to Annex VII of Regulation (EC) No 1907/2006, the eye irritating potentail of aluminium vanadium tetraoxide cannot be fully characterised.

Justification for classification or non-classification

Skin irritation/corrosion

Alumnium vanadium tetraoxide does possess a skin irritating but not corrosive potential based on in vitro tests according to OECD 439 and OECD 435, respectively. Thus, Alumnium vanadium tetraoxide does require classification as skin irritatant (Category 2) according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation

Based on results of the BCOP test(OECD 437) and the reconstructed human cornea-like epithelium test  (OECD 492), aluminium vanadium tetraoxide is not corrosive to the eye but the hazardous properties with regard to irritation (CLP Category 2) could not be characterised.