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Toxicological information

Eye irritation

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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 guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium vanadium tetraoxide
EC Number:
815-205-8
Cas Number:
13530-56-8
Molecular formula:
AlVO4
IUPAC Name:
Aluminium vanadium tetraoxide
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: greenish brown 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 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.

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.
- 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.

Results and discussion

In vitro

Results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.

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