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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:
2021-01-19 to 2021-02-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

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:
2019-06-18
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
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed signed 2019-10-23

Test material

Constituent 1
Chemical structure
Reference substance name:
Cobalt aluminate blue spinel
EC Number:
310-193-6
EC Name:
Cobalt aluminate blue spinel
Cas Number:
1345-16-0
Molecular formula:
Co(x)Al(3-x)O4 1≤x≤1,5
IUPAC Name:
Cobalt aluminium spinel
Test material form:
solid: particulate/powder
Details on test material:
- Substance name: Cobalt aluminate blue spinel
- C.I. name: Pigment blue 28
- Substance type: inorganic pigment
- Physical state: solid, blue powder, odourless
- Storage condition: Keep dry in closed containers

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
JUSTIFICATION OF THE TEST METHODS AND CONSIDERATIONS REGARDING APPLICABILITY:
This in vitro method is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS “No Category”) without further testing within a tiered testing strategy from those requiring classification and labelling (UN GHS categories 1 and 2). Therefore, it can be used for regulatory purposes as an initial step in the bottom-up approach or as one of the last steps in a top-down approach to test eye irritation/corrosion potential. It is not intended to differentiate between UN GHS “Category 1” (serious eye damage) and UN GHS “Category 2” (eye irritation) which would require additional testing. Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the MTT dye to formazan conversion by the EpiOcular™ tissue construct after topical exposure to the test substance.

RhCE TISSUE CONSTRUCT USED: EpiOcular™ (Lot No.: 30694; Standard Assay Kit and MTT-100 kit; source: MatTek Corporation (82105 Bratislava, Slovakia))
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

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): 50 mg (≙ 83.3 mg/cm2)were applied to the tissue surface. The test item was tested neat.
Duration of treatment / exposure:
6 hours
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
about 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:
PRE-TEST FOR MTT INTERFERENCE
- To test if a test item directly reduces MTT, 1 ml of a MTT solution (1 mg/mL) including 50 ± 2 mg of the test item was incubated for 180 min under (37 ± 1.5°C, 5 ± 0.5% CO2).
- 50 µL deionised water in MTT solution was used as negative control.
- After incubation the change of colour was determined by the unaided eye.

PRE-TEST FOR COLOUR INTERFERENCE
- To check the colouring potential of the test item, 50 ± 2 mg of the test item was added to 1 mL of deionised water and mixed. 1 mL of deionised water was used as control (blank). Both were incubated for 60 min (37 ± 1.5°C, 5 ± 0.5% CO2).
- In parallel, 50 ± 2mg of the test item was added to 2 mL of isopropanol and mixed. A control (2 mL of isopropanol, blank) was run concurrently. Both were incubated for 3 hours at room temperature.
- After incubation the presence of the staining was evaluated by OD measurement.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE AND COLOUR INTERFERENCE
Since the test item interfered with MTT and reduced it to formazan by itself in the second pre-experiment two additional controls in duplicates run with the main experiment – the freeze-killed tissue controls (killed controls = KC):
- Deionised water treated freeze-killed tissues (NC_KC)
- Test item treated freeze-killed tissues (TI_KC)
At the end, the following data correction procedure for Viability plus killed controls were performed:
Corrected viability (%) = TI viability – (TI_KC viability – NC_KC viability)

DETAILS OF THE TEST PROCEDURE USED
Pre-warming:
- EpiOcularTM tissues were equilibrated at room temperature for 15 min. The inserts with the tissues were transferred into 6-well-plates containing 1.0 ml assay medium and incubated for 60 minutes (37 ± 1.5°C, 5 ± 0.5% CO2). Afterwards, the medium was changed and a further pre-incubation for 17 h 53 min (37 ± 1.5°C, 5 ± 0.5% CO2) follows.
- Treatment:
After pre-warming and prior to application of the test item respectively the controls, all tissues (the freeze killed tissues also) were pre-wetted with 20 µL Ca2+Mg2+free-DPBS and incubated for 30 min (37 ± 1.5°C, 5 ± 0.5% CO2).
Concurrent negative and positive control were applied at a volume of 50 µL and for the test item 50 mg to the tissue surface and incubated for 6 h in assay medium (37 ± 1.5°C, 5 ± 0.5% CO2)
Afterwards all tissues were rinsed 3 times in 100 mL DPBS and incubated for 25 min in 5 ml assay medium at room temperature in a 12-well plate (post-exposure immersion). At the end of this incubation the tissues were transferred into a 6-well plate with 1 ml assay medium and were incubated for a post exposure incubation for 18 h (37 ± 1.5°C, 5 ± 0.5% CO2).
- MTT Assay:
The tissues were placed into the 24-well plate containing 300 µL of MTT solution and were incubated for 180 min (37 ± 1.5°C, 5 ± 0.5% CO2).
At the end of this incubation the tissues were transferred into new 24-well plates containing 2 mL isopropanol. The formazan salt was extracted with isopropanol within 2 h 10 min while shaking at room temperature.
Then, the extracts were mixed and two 200 µL aliquots formazan solution were transferred to a 96-well plate for OD measurement. 200 µL of isopropanol were added to the wells designated as blanks for 96-well plate.
-Measurement:
The optical density (OD570nm) was determined spectrophotometrically in duplicates by a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

REMOVAL OF TEST MATERIAL
Afterwards all tissues were rinsed several times in 100 ml of Ca2+/Mg2+-free PBS

TEST ACCEPTANCE CRITERIA
The test meets acceptance criteria:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%
- OD control values are not below historically established boundaries/ positive and negative control mean values and acceptance ranges based on historical data.

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.

DESCRIPTION OF DATA EVALUATION
1) The mean OD value of the two wells for each tissue and the blank control (ODBlk) was calculated (Mean [OD570] (well 1 and well 2).
2) The mean ODBlk was subtracted from each mean OD value of the two wells.
(Mean [OD570] blank corr. (well 1 and well 2)). These values were used for all further calculations below.
3) The mean OD of the two relating tissues for each test group (negative control (NC), positive control (PC)) and the test item (TI) was calculated with the blank corrected mean OD (Mean [OD570] of T1 and T2)
4) The percent viability of each test group relative to the negative control (= 100%) was calculated:
Viability (%) =100 × (mean OD_TI/PC/NC) / mean OD_NC)
5) The relative OD of each tissue per test group was calculated. 100 divided by the mean ODNC T1 and T2 x mean OD of each test group.
6) The difference of the viability values between duplicate tissues was calculated: The relative OD of T2 was subtracted from T1.

PREDICTION MODEL
If the test item-treated tissue viability is > 60% after exposure and post-exposure incubation 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).
If the test item-treated tissue viability is ≤ 60% after exposure and post-exposure incubation relative to negative control treated tissue viability, no prediction can be made for this test item.

Results and discussion

In vitro

Results
Irritation parameter:
other: mean percent tissue viability  (migrated information)
Value:
81.94
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
TEST FOR MTT INTERFERENCE
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed purple colour. Therefore, an additional test with freeze-killed tissues was necessary in the main experiment.

TEST FOR COLOUR INTERFERENCE
In the colour interference the test item not changed colour when mixed with deionised water or isopropanol, the mean OD of the test item in deionised water or isopropanol was < 0.08 . Therefore, no additional viable tissues (without MTT addition) were necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of EpiOcularTM EIT for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the fifteen proficiency chemicals listed in Table 1 of OECD TG 492. The respective proficiency certificate given by MatTek is annexed to this endpoint study record.

ACCEPTANCE OF RESULTS:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.8 (1.720)
- mean relative tissue viability of the positive control is < 50% (18.32%)
- relative tissue viability difference of replicate tissues is < 20% (0.1 p.p. to 1.23 p.p.)
- The OD control values of the positive control were within the historically established boundaries.
- positive and negative control mean values and acceptance ranges based on historical data.
The acceptance criteria were met.

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

Any other information on results incl. tables

Results after treatment with Cobalt aluminate blue spinel and controls

Treatment Group

Tissue No.

Well 1 [OD570]

Well 2 [OD570]

Mean [OD570] (Well 1 and well 2)

Mean [OD570] blank corr. (Well 1 and well 2)

Mean [OD570] of T1 and T2

Mean tissue viabil. [%]

Viabil. of T1 and T2 [%]

Diff. of viabil. between T1 and T2 [p.p.]

Blank

-

0.037

0.036

0.037

-

-

-

-

-

Negative Control

1

1.770

1.742

1.756

1.719

1.720

100.0

100.0

0.10

2

1.794

1.721

1.758

1.721

100.0

Positive Control

1

0.360

0.350

0.355

0.319

0.315

18.32

18.5

0.40

2

0.345

0.351

0.348

0.312

18.1

Test Item

1

1.492

1.423

1.458

1.421

1.411

81.94*

82.6

1.23

2

1.436

1.438

1.437

1.400

81.4

Negative Control
Freeze killed Tissues

1

0.072

0.070

0.071

0.034

0.038

2.19

2.0

0.41

2

0.079

0.077

0.078

0.041

2.4

Test Item Freeze killed Tissues

1

0.069

0.069

0.069

0.032

0.039

2.26

1.9

0.75

2

0.082

0.082

0.082

0.045

2.6

* Corrected viability (%) = TI viability – (TI_KC viability – NC_KC viability)

Historical Control Data

Positive Control; OD at 570 nm after

exposition to Methyl acetate (MatTek)

Negative Control OD at 570 nm

DPBS (MatTek)

Mean Viability

27.13%

Mean Absorption

1.70

Standard Deviation

9.54 p.p.

Standard Deviation

0.29

Range of Viabilities

6.73%–42.54%

Range of Absorbance*

1.02–2.32

Mean Absorption

0.46

* should be 0.8 - 2.8 (OECD 439)

or 1.0 - 2.5 (MatTek)

Standard Deviation

0.17

Range of Absorbance

0.08–0.79

Data of 33 sets of controls shared between 75 studies performed from June 2016 until May 2020. (p.p. – percentage points)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, in this study and under the experimental conditions reported, Cobalt aluminate blue spinel does not require classification and labelling for eye irritation or serious eye damage according to UN GHS and EU CLP.