High temperature calcination products of titanium dioxide, calcium carbonate, calcium fluoride, quartz and antimony oxide containing lewisite and wollastonite

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 December 2016 - 10 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Eye irritation is primarily defined by the extent of corneal injury; substances which damage the superficial epithelium may cause slight irritation, whereas further penetration to the corneal stroma or endothelium may induce mild or severe irritation, respectively. The EpiOcular™ Eye Irritation Test (EIT) was conducted for the test item, following equivocal results in the Bovine Corneal Opacity and Permeability Test method (OECD 437). The reconstructed human cornea-like epithelium (RhCE) test method, utilises a commercially available 3D model of the human corneal epithelium, derived from normal human epidermal keratinocytes. The MatTek EpiOcular™ Eye Irritation Test (OECD 492) has been validated extensively and is an accepted in vitro test method to detect eye corrosion/irritation (Category 1) and/or the absence of effects (not classified under CLP). Data from OECD 492 can be used for REACH Annex VII and Annex VIII requirements for serious eye damage/eye irritation. Solid and partially soluble solids, such as Uverithe, are not considered to be outside the applicability domain of the test method.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

antimony trioxide

IUPAC Name:

antimony trioxide

Constituent 2

Reference substance name:

Titanium dioxide

EC Number:

236-675-5

EC Name:

Titanium dioxide

Cas Number:

13463-67-7

Molecular formula:

O2Ti

IUPAC Name:

Titanium(IV) oxide

Constituent 3

Reference substance name:

Calcium oxide

EC Number:

215-138-9

EC Name:

Calcium oxide

Cas Number:

1305-78-8

Molecular formula:

CaO

IUPAC Name:

oxocalcium

Constituent 4

Reference substance name:

Silicon dioxide

EC Number:

231-545-4

EC Name:

Silicon dioxide

Cas Number:

7631-86-9

Molecular formula:

O2Si

IUPAC Name:

dioxosilane

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: sponsor batch# 434/08/15
- Manufacture date of the lot/batch: 04/2015
- Expiration date of the lot/batch: 2/2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Composition: Antimony oxide (Sb2O3) 38.60%; Titanium oxide (TiO) 31.15%; Calcium oxide (CaO) 26.67%; Silicon oxide (SiO2): 4.40%; and Fluorine (F-): 1.53%.
- Physical characteristics: Powder
- Storage condition of test material: At ambient temperature (10 - 25°), container kept tightly closed and stored in a dry and well-ventilated place.

Test animals / tissue source

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST MODEL
- Test model: EpiOcular ™ test system (Catalogue No. OCL-200-EIT, Lot no. 23758)
- Source: MatTek In Vitro Life Science, Mlynské Nivy 73, Bratislava, Slovakia
- Analysis: The MatTek EpiOcular™ corneal model is derived from normal human keratinocytes, cultured to form a stratified, 3D structure similar to that found in the cornea. Each batch of EpiOcular™ tissues is safety tested for HIV, Hepatitis-B, Hepatitis-C and bacteria, yeast and fungi by the manufacturer. Cells are cultured on 9 mm diameter permeable cell culture inserts.

ANALYSIS FOR TISSUE FUNCTIONALITY AND QUALITY
- Tissue viability (MTT QC assay): OD 1.339 ± 0.127 (1.1 - 3.0 acceptance range)
- Barrier function (ET50 assay): ET50 31.03 minutes (12.2 - 37.5 acceptance range)
- Sterility (Long term antibiotic and antimycotic free culture): sterile (no contamination)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C
- CO2 level: 5%

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

POSITIVE CONTROL:
- Positive control(s): Methyl acetate
- Amount(s) applied (volume or weight with unit): 50 µL
- Source: Sigma Aldrich
- Lot/batch no. (if required): BCBR1040V

NEGATIVE CONTROL:
- Negative control(s): Ultrapure water

Duration of treatment / exposure:

Exposure 6 hours ± 15 minutes

Duration of post- treatment incubation (in vitro):

- Following rinsing and drying, tissues were submerged in EpiOcular™ medium and incubated at ambient temperature for 25 ± 1 minute post-soak period, to ensure removal of the absorbed test item. Tissues were blotted dry on tissue paper, then returned to prewarmed EpiOcular™ medium and incubated for a further 18 hours prior to the MTT assay.

Number of animals or in vitro replicates:

3

Details on study design:

TEST MODEL
- Test model: EpiOcular ™ test system (Catalogue No. OCL-200-EIT, Lot no. 23758)
- Source: MatTek In Vitro Life Science, Mlynské Nivy 73, Bratislava, Slovakia

ANALYSIS FOR TISSUE FUNCTIONALITY AND QUALITY
- Analysis: The MatTek EpiOcular™ corneal model is derived from normal human keratinocytes, cultured to form a stratified, 3D structure similar to that found in the cornea. Each batch of EpiOcular™ tissues is safety tested for HIV, Hepatitis-B, Hepatitis-C and bacteria, yeast and fungi by the manufacturer. Cells are cultured on 9 mm diameter permeable cell culture inserts.
- Tissue viability (MTT QC assay): OD 1.339 ± 0.127 (1.1 - 3.0 acceptance range)
- Barrier function (ET50 assay): ET50 31.03 minutes (12.2 - 37.5 acceptance range)
- Sterility (Long term antibiotic and antimycotic free culture): sterile (no contamination)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C
- CO2 level: 5%

PREDOSE TREATMENT
- Prior to application of the test items or controls, the surface of each EpiOcular™ tissue was moistened with PBS (20 µL). The tissues were incubated for a further 30 ± 2 minutes prior to dosing.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg
- Application of test item to tissues: Solid test item was applied directly to the EpiOcular™ surface, in triplicate. Prior to dosing, a weighting trial was conducted to ensure that approximately 50 mg could be delivered accurately.

POSITIVE CONTROL:
- Positive control(s): Methyl acetate
- Amount(s) applied (volume or weight with unit): 50 µL
- Source: Sigma Aldrich
- Lot/batch no. (if required): BCBR1040V
- Application of positive control to tissues: Control items were applied with a positive displacement pipette and spread evenly over the tissue using the pipette tip, in triplicate

NEGATIVE CONTROL:
- Negative control(s): Ultrapure water
- Application of positive control to tissues: Control items were applied with a positive displacement pipette and spread evenly over the tissue using the pipette tip, in triplicate

MTT DIRECT REDUCTION TEST
Uverithe (50 mg  2 mg) was incubated in a solution of MTT in phosphate buffered saline (1 mL, 1 mg/mL) in a 12 well plate. Eugenol and water (both 50 µL) were used as positive and negative controls for MTT reduction, respectively. Formation of a purple colour was assessed visually following incubation for 3 h ± 10 min. MTT direct reduction assessment was carried out in duplicate for each test and control item. Test items that reduced MTT required additional killed tissue (non specific MTT reduction, NSMTT) controls to be run in parallel with the irritation assay. MTT reducing test chemicals were applied to three killed tissue replicates, which then underwent the entire testing procedure, to generate a (NSMTT) control. In parallel, three killed tissues were treated with the negative control (ultrapure water), to generate a “killed untreated” NSMTT value.

IDENTIFICATION OF COLOUR INTERFERING TEST ITEMS
To identify potential interference by a coloured test item, Uverithe (50 mg  2 mg) was incubated with water (1 mL) or isopropanol (1 mL) for ca 1 h. Each solvent was tested in duplicate. As the test item did not fully dissolve and particles were present in the aliquots, it was not possible to assess the optical density of the test item, therefore, a visual observation of the assessment of colour was performed instead.
Colour interfering test items required additional controls with viable tissues (in triplicate) which underwent the entire testing procedure but were incubated with medium instead of MTT solution during the MTT assay. This generated a non specific colour in living tissues (NSCliving) control. The NSCliving control was performed concurrently to the testing of the coloured test item, and on every occasion where the coloured test item was tested due to the inherent biological variability of living tissues. Test items that were identified as reducing MTT and causing colour interference required a third set of adapted controls. This was the non-specific colour in killed tissues (NSCkilled) control. In this control, the test item was applied to three killed tissue replicates (on one occasion) which underwent the entire testing procedure but were incubated with medium instead of MTT solution during the MTT assay.

TEST SYSTEM SET UP
EpiOcular™ tissues were shipped in sterile units and on arrival were transferred to 6 well plates containing EpiOcular™ Medium (1 mL). Prior to transfer, the pH and temperature indicators were checked and found to be acceptable. The EpiOcular™ tissues were incubated for ca 1 h, allowing tissues to recover. After this initial recovery period, spent media was replaced with fresh EpiOcular™ Medium (1 mL) and then tissues were incubated overnight prior to performing the EIT as described in the EpiOcular™ EIT Protocol provided by MatTek[3].
Killed tissues were previously prepared by transferring the tissues from transport agar into empty wells in a plate. The tissues then went through 2 freeze thaw cycles using a freezer set to maintain a temperature of -20°C. Following the second thaw the tissues were stored in a freezer set to maintain a temperature of -20°C until required. On the day of use the tissues were removed from the freezer, thawed to room temperature and transferred to a 6 well plate containing EpiOcular™ Medium (1 mL). Killed tissues were then treated as per living tissues.

EXPOSURE
- Exposure: 6 hours ± 15 minutes
- Rinsing procedure: The test item or control treatments were removed by rinsing (3-5 times) and then tapped dry on tissue paper.
- Post-exposure incubation: Following rinsing and drying, tissues were submerged in EpiOcular™ medium and incubated at ambient temperature for 25 ± 1 minute post-soak period, to ensure removal of the absorbed test item. Tissues were blotted dry on tissue paper, then returned to prewarmed EpiOcular™ medium and incubated for a further 18 hours prior to the MTT assay.

MTT ASSAY
- Incubation: Following exposure, rinsing and recovery, viability of all EpiOcular™ tissues was measured by the MTT assay. Tissues were blotted dry and transferred to MTT solution in 24 well plates (1 mg/mL MTT in Assay Medium; 300 µL per well). Non-specific colour control tissues were transferred to EpiOcular™ Medium (1 mL) instead of MTT solution. The tissues were then incubated for 3 hours (± 10 minutes).
- Following incubation, EpiOcular™ tissues were tapped dry on tissue paper and transferred to isopropanol (2 mL).
- Formazan extraction: Samples were protected from light and evaporation using tin foil and Parafilm® and were extracted on a rocking platform at ambient temperature for 2 h. Tissues were removed, and extraction solutions were collected. Duplicate aliquots (200 µL) of each isopropanol extract were transferred to 96 well plates and measured using a BioTek ELx808 plate reader at 562 nm. Wells containing isopropanol were read in parallel as blanks.
- Optical density (OD) was read using a BioTek ELx808 plate reader with assay parameter setup and data capture by WinKQCL Version 3.0.1.

ACCEPTANCE OF RESULTS:
The assay was deemed acceptable if:
- The mean OD value of the negative control tissues was >0.8 and <2.5, and the SD of the final viability between the three replicates was <18%.
- The mean % viability of the positive control tissues was <50%, and the variability in the % viability of the tissues was <20% or SD of the final viability between the three replicates was <18%.
The results of a test item were deemed acceptable if:
- The SD of the final viability between the three replicates was <18% and the variability in the % viability of the three tissues was <20%.

Results and discussion

In vitro

Results

Irritation parameter:

other: Viability

Run / experiment:

Mean corrected final viability (%)

Value:

86.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- MTT Direct Reduction Test/Colour interference: Uverithe did not reduce MTT in the direct reduction test, and was not considered to interfere with visual colour observations.

TEST ITEM RESULTS
- Uverithe resulted in a mean corrected final viability of 86.7% (SD: 9.7%). Thus, the test item achieved a “No category” GHS classification.
- The variability between replicates was acceptable, with an SD (of the final viability) <18%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean A562 value measured for the negative control tissues was 1.33. This was within the assay acceptance criteria values of 0.5 to 2.5, with an SD (of the final viability) <18% (SD was 3.78%).
- Acceptance criteria met for positive control: The mean calculated viability for the positive control was 20.5% (SD: 0.9%). In both cases, these were within the assay acceptance criteria of <50%, with a SD <18%.

Any other information on results incl. tables

Results of the EIT for Uverithe and controls

	Tissue ID.	Aliquot OD562		Corrected OD562		Mean OD562	Mean OD562	% Viability	Mean Final % Viability	SD	Corrected Final Viability (%)	UN GHS Classification
Tissue Treatment		1	2	1	2
Negative Control (NC)	NC-A	1.389	1.402	1.351	1.364	1.357	1.334	101.74	100.00	3.78	100.00	No Category
	NC-B	1.316	1.313	1.278	1.275	1.276		95.66
	NC-C	1.414	1.400	1.376	1.362	1.369		102.60
Positive Control (PC)	PC-A	0.321	0.327	0.283	0.289	0.286		21.41	20.51	0.90	20.51	Category 1/2
	PC-B	0.301	0.299	0.263	0.261	0.262		19.62
	PC-C	0.312	0.312	0.274	0.274	0.274		20.51
Uverithe	TT1A	1.070	1.077	1.032	1.039	1.035		77.60	86.71	9.70	86.71	No Category
	TT1B	1.189	1.172	1.151	1.134	1.142		85.62
	TT1C	1.331	1.331	1.293	1.293	1.293		96.90

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The EpiOcular™ (OECD 492) has been validated extensively and is an accepted in vitro test method to detect the absence of effects (not classified under CLP). Solids and partially soluble solids, such as Uverithe, are not considered to be outside the applicability domain of the test method. Consequently, data from the EpiOcular™ EIT can be used to fulfil Annex VII and Annex VIII requirements for serious eye damage/eye irritation potential. The mean percentage viability of the EpiOcular™ tissues treated with Uverithe was 86.7%, compared to the negative control. According to the GHS and CLP criteria, the EpiOcular™ EIT categorised the test item as “No Category”. Conducted according to the aforementioned guidelines and GLP, the study passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Executive summary:

Eye irritation is primarily defined by the extent of corneal injury; substances which damage the superficial epithelium may cause slight irritation, whereas further penetration to the corneal stroma or endothelium may induce mild or severe irritation, respectively. A reconstructed human cornea-like epithelium (RhCE) test method, using the EpiOcular™ 3D model of the human corneal epithelium, was conducted for the test item.

The test item was applied to the upper surface of the EpiOcular™ tissue, equivalent to the anterior surface of the human cornea, for 6 hours ± 15 minutes and then rinsed in phosphate buffered saline. Following post-soak and recovery periods, cell viability was assayed as a measure of ocular irritation potential, using the MTT assay. In viable cells, mitochondrial dehydrogenase activity reduces MTT (Methylthiazolyldiphenyl-tetrazolium bromide) to a bluey purple formazan metabolite. The reduction of the MTT solution was measured using a spectrophotometer.

The mean percentage viability of the EpiOcular™ tissues treated with Uverithe was 86.7%, compared to the negative control. The variability between replicates was acceptable, with an SD (of the final viability) <18%. The EpiOcular™ EIT was successfully run, with all positive and negative control acceptance criteria being met. The MatTek EpiOcular™ Eye Irritation Test (OECD 492) has been validated extensively and is an accepted in vitro test method to detect eye corrosion/irritation (Category 1) and/or the absence of effects (not classified under CLP). Conducted according to the aforementioned guidelines and GLP, the study passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).