[No public or meaningful name is available]

Endpoint summary

Description of key information

skin irritation: not irritating (OECD 439; GLP)

eye irritation: not irritating (OECD 492; GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-01-12 to 2021-01-15

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:

yes

Remarks:

OECD 439 (2020): Pre-test for colour interference was carried out using the MatTek Protocol, i.e. only performed with test chemical in water and no spectral analysis in the range of 570 nm. No historical data of pos. and neg. control are available.

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human 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:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SITor EPI-212-SIT; MatTek)
- Tissue lot number: 33786

TEST FOR MTT INTERFERENCE
Approximately 25 mg test substance was added to 1.0 mL of MTT medium in a clear glass vial. After 60 minutes of incubation at 37.2 - 37.4 °C, in an atmosphere containing 5 % CO2, the mixture was shaken and evaluated for presence and intensity of staining/coloration. A vial of MTT medium served as a control. No change was noted.

TEST FOR COLOUR INTERFERENCE
25 mg of test substance was added to 0.3 mL of deionized water, in a clear glass vial. After 60 minutes of incubation at 37.2 - 37.4 °C, in an atmosphere containing 5 % CO2, the mixture was shaken and evaluated for presence and intensity of staining/coloration. No change was noted.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE AND COLOUR INTERFERENCE
Since the test item/ water solution did not change colour and the test item did not interfere with MTT in the pre-experiments, no additional tissues were necessary in the main experiment.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.1 - 37.2 °C and ambient temperatur
- Temperature of post-treatment incubation: 37.2 - 37.3 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- The tissues were rinsed with sterile DPBS by filling and emptying the tissue insert at least 15 times to remove any residual test material. A constant stream of DPBS was to dislodge the test substance. Cotton swabs were also used to aid in removal of the residual test material.
- After the last rinse, the insert was completely submerged 3 times in a container filled with 150 mL DPBS while gently swishing. The tissue insert was rinsed inside and out with sterile DPBS one final time and the insert was gently shaken and blotted to remove excess liquid.

EXPOSURE
- After the pre-incubation at 37.1 - 37.3 °C and 5 % CO2 for 60 minutes, the inserts were transferred from the upper wells to fresh media in the lower wells of the 6-well plate and further incubated overnight for ~ 18 hours at 37.0 - 37.1 °C and 5 % CO2.
- The upper row of three, 6-well plates were filled with 0.9 mL of assay medium per well. Two plates were prepared for each test substance and one plate for the control substance. Pre-incubated plates were removed from the incubator a few minutes before exposure to the chemicals.
- The tissues were wetted with 25 µL DPBS prior to application.
- Approximately 25 mg of the test substance, and 30 μL of negative control or positive control were added to the surface of three single viable tissues. The tissues were placed in the incubator (37 °C, 5 % CO2) for 35 ± 1 minutes. Then all tissues were removed from the incubator and placed in the sterile hood at ambient temperature until a total of 60 ± 1 minutes.
- The tissue inserts were transferred to previously prepared 6-well plates containing 0.9 mL of assay medium in 3 wells (top row). The tissues were swabbed to remove moisture from the surface. The tissues were incubated for 26 hours at 37.2 - 37.3 °C and 5 % CO2. The plates were removed, and the remaining 3 lower wells were filled with 0.9 mL of fresh assay medium. The inserts were transferred to the freshly filled wells and the plates were placed back into the incubator for an additional ~20 hours at 37.2 - 37.3 °C and 5 % CO2.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT
- MTT concentration: 1 mg/mL
- Incubation time: ~3 hours (37.2 - 37.3 °C, 5 % CO2)
- Inserts were removed from the 6-well plates, and residual media was blotted as needed and transferred into appropriately labelled wells of a 24-well plate containing 300 μL of prepared MTT medium or a blank well. The plates were incubated.
- After the 3 hour, the MTT medium was aspirated from all the wells and discarded appropriately. The wells were rinsed with DPBS three times. After the final rinse was aspirated, the inserts were transferred to a fresh 24-well plate. One mL of isopropanol (extraction solution MTT-100-EXT) was added to each insert and the plate was sealed with Parafilm®. Extraction was performed for 2 hours at room temperature on a shaker (120 rpm). The tissues were discarded and an additional 1 mL of isopropanol was added to the well. The extract in each well was mixed and then duplicate 200 μL aliquots of each were transferred into a 96-well flat bottom microtiter plate using the plate design below for OD measurement. Isopropanol was used as blanks.

NUMBER OF REPLICATE TISSUES:
- Three tissue replicates for test item and controls

PREDICTION MODEL / DECISION CRITERIA
Optical density (OD) was measured in a 96-well plate spectrophotometer using a wavelength of 570 nm, without a reference filter.
Using the resulting OD, viability of the tissue for each treatment group was determined:
Relative viability TS (%) = [ODTS / Mean of ODNC] x 100
Relative viability NC (%) = [ODNC / Mean of ODNC] x 100
Relative viability PC (%) = [ODPC / Mean of ODNC] x 100
For each test substance, negative control, and positive control, the mean relative viability of the tissues was calculated and used for classification. If the mean tissue viability is ≤ 50%, the test substance is predicted to be an irritant (GHS Category 2). If the mean tissue viability is >50%, a non-irritant is confirmed.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: Three tissue replicates were wetted with 25 µL of DPBS prior to application and then 25 mg of the neat test substance was added to the viable tissue surface.

VEHICLE
- Amount(s) applied: not applicable

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

~46 hours

Number of replicates:

Number of EpiDerm tissues per group: triplicates

Irritation / corrosion parameter:

% tissue viability

Value:

104.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

TEST FOR MTT INTERFERENCE
In the pre-experiment for MTT interference the test item did not reduce MTT to Formazan salt.

TEST FOR COLOUR INTERFERENCE
In the pre-experiment for colour interference the test item did not change colour when mixed with deionised water.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of EpiDermTM for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the ten proficiency chemicals listed in Table 1 of OECD TG 439. The respective Laboratory Technical Proficiency Data are attached in the field "Overall remarks, attachments" below.

ACCEPTANCE OF RESULTS:
- the mean OD570 of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 (1.994) in accordance with OECD TG 439.
- the viability of the tissue replicates treated with the positive control is ≤ 20% (2.6).
- the standard deviation from individual % tissue viablitiies calculated from 3 identically treated replicates is ≤ 18 (0.13 - 5.29).

Optical density and viability results I

Treatment Group	Tissue No.	Raw data OD 570 nm		Blank Corrected data OD 570 nm		Mean OD of aliquots	% of viability
		Aliq. 1	Aliq. 2	Aliq. 1	Aliq. 2
Blank		-	-	-	-	0.0872	-
Negative Control	1	2.0752	2.1466	1.988	2.059	2.024	101.5
	2	1.9928	2.0178	1.906	1.931	1.918	96.2
	3	2.1368	2.1205	2.050	2.033	2.041	102.4
Positive Control	1	0.1404	0.1417	0.053	0.055	0.054	2.7
	2	0.1356	0.136	0.048	0.049	0.049	2.4
	3	0.1376	0.1381	0.050	0.051	0.051	2.5
Test Item	1	2.182	2.1544	2.095	2.067	2.081	104.3
	2	2.0709	2.0822	19.984	1.995	1.989	99.7
	3	2.276	2.2976	2.189	2.210	2.200	110.3

Optical density and viability results II

Tissue-Type	Substance	Average OD	SD (OD)	Average % viability	SD (viability)	CV (%)
viable	Negative Control (DPBS)	1.994	0.067	100.0	3.34	3.34
	Positive Control (5% SDS)	0.051	0.003	2.6	0.13	5.18
	Test Item	2.090	0.105	104.8	5.29	5.04

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this study and under the experimental conditions reported, Titanium, iron and aluminium pseudobrookite and rutile is non-irritant to skin and does not require classification and labelling for skin irritation or corrosivity according to UN GHS and EU CLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-01-13 to 2021-01-14

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:

2019-06-18

Deviations:

yes

Remarks:

In the pre-test for Colour Interference the absorption of the test item water solution in the range of 570 nm was not evaluated by OD measurement. No historical data of pos. and neg. control are available.

GLP compliance:

yes

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.:31770; OCL-200-EIT and MTT-100 assy 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.

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 of the neat test substance was applied to the tissue surface. All tissues were pre-wetted with 20 µL DPBS prior application and incubated for 30 min at 37.2 °C.

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 per group: duplicates

Details on study design:

PRE-TEST FOR MTT INTERFERENCE
Approximately 50 mg of test substance or 50 μL deionized water (control) was added to 1 mL aliquot of MTT medium. After ~3 hours of incubation (37.2 - 37.4 °C, 5% CO2), the mixtures were evaluated for change in coloration to purple/blue.

PRE-TEST FOR COLOUR INTERFERENCE
Approximately 50 mg of the test substance was added to both 1 mL of deionized water and 2 mL of isopropanol. After 60 minutes of incubation (37.2 - 37.4 °C, 5% CO2) in deionized water, or 3 hours at ambient temperature in isopropanol. The water solution was evaluated for the presence and intensity of staining/coloration. For the isopropanol solution the absorption in the range of 570±20 nm was evaluated by OD measurement.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE AND COLOUR INTERFERENCE
Since the test item/ water and test item/ isopropanol solutions did not change colour and the test item did not interfere with MTT, no additional tissues were necessary in the main experiment.

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 (37.2 °C, 5 % CO2). Afterwards, the medium was changed and a further pre-incubation for approximately 16 hours (37.0 - 37.3 °C, 5 % CO2) follows.
-Pre-treatment:
After pre-warming and prior to application of the test item respectively the controls, all tissues were pre-wetted with 20 µL DPBS and incubated for 30 min (37.2 °C, 5 % CO2).
- Treatment/Exposure:
Concurrent negative and positive control were applied at a volume of 50 µL and for the test substance 50 mg to the tissue surface and incubated for 6 h in assay medium (37.1 - 37.3 °C, 5 % CO2)
- Rinsing:
Afterwards all tissues were rinsed 3 times in 100 - 150 mL DPBS.
- Post-exposure immersion:
The inserts were transferred to new 12-well plates containing 5 ml assay medium and incubated for 35 min at room temperature and allowed to soak.
Post-exposer incubation:
The tissues were transferred into a 6-well plate with 1 ml assay medium and were incubated for a for about 18 h (37.3 °C, 5 % CO2).
- MTT Assay:
The tissues were placed into the 24-well plate containing 300 µL of MTT (1 mg/ mL) solution and were incubated for 3 hours (37.2 °C, 5 % CO2).
The inserts were transferred to 6-well plates containing 1 mL of isopropanol in each well. Samples were shaken for 2 hours at room temperature on a plate shaker. A volume of 1 mL of isopropanol was added to the extraction solution and the solution was mixed in each well. Duplicate 200 μL aliquots of each were transferred into a 96-well plate. Isopropanol was used as blanks.
-Measurement:
Optical density (OD) was read in a 96-well plate spectrophotometer using a wavelength of 570 nm without using a reference filter.

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%

DESCRIPTION OF DATA EVALUATION
Optical density (OD) was read in a 96-well plate spectrophotometer using a wavelength of 570 nm without using a reference filter.
Blank corrected OD values were calculated by subtracting the OD of the blank wells from the OD of all other measured wells. The mean OD value of each pair of aliquots was then calculated for each tissue.
The mean corrected OD for treated viable tissues was calculated. The resulting mean OD for the negative control treated tissue corresponds to 100% viability for the assay.

Viability [%] = corrected test article OD / corrected mean negative control OD x 100

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, the test is identified as irritant to eyes and requires classification and labelling according to UN GHS (Category 2).

Irritation parameter:

mean percent tissue viability 

Value:

65.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

TEST FOR MTT INTERFERENCE
In the pre-experiment for MTT interference no significant colour change was noted, so the test item did not reduce MTT to Formazan salt.

TEST FOR COLOUR INTERFERENCE
In the pre-experiment for colour interference the test item did not change colour when mixed with deionised water or isopropanol. The connected OD of the test item isopropanol solution was well below 0.08.

ACCEPTANCE OF RESULTS:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.8 (1.850)
- mean relative tissue viability of the positive control is < 50% (47.5%)
- relative tissue viability difference of replicate tissues is < 20% (9.21 - 19.64)

Optical density and viability results I

Treatment Group	Tissue No.	Raw data OD 570 nm		Blank Corrected data OD 570 nm		Mean OD of aliquots	% of viability
		Aliq. 1	Aliq. 2	Aliq. 1	Aliq. 2
Blank		-	-	-	-	0.0866	-
Negative Control	1	1.8371	1.8662	1.751	1.780	1.765	95.4
	2	2.0065	2.0375	1.920	1.951	1.935	104.6
Positive Control	1	0.8543	0.8567	0.768	0.770	0.769	41.6
	2	1.0846	1.0658	0.998	0.979	0.989	53.4
Test Item	1	1.1152	1.1261	1.029	1.040	1.034	55.9
	2	1.4841	1.4839	1.398	1.397	1.397	75.5

Optical density and viability results II

Substance	Average Optical Density reading	SD (Optical Density)	Average % Viability	SD (Viability)
Test Item	1.216	0.363	65.7	19.64
Negative Control (deionized water)	1.850	0.170	100.0	9.21
Positive Control (methyl acetate)	0.879	0.220	47.5	11.87

 

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this study and under the experimental conditions reported, Titanium, iron and aluminium pseudobrookite and rutile is non-irritant to eyes and does not require classification and labelling for eye irritation or serious eye damage according to UN GHS and EU CLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The substance was not observed to be a skin irritant in a reliable in vitro skin irritation study according to OECD 439.

Eye irritation

The substance was not observed to be an eye irritant in a reliable in vitro eye irritation study according to OECD 492.

Justification for classification or non-classification

Skin irritation:

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

Eye irritation:

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