[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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 material

Test material information

Constituent 1

Reference substance name:

titanium, chromium, iron, zinc oxide

Molecular formula:

x(Zn, Ti, Cr, Fe)3O4 * yTiO2 * z(Fe, Cr, Zn, Ti)3O5

IUPAC Name:

titanium, chromium, iron, zinc oxide

Test material form:

solid: particulate/powder

Details on test material:

- Chemical description: Chromium, iron, titanium and zinc spinel and rutile
- Physical state: Solid, brown powder, odourless
- Crystal Structure: Spinel and rutile
- Storage condition of test material: Kept dry in closed containers
- Purity : ca 98%

In vitro test system

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, 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 temperature
- 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

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

101.8

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 - 3.34).

Any other information on results incl. tables

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	2.1997	2.1852	2.113	2.098	2.105	105.6
	2	2.0212	1.998	1.934	1.911	1.922	96.4
	3	2.1513	2.1515	2.064	2.064	2.064	103.5

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	2.031	0.096	101.8	4.81	4.72

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this study and under the experimental conditions reported, Chromium, iron, titanium and zinc spinel 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.