Zircon, cadmium yellow

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-01-26 to 2021-02-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Reference 2

Reference Type:

other: study report amendment

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2020-06-26

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2019-02-10

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2019-10-23

Test material

Test material information

Constituent 1

Reference substance name:

Zirconium zircon with encapsulated cadmium sulphide

Cas Number:

72968-34-4

Molecular formula:

ZrSiO4.xCdS 0,03≤x≤0,3

IUPAC Name:

Zirconium zircon with encapsulated cadmium sulphide

Test material form:

solid: particulate/powder

Details on test material:

- Test material name: Cadmium sulphur zirconium silicon zircon
- new EC name: Zirconium zircon with encapsulated cadmium sulphide
- C.I. name: Zircon, cadmium yellow
- Substance type: inorganic pigment
- Physical state: solid, odourless intense yellow powder
- Storage conditions: At room temperature, under moisture protection

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:

other: Dulbecco's Phosphate Buffered Saline (DPBS)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT; MatTek)
- Tissue lot number: 34122

TEST FOR MTT INTERFERENCE
- To test if a test item directly reduces MTT, 1 ml of a MTT/DMEM solution (1 mg/mL) including 25±2 mg of the test item was incubated for 1 hour (37 ± 1.5°C, 5 ± 0.5% CO2).
- Untreated MTT/DMEM solution (1 mg/mL) was used as negative control.
- After incubation the change of colour was determined by the unaided eye.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25±2 mg of the test item was added to 300 µl of deionised water and mixed. 330 µl of deionised water was used as control (blank). Both were incubated for 60 min (37 ± 1.5°C, 5 ± 0.5% CO2).
- In parallel, 25±2 mg 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 1 hour at room temperature.
- After incubation the change of colour was determined by the unaided eye.

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 significantly 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 ± 1.5 °C for 35 minutes followed by incubation at room temperature until the 60-minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- At the end of the treatment interval the tissues were gently rinsed with PBS several times in order to remove any residual test material and controls.

EXPOSURE
- After pre-warming of the EpiDerm™ 25 ± 2 mg (39.7 mg/cm2) of the test item and 30 μL of the positive and negative control, respectively, were applied onto the surface of the tissue. The tissues were wetted with 25 µL DPBS prior to application. Within the exposure time of 60 minutes the 6-well plates were placed in the incubator for 35 minutes at 37 ± 1.5 °C and the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
- At the end of the treatment interval the tissues were gently rinsed with PBS (in-house) several times in order to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.
- Afterwards, the tissues were incubated in 0.9 mL of fresh assay medium for 23 h 38 min. After this incubation period the medium was changed, and the tissues were incubated for another 18 h 15 min at standard incubation conditions. The complete incubation time was 41 h 53 min.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes (37 ± 1.5°C, 5 ± 0.5% CO2)
- After 3 hour ± 5 minutes incubation the tissues were rinsed with PBS and carefully dried with blotting paper. The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for 2 hours 25 min while shaking at room temperature. At the end of the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken.
- Per each tissue, 3 x 200 µL aliquots of the formazan solution were transferred into a 96-well, flat bottom, microtiter 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).

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Attached background material" below.

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

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponded to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [(mean ODtest item / positive control) / ODmean of negative control] * 100
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control. The test item is considered to be irritant to skin or serious eye damaging in accordance with Regulation EC No. 1272/2008 (UN GHS “Category 1 or 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. In this case further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification. The test substance may be considered as non-irritant to skin in accordance with Regulation EC No. 1272/2008 (UN GHS “No Category”) if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: The test item was tested neat. Three tissue replicates were wetted with 25 µL of DPBS prior to application and then 25 ± 2 mg (39.7 mg/cm²) of the test item were applied uniformly to the epidermis surface.

VEHICLE
- Amount(s) applied: 25 µL of DPBS

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):

41 h 53 min

Number of replicates:

triplicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

101.51

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
In the pre-experiment for MTT interference the test item did not reduce MTT to Formazan salt after optical evaluation.

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. No colouring was detectable by unaided eye-assessment.

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 "Attached background material" below.

ACCEPTANCE OF RESULTS:
- the mean OD570 of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 (1.617) in accordance with OECD TG 439.
- the viability of the tissue replicates treated with the positive control is ≤ 20% (3.47).
- the standard deviation calculated from 3 identically treated replicates is ≤ 18 (0.001 - 1.5).
- Concurrent negative controls and positive controls demonstrate that viability (with the NC), barrier function and resulting tissue sensitivity (with the PC) were within the defined historical acceptance ranges. The results of the positive and negative controls demonstrate reproducibility over time.

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

Any other information on results incl. tables

 Results after treatment with Cadmium sulphur zirconium silicon zircon and the controls

Treatment Group	Tissue No.	OD 570 nm			Mean OD of 3 Wells	Mean OD of 3 Wells blank corrected	Mean OD of 3 tissues	Rel. Viability [%] Tissue 1, 2 + 3	Standard Deviation	Mean Rel. Viability [%]
		Well 1	Well 2	Well 3
Blank		0.037	0.038	0.037	0.037	-	-	-	-	-
Negative Control	1	1.699	1.633	1.640	1.657	1.620	1.617	100.205	0.019	100.0
	2	1.654	1.676	1.682	1.671	1.634		101.046
	3	1.649	1.632	1.620	1.633	1.597		98.749
Positive Control	1	0.096	0.093	0.094	0.094	0.057	0.056	3.552	0.001	3.47
	2	0.092	0.095	0.092	0.093	0.056		3.466
	3	0.092	0.092	0.092	0.092	0.055		3.400
Test Item	1	1.728	1.696	1.693	1.706	1.669	1.641	103.225	1.5	101.51
	2	1.653	1.672	1.663	1.663	1.626		100.572
	3	1.666	1.663	1.668	1.666	1.629		100.743

Historical Control Data

Positive Control; OD at 570 nm after exposition to 5% SDS solution in deionized water (MatTek)		Negative Control OD at 570 nm DPBS (MatTek)
Tissue Viability [%]	3.93	Mean OD	1.71
Standard Deviation	0.95 % points	Standard Deviation	0.18
Range of Viabilities	2.24% - 6.19 %	Range of OD*	1.28 - 2
Mean OD	0.07	* should be ≥ 0.8 and ≤ 2.8 (OECD 439/ MatTek)
Standard Deviation	0.02
Range of OD	0.03 – 0.11
Data of 60 sets of controls shared between 226 studies performed from August 2015 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, Cadmium sulphur zirconium silicon zircon is non-irritant to skin and does not require classification and labelling for skin irritation according to UN GHS and EU CLP.