Zircon, cadmium yellow

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:

2021-01-26 to 2021-02-12

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:

2020-06-26

Deviations:

no

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 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

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.

 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

 

 

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.

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:

2021-01-19 to 2021-02-04

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:

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 2019-10-23

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.

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 OD of the test item/ water and test item/ isopropanol was < 0.08 and the test item did not interfere with MTT in the pre-experiments, 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 ± 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 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 about 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
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.

Irritation parameter:

mean percent tissue viability 

Value:

73.87

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 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 5.6 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.

Results after treatment with Cadmium sulphur zirconium silicon zircon and the controls for 6 hours

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	1.278	1.240	1.259	1.222	1.271	73.87	71.1	5.60
	2	1 .351	1.360	1.355	1.319			76.7

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)

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this study and under the experimental conditions reported, Cadmium sulphur zirconium silicon zircon 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.