Olivine, cobalt silicate blue

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-11-06 to 2019-11-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

2019-06-18

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:

2017-07-11

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2019-10-23

Test material

Test material information

Constituent 1

Reference substance name:

Olivine, cobalt silicate blue

EC Number:

269-093-5

EC Name:

Olivine, cobalt silicate blue

Cas Number:

68187-40-6

Molecular formula:

Co2SiO4

IUPAC Name:

silicon(4+) bis(λ²-cobalt(2+)) tetraoxidandiide

Test material form:

solid: particulate/powder

Details on test material:

- Test item identification: Olivine, cobalt silicate blue (Pigment Blue 73)
- Substance type: inorganic pigment
- Storage condition of test material: Keep dry and container tightly closed. Keep container in an adequately ventilated storage

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: 30837

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
Since the test item interfered with MTT and reduced it to formazan by itself in the second pre-experiment two additional controls in duplicates run with the main experiment – the freeze-killed tissue controls (killed controls = KC):
- Negative control/ deionised water treated freeze-killed tissues (NC_KC)
- Test item treated freeze-killed tissues (TI_KC)
At the end, the following data correction procedure for viability plus Killed Control tests was performed::
Corrected viability (%) = TI viability – (TI_KC viability – NC_KC viability)

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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- 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 within about 3 hours 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, and the insert were discarded.
- 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
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues were calculated.
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with Regulation EC No. 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance. The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 and 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 mg (39 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):

approx. 42 hours

Number of replicates:

triplicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

93.39

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
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed purple colour. Therefore, an additional test with freeze-killed tissues was necessary for the quantitative correction of the test item viability.

TEST 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. Therefore, an additional test with viable tissues was not necessary.

ACCEPTANCE OF RESULTS:
- the mean OD570 of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 (1.953) in accordance with OECD TG 439.
- the viability of the tissue replicates treated with the positive control is ≤ 20% (4.35%) .
- the standard deviation calculated from 3 identically treated replicates is ≤ 18 (0.6 – 8.1).
- Concurrent negative controls (NC) and positive controls (PC) were used in each run to demonstrate that viability (with the NC), barrier function and resulting tissue sensitivity (with the PC) of the tissues were within a defined historical acceptance range.

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 Cobalt silicate olivine 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		0.040	0.041	0.053	0.044
Negative Control	1	2.041	1.986	2.000	2.009	1.965	1.953	100.591	1.0	100.0
	2	2.019	1.953	1.951	1.974	1.930		98.803
	3	2.063	1.995	1.970	2.009	1.965		100.606
Positive Control	1	0.154	0.143	0.132	0.143	0.098	0.085	5.036	0.6	4.35
	2	0.124	0.122	0.121	0.122	0.078		3.988
	3	0.123	0.122	0.123	0.123	0.078		4.014
Test Item (TI)	1	1.713	1.681	1.674	1.689	1.645	1.809	84.211	8.1	93.39*
	2	1.918	1.843	1.841	1.867	1.823		93.317
	3	2.033	1.995	1.981	2.003	1.959		100.280
Blank		0.038	0.039	0.039	0.039
Negative Control Freeze killed Tissues (NC_KC)	1	0.115	0.113	0.113	0.114	0.075	0.072	3.835	0.2	3.69
	2	0.109	0.108	0.108	0.108	0.069		3.548
Test Item Freeze killed Tissues (TI_KC)	1	0.097	0.096	0.097	0.097	0.058	0.057	2.963	0.1	2.90
	2	0.095	0.094	0.095	0.094	0.056		2.847

* Corrected viability (%) = TI viability – (TI_KC viability – NC_KC viability)

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 [%]	3.98	Mean OD	1.69
Standard Deviation	1.01 p.p.	Standard Deviation	0.19
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 51 sets of controls shared between 197 studies performed from August 2015 until June 2019 (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, Cobalt silicate olivine is non-irritant to skin and does not require classification and labelling for skin irritation according to UN GHS and EU CLP.