Cobalt zinc silicate blue phenacite

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-28 to 2015-08-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

adopted 2015-07-28

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Rev. 3/26/2012

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt zinc silicate blue phenacite

EC Number:

270-208-6

EC Name:

Cobalt zinc silicate blue phenacite

Cas Number:

68412-74-8

Molecular formula:

Zn(2-x)Co(x)SiO4 0,7≤x≤1,1

IUPAC Name:

cobalt zinc silicate blue phenacite

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): cobalt zinc silicate blue phenacite, Pigment Blue 74
- Molecular formula: CoxZnySiO4 (x = 1.079, y = 0.827)
- Physical state: odourless intense blue powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: keep dry in closed container at room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

not applicable

Justification for test system used:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

other: Dulbecco's phosphate buffered saline (DPBS)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin model (source: MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue lot number: 21684
- Delivery date: 2015-08-11

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37 ± 1.5 °C (23 hours)
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 minutes, room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
Tissues were rinsed with DPBS at least 15 times in order to remove any residual test material.

After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were again rinsed with DPBS. The tissues were then transferred into plates with fresh assay medium. Tissues were incubated for nearly 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation the inserts were transferred into new plates containing fresh medium. Thereafter tissues were incubated for another 19 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was about 42 hours. Following incubation, the tissue viablity was measured.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT/ EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/ well)
- Incubation time with MTT: 3 hours
- Extraction of Formazan: after the incubation period, MTT solution was aspirated from the wells, and the wells were rinsed three times with DPBS. Inserts were transferred onto new plates and immersed into extractant solution (isopropanol). Tissues were completely covered from both sides and the plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for 22 hours without shaking in the refrigerator in the dark.
After the extraction period was completed, the inserts were pierced to allow the extract to run into the well from which the insert was taken and the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. The optical density was determined with a spectrophotometer. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm
- Filter bandwidth: 1 nm

TEST FOR COLOUR INTERFERENCE
Prior to the start of the test, the test item’s colour interference potential was evaluated. About 25 mg of the test item were mixed with 300 µL of deionised water. This mixture was incubated for 60 minutes at 37 ± 1.5 °C (5 ± 0.5% CO2; 95% RH). Deionized water was used as control.
The test item dyed water, therefore an additional control had to be performed. The coloured test item and the negative control were applied on each one viable tissue, which underwent the entire skin irritation test but was incubated with medium instead of MTT solution during the MTT incubation step. The true tissue viability was calculated as the difference between the OD at 570 nm obtained with living tissue incubated with MTT solution and the OD obtained with living tissue incubated with medium without MTT, and subsequently divided by the OD of the negative control performed in the same experiment.

TEST FOR DIRECT MTT REDUCTION
For correct interpretation of results, the ability of the test item to directly reduce MTT was assessed. For this purpose, approx. 25 mg of the test item were added to 1 mL of MTT solution (resulting: 1 mg/mL). This mixture was incubated in the dark at 37 ± 1.5 °C for 60 minutes. MTT/MTT diluent was used as control. If the colour of the MTT turned blue/purple, the test item is persumed to have reduced MTT.

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.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds 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 OD test item or porsitive control/ mean OD negative control) x 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model: if the mean relative tissue viability of three individual tissues is less or equal to 50% of the negative control, the test item needs to be classified and labeled for its skin irritauion potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 25 mg of the test item, wetted with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL DPBS

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% Sodium Lauryl Sulfate (SLS) solution

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

approx. 42 hours

Number of replicates:

Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

(corrected mean)

Value:

89.2

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:

- OTHER EFFECTS:
- Colour interference with MTT: colour of test item/water mixture changed during the incubation period. Therefore, the OD570 value of the test item in the normal test procedure had to be corrected by the OD570 value gained in the additional test without MTT incubation with one viable tissue.
- Direct-MTT reduction: the colour did not turn blue/purple and the test item was not considered to be a MTT reducer. An additional test with freeze-killed tissues for data correction did not have to be performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control, the absorbance values (1.869, 1.894, and 1.949 (mean: 1.904)) were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.9 % (acceptability criterion: positive control is ≤ 20 %).
- Acceptance criteria met for variability between replicate measurements: the relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test was 18% at the highest (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%). The acceptance criteria were closely not met for the positive control (18 %) for its very low absorption values. Nevertheless, the study was valid due to the clearly positive result of the positive control.
Please refer to the field "Any other information on results incl. tables" below

Any other information on results incl. tables

HISTORICAL DATA

Positive Control		Negative Control [OD570]
Mean Viability	5.6%	Mean Absorption	1.796
Rel. Standard Deviation	2.4%	Rel. Standard Deviation	0.281%
Range of Viabilities	2.9% - 11.3%	Range of Absorbance	1.397 – 2.651
Mean Absorption	0.098
Rel. Standard Deviation	0.038%
Range of Absorbance	0.030 - 0.194

Data of 125 studies performed from November 2011 until May 2014

Table 1: Results after treatment with cobalt zinc silicate blue phenacite and controls

Dose Group	Treatment Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Mean Rel. Absorbance [% of Negative Control]**
Negative Control	60 min	1.869	1.894	1.949	1.904	100.0
Positive Control	60 min	0.102	0.105	0.074	0.094	4.9
Test Item	60 min	1.699	1.772	1.628	1.700	89.3

	Test with Viable Tissue without MTT Incubation / Calculation of corrected Mean Rel. Absorbance Value
	Treat-ment Interval	Mean Absorbance of 3 Tissues (main experiment)	Mean Absorbance of 3 Wells per tissue (Functional Test without MTT)	ODMTT– ODno MTT	Corrected Mean Rel. Absorbance [% of Negative Control]**
Negative Control	60 min	1.904	0.003	1.901	100.0
Test Item	60 min	1.700	0.005	1.695	89.2

* mean of three replicate wells after blank correction

** relative absorbance per treatment group [rounded values]: 100 x (mean absorbance_{testitem/positive control})/(mean absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not irritating to the skin.