Synthetic wollastonite

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 February 2021 - 20 February 2021

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Synthetic wollastonite

EC Number:

952-026-5

Molecular formula:

CaSiO3

IUPAC Name:

Synthetic wollastonite

Test material form:

solid: bulk

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Imerys
- Lot/batch number of test material: 2011 20
- Purity, including information on contaminants, isomers, etc.: 100%
- Molecular weight: 116.16 g/mol
- Expiry date: 27 November 2030

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was used as supplied.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Supplier: MatTek In Vitro Life Sciences Laboratories
- Model used: EPIDERM™ Reconstructed Human Epidermis
- Tissue batch number(s): 34123
- Delivery date: 17 February 2021
- Date of initiation of testing: 17 February 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 Deg C
- Temperature of post-treatment incubation: 37 Deg C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert approximately 15 times using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were then submerged 3 times in 150 ml of DPBS without Ca++ and Mg++.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE: yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)
- MTT concentration: 1.0 mg/mL
- Incubation time: 1 hour
- Spectrophotometer: Labtech LT-4500 microplate reader
The upper limit of accuracy for measured absorbance of MTT Formazan at 570 nm (OD570),
filter band pass 10 nm, was determined to be at an optical density of 2.6. (Warren, N; 2017).
- Linear OD range of spectrophotometer: OD570

NUMBER OF REPLICATE TISSUES: Triplicate

PREDICTION MODEL / DECISION CRITERIA
Data Evaluation
Quantitative MTT Assessment (Percentage Tissue Viability)
For the test item the relative mean tissue viabilities obtained after the 60-Minute exposure period followed by the 42-Hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (mean OD570 of test item/mean OD570 of negative control) x 100

Classification of irritation potential is based upon relative mean tissue viability following the
60-Minute exposure period followed by the 42-Hour post-exposure incubation period
according to the following:

Relative mean tissue viability is ≤50% = Corrosive or Irritant - H314 or H315 Category 1 or 2
Relative mean tissue viability is >50% = Non-irritant - Not classified for irritation

If the relative mean tissue viability is ≤50%, differentiation between EU CLP/UN GHS Category 1 and Category 2 will not be possible based on the results of this study in isolation.

EU CLP/UN GHS:
Hazard Category 1 - Code H314; Statement “Causes severe skin burns and eye damage”
Hazard Category 2 - Code H315; Statement “Causes Skin Irritation”

Acceptance Criteria
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤20% relative to the negative control treated tissues.

Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is equal or greater than 0.8 and ≤2.8.

Standard Deviation:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18% for each testing or control group.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- Concentration (if solution): The test item was used as supplied.

NEGATIVE CONTROL: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): Used as supplied.

POSITIVE CONTROL: Sodium dodecyl sulphate
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): Prepared as a 5% w/v aqueous solution.

MTT SOLUTION
- Amount(s) applied (volume or weight): 1 mL
- Concentration (if solution): 1.0 mg/mL MTT solution.

Duration of treatment / exposure:

60 +/- 1 minutes

Duration of post-treatment incubation (if applicable):

1st period: 24 +/- 2 hours, 2nd period 18 +/- 2 hours

Number of replicates:

Triplicate

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-Minute exposure period and 42-Hour post-exposure incubation period

Value:

100.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

It was considered unnecessary to perform IL-1a analysis on the assay medium, retained from
the wells of the 24 ±2 hour post exposure incubation plate, as the results of the MTT test
were unequivocal.

Any other information on results incl. tables

Direct MTT Reduction
The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

Assessment of Color Interference with the MTT endpoint
The solutions containing the test item were colorless. It was therefore unnecessary to run color correction tissues.

Acceptance Criteria
The relative mean tissue viability for the positive control treated tissues was 4.0% relative to the negative control treated tissues. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 1.813. The negative control acceptance criteria were therefore satisfied.
The standard deviation between the three tissue replicates of each treatment group did not exceed 18%. The acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study and under the experimental conditions reported, the test item was classified as non-irritant.