High temperature calcination products of titanium dioxide, calcium carbonate, calcium fluoride, quartz and antimony oxide containing lewisite and 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:

19 July 2016 - 12 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Elicited via a disturbance of the desquamation process and an inflammatory response (i.e. papules, vesicles, bullae and oedema), skin irritation requires penetration of the stratum corneum and elicitation of a biological response. According to Annex VII of the REACH Regulation if new test data are required, these must be derived from in vitro methods only. The EpiDerm™ human skin model (OECD 439) has been validated extensively and is an accepted in vitro test method to detect skin corrosion/irritation (Category 1 or 2) and/or the absence of effects (not classified under CLP).

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 28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No. 640/2012, Method B.46: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, adopted July 06, 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

antimony trioxide

IUPAC Name:

antimony trioxide

Constituent 2

Reference substance name:

Titanium dioxide

EC Number:

236-675-5

EC Name:

Titanium dioxide

Cas Number:

13463-67-7

Molecular formula:

O2Ti

IUPAC Name:

Titanium(IV) oxide

Constituent 3

Reference substance name:

Calcium oxide

EC Number:

215-138-9

EC Name:

Calcium oxide

Cas Number:

1305-78-8

Molecular formula:

CaO

IUPAC Name:

oxocalcium

Constituent 4

Reference substance name:

Silicon dioxide

EC Number:

231-545-4

EC Name:

Silicon dioxide

Cas Number:

7631-86-9

Molecular formula:

O2Si

IUPAC Name:

dioxosilane

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: sponsor batch# 434/08/15
- Expiration date of the lot/batch: March 2020
- Purity test date: 21 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Composition: Antimony oxide (Sb2O3) 38.60%; Titanium oxide (TiO) 31.15%; Calcium oxide (CaO) 26.67%; Silicon dioxide (SiO2) 4.40%; Fluorine (F-) 1.53%.
- Physical characteristics: Powder
- Storage condition of test material: At ambient temperature (10 - 25°), container kept tightly closed and stored in a dry and well-ventilated place.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The optical properties of the test item were evaluated under aqueous conditions (25 mg Uverithe was mixed with 300 μL deionized water1 and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes). No discolorations were noted. The test item was also evaluated for its potential to interfere with the MTT assay reagent, via reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt. Therefore, 25 mg Uverithe was added to 1 mL MTT solution and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 minutes. Untreated MTT solution was used as control. No change of colour was noted. The test substance was not found to interact with the MTT measurement, no additional testing was performed.

OTHER SPECIFICS:
Administration of the test, negative and positive reference item: As a fine powder, 25 mg of test item was applied to the skin model with a surface area of 0.63 cm2 moistened with 25 mL of Dulbecco’s phosphate buffered saline to uniformly cover the skin surface.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: Differentiated epidermal keratinocytes

Cell source:

other: Keratinocyte strain 00267

Source strain:

other: All cells used to produce EpiDerm tissue are purchased or derived from tissue obtained from accredited institutions, from the donor or the donor's legal next of kin for use of the tissues or derivatives of the tissue for research purposes

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Human
- Tissue: normal epidermal keratinocytes

Justification for test system used:

The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis
- Source: MatTek in vitro Life Sceince Laboratories (Bratislava, Slovakia)
- Tissue batch number(s): 23349
- Date of analysis of tissue functionality and quality: 10 August 2016
- Date of initiation of testing: August 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: DPBS
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentrate: MatTek Corporation batch no# 072616ISA
- MTT diluent: MatTek Corporation batch no# 1737674
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 6.4
- Wavelength: 540 nm
- Replicates: The spectrophotometer measurements were made for each of the three tissues in two replicates.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Manufacturer MTT assay, 2.141 ± 0.124 OD (acceptable range: 1.0 to 3.0)
- Barrier function: Manufacturer ET-50 assay, 6.16 hrs (acceptable range: 4.77 to 8.72 hrs)
- Contamination: Manufacturer long term antibiotic and antimycotic free culture, sterile (acceptable criteria: no contamination)

NUMBER OF REPLICATE TISSUES: 3

ASSAY ACCEPTABILITY CRITERIA
- Negative control: The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 1.0 and ≤ 2.5.
- Positive control: A 5% SDS (in H2O) solution was used as a positive control (PC) and tested concurrently with the test chemicals. Concurrent means here that the PC has to be tested in each assay, but only one PC is required per testing day. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%.
- Standard deviation: Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%.

PREDICTION MODEL / DECISION CRITERIA
- Justification for the selection of the cut-off point(s): According to the EU and GHS classification (H314 or H315 / Category 1/2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.
mean tissue viability ≤ 50% Irritant (I), (H314 or H315 or GHS Category 1 or 29)
mean tissue viability > 50% non-irritant (NI).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of the test item was applied to 0.63 cm2 skin model

NEGATIVE CONTROL
- Negative control: Dulbecco’s Phosphate Buffered Saline (D-PBS)
- Source and lot/batch number: GIBCO Invitrogen GmbH (76131, Germany) batch no# 1782127
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Positive control: Sodium dodecyl sulphate
- Source and lot/batch number: MatTek Corporation batch no# 012616TMB
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% aqueous solution

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hour

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

results presented as decimal figure (i.e. 100% = 1)

Run / experiment:

Mean % optical density (OD540) relative to negative controls (n= 3)

Value:

1.029

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

other: absolute optical density (OD 540)

Run / experiment:

Tissue 1

Value:

1.884

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

other: absolute optical density (OD 540)

Run / experiment:

Tissue 2

Value:

1.636

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

other: absolute optical density (OD 540)

Run / experiment:

Tissue 3

Value:

1.907

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:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean optical density (OD) of 3 negative control tissues was 1.758 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.
- Acceptance criteria met for positive control: The viability of cells treated with the positive reference item, 5% SDS, was 5.1% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation determined for all triplicates was below the limit of acceptance of 18%.
- Range of historical values if different from the ones specified in the test guideline: Results for positive and negative control were in line with historical data. All acceptance criteria required were fulfilled.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, Uverithe was non-cytotoxic to in an experiment employing an artificial three-dimensional model of human skin. The test item did not show irritant properties and should not be classified as irritant (UN GHS no category).

Executive summary:

Skin irritation of the test item was evaluated with the EpiDerm Reconstructed Human Epidermis Model. Cell viability of the multi-layered tissue culture of highly differentiated epidermal keratinocytes topically exposed to the test substance was evaluated using the MTT assay, which measures the conversion of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt.

Undiluted test item was applied to the EpiDerm tissue for 60 minutes, alongside a negative and positive control. The mean relative absorbance value of the test item, corresponding to the cell viability did not significantly decrease (102.9 %; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

The test item passed the MTT- and the Colour Interference pre-tests. Conducted according to OECD Test Guideline 439 and GLP, the study is considered to be reliable without restriction (Klimisch 1).