Reaction mass of zinc oxide and zinc peroxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01/2015 - 03/2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Human Skin Model Test (Updated Guideline adopted September 26, 2014).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Zinc peroxide

EC Number:

215-226-7

EC Name:

Zinc peroxide

Cas Number:

1314-22-3

Molecular formula:

O2Zn

IUPAC Name:

zinc peroxide

Constituent 2

Reference substance name:

Zinc oxide

EC Number:

215-222-5

EC Name:

Zinc oxide

Cas Number:

1314-13-2

Molecular formula:

OZn

IUPAC Name:

oxozinc

Test material form:

solid: particulate/powder

Remarks:

Fine powder

Specific details on test material used for the study:

Identification: Zinc Peroxide
Appearance: Solid, pale yellow

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch: 1076945
- Expiration date of the lot/batch: 30.09.2015
- Purity test date: Man. / Release: 09/2013

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle: Not stable in water
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Skin corrosion refers to the production of irreversible tissue damage in the skin following the application of a test material [as defined by the Globally Harmonised System for the Classification and Labelling of Chemical Substances and Mixtures (GHS)].
Validation studies have shown that tests employing human skin models are able to discriminate between known skin corrosives (Optional sub-category 1A, optional sub-category 1B and 1C according to GHS) and non-corrosives. The test protocol may also enable the distinction between severe and less severe skin corrosives.

Vehicle:

water

Details on test system:

Cell Culture: EpiDermTM Kits and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia).
The EpiDermTM tissue consisted of normal, human-derived epidermal keratinocytes cultured to form a multilayered, highly differentiated model of the human epidermis.

SKIN DISC PREPARATION
- Procedure used:
Pre-Warming of EpiDerm Tissues: At least 1 hour before dosing, EpiDerm tissues were removed from the refrigerator.
Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed assay medium.
A 24-well plate was prepared as holding plate containing 300 uL assay medium. The holding plate was pre warmed in an incubator (37 +/- 1.5 °C, 5 +/- 0.5 % CO2) until use.
- Quality control for skin discs:
Tissue viability; MTT QC assay, 4 hours, n=3. Acceptance Critera: OD (540-570 nm) [1.0-3.0]. Result and QA Statement: 1.604 +/- 0.131, Pass
Barrier function; ET-50 assay,100 µL 1% Triton X-100, 4 time points, n=3, MTT assay. Acceptance Critera: ET-50 [4.77-8.72 hrs]. Result and QA Statement: 6.60 hrs, Pass
Sterility; Long term antibiotic and antimycotic free culture. Acceptance Critera: No contamination. Result and QA Statement: Sterile, Pass

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 +/- 1.5 °C, 5 +/- 0.5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing PBS to remove any residual test material (20 times).
Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.


DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1))
- Wavelength: 570 nm


NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA
Interpretation of results: The mean OD value obtained for the duplicate tissues per test item was used to calculate a percent viability relative to the negative control, which is arbitrarily set at 100%.
Viability measured after exposure time points --> Prediction to be considered
< 50% after 3 minutes exposure --> Corrosive: Optional Sub-category 1A
>= 50% after 3 minutes exposure AND < 15% after 60 minutes exposure --> Corrosive: Optional Sub-category 1B and 1C
>= 50% after 3 minutes exposure AND >= 15% after 60 minutes exposure --> Non-corrosive

Acceptability of the Assay
An assay met the acceptance criterion if
- the mean OD of the tissue replicates treated with the negative control is >= 0.8 and <= 2.8 for every exposure time
- the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control
- the Coefficient of Variation (CV) in the range 20 -- 100% viability between tissue replicates is <=30%
The quality certificate of the supplier of the test kit demonstrating its robustness (treatment with 1% Triton X-100: 4.08 hours <= ET50 <= 8.7 hours) is annexed to the report.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

other: Optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water / Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent

Amount/concentration applied:

TEST MATERIAL
- Preparation of the Test Item: The test item was not crushed and ground in a mortar, since its consistency was sufficient for even application.
- Amount(s) applied (volume or weight with unit): 25 uL of deionised water and 25 mg (2 times) and about 29 mg (1 time) of the test item were applied evenly onto the surface of duplicate EpiDermTM tissue (39.7 — 46.0 mg/cm2).
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 uL of deionised water
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Deionised water (produced in-house) was used as negative control. 50 uL were applied to each set of duplicate tissues for the 3 min and 1 hour exposure periods.
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 8.0 N Potassium Hydroxide (Sigma) was used as positive control. 50 µL were applied to each set of duplicate tissues for the 3 min and 1 hour exposure periods.
- Concentration (if solution):

Duration of treatment / exposure:

- Test Item: 3 +/- 0.5 minutes, 60 +/- 5 minutes
- Negative Control: 3 +/- 0.5 minutes, 60 +/- 5 minutes
- Positive Control: 3 +/- 0.5 minutes, 60 +/- 5 minutes

Duration of post-treatment incubation (if applicable):

approximately 17.5 hours for the 3 minutes exposure and nearly 17 hours for the 1 hour exposure

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

85.6

Negative controls validity:

valid

Remarks:

mean OD570 = 1.162

Positive controls validity:

valid

Remarks:

30.8% viability

Remarks on result:

other: not corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h exposure

Value:

116.6

Negative controls validity:

valid

Remarks:

mean OD570 = 1.106

Positive controls validity:

valid

Remarks:

4.7% viability

Remarks on result:

other: not corrosive

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity ofthe test item after 1 hour incubation with MTT-reagent did not show blue colour.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The quality certificate of the supplier of the test kit demonstrated its robustness (treatment with 1% Triton X-100: 4.08 hours
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: please see attached report

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, Zinc Peroxide was non corrosive to skin according to EU CLP and UN GHS.

Executive summary:

An in vitro study was performed to assess the corrosive potential of Zinc Peroxide by means of the Human Skin Model Test with EpiDermTM tissues models.

The test item did not prove to be a MTT reducer (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Independent duplicate tissues of EpiDerm^TM were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour,

respectively.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD₅₇₀ >= 0.8 and <= 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 — 100% viability between the tissue replicates is <= 30%, thus the validity of the test system and the specific batch ofthe tissue models is confirmed.

After exposure of the tissues to the test item the relative absorbance value decreased to 85.6% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was not reduced (116.6%). Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive.

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item Zinc Peroxide was non corrosive to skin according to EU CLP and UN GHS.