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Diss Factsheets

Administrative data

Description of key information

not irritating to skin (97.1% relative tissue viability; OECD guideline 439, GLP, KL1)

not irritating to eye (89.6% mean relative absorbance, OECD guideline 492, GLP, KL1)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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
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)
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:
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
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
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
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 EpiDermTM 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.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05/2015 - 08/2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted July 26, 2013; and as described in detail in the Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200), Rev. 3/26/2012.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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:
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
water
Details on test system:
SKIN DISC PREPARATION
- Procedure used: Epi-200 SIT kits and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing ntercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts.
EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate and reached the testing facility on June 23, 2015.
On day of receipt the preincubation phase of the EpiDerm™ tissues started.
- 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 an d QA Statement: 1.945 +/- 0.155, 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: 6.62 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: ca. 25 min RT, ca. 35 min 37 ± 1.5 °C, 5 ± 0.5 % CO2.
- 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: tissues were gently 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 once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper.
- Observable damage in the tissue due to washing:

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm
- Filter: 570 ± 1 nm

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential (see OECD TG 439) and is used for the purpose of classification as irritating or non-irritating according to chemicals law (EU CLP, UN GHS). Depending on the regulatory framework and applicability of the test guideline, chemicals that produce cell viabilities above the defined threshold level, are considered non-irritants. The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
other: Prior to the start of the test, the test item’s colour interference potential was evaluated. The ability of the test item to directly reduce MTT was also tested..
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Each approximately 25 - 26 mg (~ 39 mg/cm² according to guideline) of the test item were applied to the tissues, wetted with 25 µL DPBS, and spread to match the surface of the tissue.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS (MatTek) was used as negative control per tissue.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% SLS solution in deionised water (MatTek) was used a positive control per tissue.
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min
Value:
ca. 97.1
Negative controls validity:
valid
Remarks:
mean OD570 = 1.792
Positive controls validity:
valid
Remarks:
5.45 tissue viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system:
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 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: positive control induced a decrease in the relative absorbance as compared to the negative control to 5.4% thus ensuring the validity of the test system
- Acceptance criteria met for variability between replicate measurements: below 14% (<18% required)

- Range of historical values if different from the ones specified in the test guideline:
Positive Control:
Mean Viability 5.6%
Rel. Standard Deviation 2.4%
Range of Viabilities 2.9% - 11.3%
Mean Absorption 0.098
Rel. Standard Deviation 0.038%
Range of Absorbance 0.030 - 0.194

Negative Control [OD570]
Mean Absorption 1.796
Rel. Standard Deviation 0.281%
Range of Absorbance 1.397 – 2.651
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, Zinc Peroxide is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

An in vitro study was performed to assess the irritation potential of Zinc Peroxide by means of the Human Skin Model Test.

The test item did not reduce MTT (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.

Each three tissues of  the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes.

Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of the tissue.

30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue.

After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control  for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item Zinc Peroxide the mean relative absorbance value decreased irrelevantly to 97.1% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Zinc Peroxide is not irritant to skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
July, 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.
The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg (tissues were pre-wetted with 20 µL of Ca++ Mg++ free-DPBS)

Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
18 h
Number of animals or in vitro replicates:
2
Details on study design:
- EpiOcular Kit: MatTek Corporation, Lot No.: 23771

Assessment of Direct MTT Reduction by the Test Item: - approximately 50 mg of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control of 50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution was run concurrently.

Assessment of Coloured or Staining Materials: Since the test item was non-coloured additional tests had to be performed to assess, if it became colorant after contact with water or isopropanol. For this purpose each approximately 50 mg of the test item was added to 1.0 mL of water and to 2 mL isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for at least one hour, the isopropanol mixture or for 2 to 3 hours at room temperature

Experimental Performance:
After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++ Mg++ free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
Test item exposure: After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control item were tested by applying approximately 50 mg of test item or 50 µL of the controls topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 6 hours.
At the end of the 6 hours treatment time, the test item was removed by extensively rinsing of the tissues with Ca++ Mg++ -free DPBS brought to room temperature. Since it was not possible to completely remove a visible remainder of the test item, this was noted in the study file. No further rinsing was done.
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium (room temperature) in a pre-labelled 12-well plate for 25 minutes immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item or control absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for about 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) or a standard plate sealer, and were immediately extracted (shaken for 2.5 hours at room temperature). The tissues were not be pierced. The corresponding negative, positive, and additional viable tissues (without MTT addition) were treated identically without piercing. For this procedure it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6-well plates.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate(s).
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.
Irritation parameter:
other: Mean Rel. Absorbance [% of Negative Control]
Value:
89.6
Negative controls validity:
valid
Remarks:
OD values 1.059 and 1.110
Positive controls validity:
valid
Remarks:
37.0% rel. absorbance
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not led to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour. Therefore, an additional test with freeze-killed tissues was not necessary.

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
The negative control OD values were determined to be 1.059 and 1.110, thus ranging within the limits of acceptance of > 0.8 and < 2.5.
The mean relative viability of the positive control was 37.0%, i.e. below 50% of the negative control viability.
The values for the difference of viability between the two relating tissues of a single item in the same run were determined to be between 3.3% to 4.9% for positive and negative control tissues and tissues of single test items. Hence, they were within the acceptance criteria of <20%.

Results after treatment for 6 hours with Zinc Peroxide and the controls

Dose Group

Ab-sorbance
Well 1
(Tissue 1/2)

Ab-sorbance
Well 2 (Tissue 1/2)

Mean Absor-bance* (Tissue 1/2)

Mean Absorbance* Tissue 1 and 2 minus Mean Blank

Mean Absorbance of
2 Tissues*

Rel. Absorbance [%]
Tissue 1 and 2**

Absolute Value of the Difference of the Rel. Absorbances [%]
Tissue 1 and 2

Mean Rel. Absorbance

[% of Negative Control]**

Blank

0.039

0.039

0.039

0.000

 

 

 

 

Negative Control

1.079

1.038

1.059

1.020

1.045

97.5

4.9

100.0

1.103

1.117

1.110

1.071

102.5

Positive Control

0.440

0.446

0.443

0.404

0.387

38.6

3.3

37.0

0.404

0.412

0.408

0.369

35.3

Test Item

0.993

1.004

0.998

0.960

0.936

91.8

4.4

89.6

0.957

0.948

0.952

0.913

87.4

*         Mean of two replicate wells after blank correction
**
       Relative absorbance [rounded values]:

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, Zinc Peroxide did not exhibit any eye irritating potential.
Executive summary:

This in vitro study was performed to assess the eye irritation potential of Zinc Peroxide in the Human Cornea Model Test. Additional tests with viable or freeze-killed tissues were not performed, since the test item was not coloured, did not dye water or isopropanol, and did not prove to be a MTT reducer.

About 50 mg of the test item and each 50 µL of the controls, respectively, were applied to either one of duplicate EpiOcular issue for 6 hours.

As compared to the negative control, treatment with the positive control induced a decrease in the mean relative absorbance to 37.0%. Thus, the validity of the test system is ensured.

The acceptance criteria were met.

Since the viability value of the tissues exposed to the test item did not decrease below 60%, the test item is not considered to possess an eye irritating potential.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

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 EpiDermTM 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 conrming 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 specic batch ofthe tissue models is conrmed.

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 dened 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.

 

An in vitro study was performed to assess the irritation potential of Zinc Peroxide by means of the Human Skin Model Test.

The test item did not reduce MTT (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.

Each three tissues of  the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes.

Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of the tissue.

30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue.

After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item Zinc Peroxide the mean relative absorbance value decreased irrelevantly to 97.1% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Zinc Peroxide is not irritant to skin.

 

Eye irritation

An in vitro study was performed to assess the eye irritation potential of Zinc Peroxide in the Human Cornea Model Test. Additional tests with viable or freeze-killed tissues were not performed, since the test item was not coloured, did not dye water or isopropanol, and did not prove to be a MTT reducer.

About 50 mg of the test item and each 50 µL of the controls, respectively, were applied to either one of duplicate EpiOcular issue for 6 hours.

As compared to the negative control, treatment with the positive control induced a decrease in the mean relative absorbance to 37.0%. Thus, the validity of the test system is ensured. The acceptance criteria were met.

Since the viability value of the tissues exposed to the test item did not decrease below 60%, the test item is not considered to possess an eye irritating potential.

Justification for classification or non-classification

Skin irritation

Based on reliable, adequate and relevant data, Zinc peroxide does not need to be classifiedfor skin irritation according to regulation (EC) 1272/2008.

 

Eye irritation

Based on reliable, adequate and relevant data, Zinc peroxide does not need to be classified for eye irritation according to regulation (EC) 1272/2008.