Reaction mass of zinc oxide and zinc peroxide

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

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)

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

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

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%.

Any other information on results incl. tables

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]:

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 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.