[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-29 to 2018-03-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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:

adopted 28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Committee for Proprietary Medicinal Products (CPMP) Note for Guidance on non-clinical local tolerance testing of medicinal products (CPMP/SWP/2145/00)

Version / remarks:

adopted 1 March 2001

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

EC Number:

814-835-0

Cas Number:

1310672-91-3

Molecular formula:

C18H16O7

IUPAC Name:

[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Batch No.: A-C012967 (Galderma Number) / 854E3F0002 (supplier lot number)
- Purity (HPLC): 99.1%
- Appearance: White Powder
- Expiry date/ reanalysis date: February 28, 2020 / June 20, 2018
- Storage conditions / packaging: Between +2 and +8°C / away from light

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The RhCE tissues are reconstructed from primary human cells, which have been cultured for several days to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in the human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

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

Duration of treatment / exposure:

6 hours ± 15 minutes.

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

Post-incubation post-soak plate: 25 ± 2 min at 37 ± 1 °C
Post-incubation post-treatment plate: 18 hours ± 15 minutes at 37 °C, 5% CO2, saturated hygrometry

Number of animals or in vitro replicates:

2 tissues per group

Details on study design:

Pre-incubation
On the day of receipt, the tissues were equilibrated (in its 24-well shipping container) to room temperature for about 15 minutes. An appropriate volume of EpiOcular™ Assay Medium was warmed to approximately 37 °C. 1 mL of Assay Medium was aliquoted into the appropriate wells of pre-labeled 6-well plates. Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and preincubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at 37 °C, 5% CO2, saturated hygrometry overnight (16 – 24 hours).
Application/Treatment of SOLID test items, control positive and control negative
Pre-Treatment
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+free-DPBS. If the Ca2+Mg2+free-DPBS does not spread across the tissues, the plate may be tapped to assure that the entire tissue surface is wetted. The tissues were incubated at 37 °C, 5% CO2, saturated hygrometry for 30 ± 2 minutes.
After the 30 ± 2 minutes Ca2+Mg2+free-DPBS pre-treatment, each solid test and control item was tested by applying 50 mg topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 6 hours ± 15 minutes. The insert was shaken gently from side to side to ensure that tissue was completely covered by the test item. The test was performed on 2 tissues for the test item, 2 tissues for the negative control and 2 tissues for the positive control. One plate was used per test item, one plate for the control positive and one plate for the control negative to prevent any adjacent effects.
At the end of the 6 hours ± 15 minutes treatment time, the test articles were rinsed. Three clean plastic beakers containing a minimum of 100 ml each of Ca2+Mg2+free DPBS were used per test item. The tissues were rinsed two at a time by holding replicate inserts together by their collar using forceps. The tissues was dipped into the first beaker, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out. This process was performed two additional times in the first beaker, then in the same way in the second and the third beaker.

Post-Soak
After rinsing, the tissues were immediately transferred to and immersed in 5 ml of previously warmed EpiOcular™ Assay Medium (37 °C ± 1 °C) in a pre-labeled 12-well plate for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. This incubation in EpiOcular™Assay Medium was intended to remove any test article absorbed into the tissue.

Post-incubation
At the end of the Post-Soak immersion, each insert was removed from the EpiOcular™ 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-labeled 6-well plate containing 1 ml of warm EpiOcular™ Assay Medium. The tissues were incubated for 18 hours ± 15 minutes at 37 °C, 5% CO2, saturated hygrometry (Post-treatment Incubation).

METHODS FOR MEASURED ENDPOINTS:
Cell viability Measurement
Incubation in MTT solution
After incubation, cell culture inserts were dried on sterile absorbent paper or gauze to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 300 μL MTT ready-to-use solution. The tissues were incubated for 3 hours ± 10 minutes at 37 °C, 5% CO2, saturated hygrometry.
Formazan extraction
After incubation the tissues were dried on sterile absorbent paper or gauze. The tissues were transferred in a 6-well plate prefilled with 2 mL of isopropanol. Plates were covered by stretching 3 parafilm layers over the plate to avoid any evaporation and the lid of the plate was added. Plates were then protected from direct light by wrapping in aluminum paper. Two to three hours incubation were performed at room temperature under gentle agitation (about 150 rpm).

Optical Density Measurement
The extraction solution was homogenized by pipetting up and down. Then, 2 x 200 μL of extraction solution was transferred (2 wells per tissue i.e. 2 replicates per tissue) into a 96 well plate. Isopropanol was used as blank (8 replicates). Optical densities (OD) was measured at 570 ± 30 nm using Synergy2 microplate reader (Biotek) and validated Gen5 Secure software (Biotek).

Acceptability of assay
The in vitro eye irritation test was considered acceptable if it meets the following criteria:
- The OD value for the negative control should be > 0.8 with an upper acceptance limit of 2.5
The mean tissue viability of the positive control should be:
- 6 hours exposure ≤ 50% relative to the mean tissue viability of the negative control (which is set at 100%)
- The difference of viability between the two relating tissues of a single chemical is < 20% in the same run (for positive and negative control tissues and tissues of single chemicals).
- The absolute mean OD of the two tissues of the negative control and the positive control should be within the laboratory historical control data range.

Data Evaluation
A test item was considered irritant in the in vitro eye irritation test if:
-The relative mean tissue viability of three individual tissues after exposure to the test item is ≤ 60% ± 5% of the mean viability of the negative controls.
A test item was considered non-irritant in the in vitro eye irritation test if:
-The relative mean tissue viability of three individual tissues after exposure to the test item is > 60% ± 5% of the mean viability of the negative controls.

Results and discussion

In vitro

Results

Irritation parameter:

other: Relative mean tissue viability

Run / experiment:

Mean of two replicates

Value:

126.8

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:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

For individual results see Table 1 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Preliminary assessment

CD08467 (test substance) were checked for possible direct MTT reduction by adding the test substances to MTT medium. Because no colour change was observed, it was concluded that CD08467 (test substance) did not interact with MTT. CD08467 (test substance) was checked to identify potential interference by test item absorbing light in the same range as MTT formazan (naturally or after treatment) by adding the test substance to water and ispopropanol. Optical densities measured after mixing CD08467 with water or isproponal were lower than 0.08, showing no interference.

Table 1: Mean absorption in the in vitro eye irritation test with test item, and positive and negative controls

Blank			Difference of viability
Negative control	Mean OD	1.187	0.0%
	Mean Viability	100%
Positive Control	Mean OD	0.431	5.2%
	Mean Viability	36.3%
CD08467	Mean OD	1.505	34.4%
	Mean Viability	126.8%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item CD08467 showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category” for eye irritation.

Executive summary:

In the present study the eye irritation potential of CD08467 (99.1% purity) was analysed according to OECD 492 using the three-dimensional RhCE tissue, consisting of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Hereby, 50 mg of the test item was applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 6-hour exposure and 18-hour post-incubation period and compared to those of the concurrent negative controls. CD08467 (test substance) was checked for possible direct MTT reduction by adding the test substances to MTT medium. Since no colour change was observed, it was concluded that the test substance did not interact with MTT. In addition, the test substance was checked to identify potential interference by absorbing light in the same range as MTT formazan (naturally or after treatment). The test substance was added to water and isopropanol and the measured optical densities were lower than 0.08, showing no interference.

The mean cell viability of positive control was less than 50% and the difference in viability between the two tissues was less than 20 %, indicating that the test system functioned properly. The relative mean tissue viability (126.8%) obtained after 6 hours of treatment with the test substance compared to the negative control tissues (100%) showed no irritation. In conclusion, under the experimental conditions of this in vitro eye irritation test, the test item is considered to be non-irritating to the eye in accordance with UN GHS “No Category”.