[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

Endpoint summary

Description of key information

The potential of CD08467 to induce skin irritation (OECD 439) and eye irritation (OECD 492) was tested in suitable in vitro test methods. Based on the results, the substance can be considered non-irritating to the skin and eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-31 to 2017-07-19

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 28th July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Union No.L220, 24 August 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: CPMP/SWP/2145/00

Version / remarks:

01 March 2001

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Batch no. I
- Appearance: white powder
- Storage conditions: Between +2 and +8 °C/ away from light
- Purity (HPLC): 99.4%

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The SKINETHIC(TM) RHE model consists of normal human keratinocytes cultured for 17 days on an inert 0.5 cm2 polycarbonate filter at the air-liquid interface. On Day 17, a highly differentiated and stratified epidermis model is obtained comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. The RHE model presents a histology comparable to the in vivo human tissue.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SKINETHIC(TM) RHE model
- Tissue batch number: 7-RHE-015

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, tissues were thoroughly rinsed with sterile phosphate buffered saline by distributing 1 mL 25 times with a multi-pipette to remove residual test items. If the nylon mesh was not removed after the rinsing step, it was removed with fine forceps.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 hours
- Spectrophotometer: Synergy2 microplate (Biotek) with validated Gen5 Secure software (Biotek)
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3 tissues for each, test item, negative control and positive control

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 42 minutes of exposure to the test items and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 42 minutes of exposure to the test items and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg ± 2 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): not specified

POSITIVE CONTROL
- Concentration (if solution): 5%

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 tissues each of test item, negative control and positive control

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

95.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

For detailed results see "Any other information on results" Table 3.

Preliminary assessment:

Direct MTT reduction by adding the test substance with MTT solution: No colour change was observed. It is concluded that CD08467 did not interact with MTT.

Potential interference by test item absorbing in the same range as MTT formazan by adding the test item to water or isopropanol: No interference with MTT formazan was observed (optical density was < 0.08)

Results of the main experiment:

Table 3: Absorption values in the in vitro skin irritation test with CD08467, positive and negative control.

Blank	Mean + SD	0.038	0.001
Negative control	Tissues RHE	Negative control RHE 1			Negative control RHE 2			Negative control RHE 3
	OD corrected	1.382	1.364	1.411	1.571	1.558	1.557	1.570	1.541	1.539
	OD mean/tissue RHE+SD	1.386	0.024		1.562	0.008		1.550	0.017
	OD mean+SD	1.499	0.099
	Viability	100%			100%			100%
	Mean viability+SD	100%	0%
Positive control	Tissues RHE	Positive control RHE 1			Positive control RHE 2			Positive control RHE 3
	OD corrected	0.018	0.019	0.018	0.017	0.017	0.018	0.018	0.018	0.017
	OD mean/tissue RHE+SD	0.019	0.001		0.018	0.001		0.018	0.001
	OD mean+SD	0.018	0.001
	Viability + SD	1.2%			1.2%			1.2%
	Mean viability+SD	1.2%	0.0%
CD08467 (test item)	Tissues RHE	Test item RHE 1			Test item RHE 2			Test item RHE 3
	OD corrected	1.284	1.314	1.326	1.468	1.485	1.510	1.478	1.494	1.514
	OD mean/tissue RHE+SD	1.308	0.022		1.488	0.021		1.496	0.018
	OD mean+SD	1431	0.106
	Viability + SD	87.2%			99.2%			99.7%
	Mean viability+SD	95.4%	7.1%

OD = optical density, SD = standard deviation; Values were corrected for background absorption (0.038). Isopropanol was used to measure the background absorption.

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 therefore classified as “non-irritant” in accordance with UN GHS “No Category” for skin irritation.

Executive summary:

In the present study the skin irritant potential of the test item CD08467 (purity 99.4%) was analysed according to OECD 439 using the SKINETHIC™ RHE model, consisting of normal human keratinocytes to distinguish between UN GHS skin irritating test substances ("Category 1 or 2") and not categorised test substances (“No Category”) which may be considered as non-irritant.

The test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 42-min exposure and 42-h post-incubation period and compared to those of the concurrent negative and positive controls.

In this study under the given conditions the test item showed no irritant effects (95.4 % mean relative tissue viability). As the relative mean tissue viability after 42-min of exposure and 42-h post-incubation was > 50%, the test item CD08467 is therefore classified as “non-irritant” in accordance with UN GHS “No Category” for skin irritation.

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

Study period:

2017-06-29 to 2018-03-27

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:

adopted 28 July 2015

Deviations:

no

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

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

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.

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.

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".

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%

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”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential of CD08467 to induce skin irritation (OECD 439) and eye irritation (OECD 492) was tested in suitable in vitro test methods. Based on the results, the substance can be considered non-irritating to the skin and eye.

Justification for classification or non-classification

Based on available data, no classification for skin and/or eye irritation is warranted based on the results from suitable in vitro tests (OECD 439 and 492).