[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: CPMP/SWP/2145/00

Version / remarks:

01 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

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%

In vitro test system

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

Results and discussion

In vitro

Results

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.

Any other information on results incl. tables

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.

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 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.