Dibutyl peroxydicarbonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-17 to 2017-05-26

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl peroxydicarbonate

EC Number:

240-344-0

EC Name:

Dibutyl peroxydicarbonate

Cas Number:

16215-49-9

Molecular formula:

C10H18O6

IUPAC Name:

1-({[(butoxycarbonyl)peroxy]carbonyl}oxy)butane

Test material form:

liquid

Details on test material:

- Test material form: liquid
- Batch No.: 1230518-02
- Colour: clear
- Density: 0.88 g/cm³
- Active Components: 50.6% Peroxide
- Purity: not specified
- Expiry Date: not provided by the sponsor
- Storage Conditions: < -20 °C

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM™ (SkinEthic)
- Tissue batch number(s): 17-EKIN-014

SkinEthic Kit:
- EPISKIN-SM™ plate containing 12 reconstructed epidermis units (area: 0.38 cm²); each reconstructed epidermis is attached to the base of a tissue culture insert with an O-ring set and maintained on nutritive agar for transport (Lot No.: 17-EKIN-014):
1x 12-well assay plate
1x flask of sterile maintenance medium (basic medium for incubations, Lot No.: 17-MAIN3-013)
1x flask of sterile assay medium (basic medium for use in MTT assays, Lot No.: 17-ESSC-013)
- Validity controls as provided by the supplier (SkinEthic):

Morphology:
- Histology scoring (HES stained vertical paraffin sections, n=6):
Specification ≥ 19.5
Result: 22.3 ± 1.0, CV = 4.7 %
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.

Barrier function:
- IC50 determination (SDS concentration, MTT test, n=14):
Specification 1.5 mg/mL < IC50 < 3 mg/mL
Result: 2.2 mg/mL


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C, 5.0 % CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- washed with DPBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per dose group

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is greater than 50%.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL (undiluted)

VEHICLE
- Amount(s) applied (volume or weight with unit): 10 µL DPBS
- Lot/batch no. (if required): Lot No.: 1838067

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL SDS solution
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 min ± 0.5 miin

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3 tissues per dose group

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

69.1

Vehicle 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 1

Any other information on results incl. tables

Results of the  Pre-Experiments

The mixture of 10 μL test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixtures of 10 μL of the test item per 90 μL aqua dest. and per 90 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSCliving equalled 0%.

Results of the main experiment

Table 1: Results of the test item

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	0.573	0.598	0.598	0.095	0.070	0.126	0.408	0.492	0.396
	0.598	0.660	0.632	0.096	0.072	0.138	0.422	0.510	0.380
OD570 (Blank corrected	0.529	0.554	0.554	0.051	0.026	0.082	0.365	0.448	0.352
	0.554	0.616	0.589	0.053	0.028	0.094	0.379	0.467	0.336
Mean OD570 of the Duplicates (Blank corrected)	0.542	0.585	0.571	0.052	0.027	0.088	0.372	0.457	0.344
Total Mean OD570 of 3 Replicate Tissues (Blank corrected)	0.566*			0.056			0.391
SD OD570	0.022			0.031			0.059
Relative Tissue Viabilities [%]	95.7	103.4	100.9	9.2	4.8	15.5	65.7	80.8	60.8
Mean Relative Tissue Viabilities [%]	100.0			9.8**			69.1
SD Tissue Viabilities [%]	3.9			5.4			10.4
CV [% Viability]	3.9			55.0			15.1

* Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is ≤ 40%.

Table 2: Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean OD570 nmBlank	0.044	< 0.1	pass
Mean Absolute OD570 nmNK	0.610	0.6 ≤ NK ≤1.5	pass
Mean Relative Viability [%] PC	9.8	≤ 40%	pass
Max. SD of % Viability [%]	10.4	≤ 18%	pass

 

Table 3: Historical Data

	OD570± 30 nmblank	Absolute OD570± 30 nm NK	Relative Viability [%] PC	SD of Viability [%]
Mean	0.043	0.839	11.3	7.8
SD	0.002	0.128	7.8	14.1
n	81	80	81	324

Historical control data were generated from 2008 – 2016.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

In the present study the skin irritant potential of Dibutyl peroxydicarbonate was analysed according to OECD 439 using the EPISKIN-Standard Model™ (EPISKIN-SM^TM), a reconstituted three-dimensional human epidermis model to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

In this study under the given conditions the test item showed no irritant effects (69.1 % mean relative tissue viability). As the relative mean tissue viability after 15 min of exposure and 42 h post-incubation was > 50%, the test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.