Dibutyl peroxydicarbonate

Endpoint summary

Description of key information

Relevant data are available to assess the skin and eye irritation potential of the target substance dibutyl peroxydicarbonate. In a primary dermal vitro irritation study conducted according to OECD guideline 439, the target substance showed no irritant effects. Contrasting results were obtained from a in vivo skin irritation study. As recommended in ECHA “Guidance on the Application of the CLP Criteria” the test design can only be used to apply the CLP criteria in case of negative results. By assessing the available data in a weight-of-evidence approach classification for skin irritation is not warranted based on the results of the OECD 439 guideline study.

In a primary eye irritation study conducted similar to OECD 405, 0.1 mL of the target substance was instilled into one eye of each of six New Zealand White Rabbits. Under the experimental conditions reported, the test substance showed only slight irritant effects in the eye of New Zealand White rabbits. Based on the results of the studies, the target substance can be considered non-irritant 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:

2017-03-17 to 2017-05-26

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)

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 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

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

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.

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”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 1976

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: FDA Fed. Reg. 28 (119), 5582, 1963

Deviations:

no

GLP compliance:

no

Specific details on test material used for the study:

- Name of the test material: Trigonox NBP-C 50 (di-n. butyl peroxydicarbonate)
- Appearance: liquid
- Purity: 50% solution in Shellsol T
- Storage condition of test material: stored at -20 ˚C until use

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Housing: Animals are caged individually and receive no hay or extraneous material that might enter the eye

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL of the test substance

Duration of treatment / exposure:

The test substance is applied to the uppler and lower eye lid, which are then carefully closed and subsequently held together for at least one second. The eyes are not washed following the application of the test material and the animals are released immediately.

Observation period (in vivo):

The eyes are examined at 24, 48, 72 h and 7 days after instillation of the test material.

Number of animals or in vitro replicates:

6 per test material

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The eyes are not washed following the application of the test material and the animals are released immediately

SCORING SYSTEM:
- The test is considered positive if four or more of the animals in the test group of six rabbits exhibit a positive reaction
- If one animal exhibits a positive reaction, the test is regarded as negative
- If two or three animals exhibit a positive reaction, the test is repeated, using a different group of six animals.
- The second test is considered positive if three or more of the animals exhibit a positive reaction.
- If only one or two animals in the second test exhibit a positive reaction, the test is again repeated with a different group of six animals.
- If a third test is needed, the substance will be regarded as an irritant if two or more animals exhibit a püositive response.

TOOL USED TO ASSESS SCORE: binocular

Irritation parameter:

iris score

Basis:

mean

Remarks:

of six animals

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.667

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

3

Reversibility:

not fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

not fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

not fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

6 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

For detailed results please refer to Table 1 in box "Any other information on results incl tables".

Other effects:

- Lesions and clinical observations: The eye lesions caused by the test item was of a slight degree.

Table 1: Individual scores awarded to the ocular lesions elicited by Trigonox NBP-C 50

Animal No.	Cornea	Iris	Conjunctivae
			Redness	Chemosis
After 24 hours
1	0	0	2	3
2	0	0	1	1
3	0	0	1	1
4	0	0	1	0
5	0	0	1	0
6	0	0	>1	1
After 48 hours
1	0	0	1	1
2	0	0	0	0
3	0	0	1	0
4	0	0	0	0
5	0	0	1	0
6	0	0	1	1
After 72 hours
1	0	0	1	0
2	0	0	0	0
3	0	0	1	1
4	0	0	0	0
5	0	0	1	0
6	0	0	1	1
After 7 days
1	0	0	1	0
2	0	0	0	0
3	0	0	0	0
4	0	0	0	0
5	0	0	0	0
6	0	0	1	0

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions reported, the test substance Trigonox NBP-C 50 showed only slight irritant effects in the eye of New Zealand White rabbits. Based on the results and in accordance with the CLP criteria, no classification is warranted.

Executive summary:

In a primary eye irritation study conducted similar to OECD test guideline 405, 0.1 mL of Trigonox NBP-C 50 was instilled into the upper and lower eye lid of one eye of each of six New Zealand White rabbits. Animals were observed for a total of 7 days. Scoring was conducted 24 h, 48 h, 72 h and 7 days after test substance administration. Eye lesions were scored by the method of Draize. Only slight irritant effects were observed, which were fully reversible within 7 days at latest. Only in two animals a conjunctivae redness score of 1 was still recorded after 7 days. Based on the results and in accordance with the CLP criteria, no classification is warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Relevant data are available to assess the skin and eye irritation potential of the target substance dibutyl peroxydicarbonate. In a primary dermal vitro irritation study conducted according to OECD guideline 439, the target substance showed no irritant effects. Contrasting results were obtained from a in vivo skin irritation study. As recommended in ECHA “Guidance on the Application of the CLP Criteria” the test design can only be used to apply the CLP criteria in case of negative results. By assessing the available data in a weight-of-evidence approach classification for skin irritation is not warranted based on the results of the OECD 439 guideline study.

In a primary eye irritation study conducted similar to OECD 405, 0.1 mL of the target substance was instilled into one eye of each of six New Zealand White Rabbits. Under the experimental conditions reported, the test substance showed only slight irritant effects in the eye of New Zealand White rabbits. Based on the results of the studies, the target substance can be considered non-irritant to the skin and eye. 

Justification for classification or non-classification

Based on the available data, no classification for skin and/or eye irritation is warranted for the target substance.