Dibutyl peroxydicarbonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-09-29 to 2017-07-17

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

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

Specific details on test material used for the study:

- Test material form: liquid
- Batch No.: 1228974
- Density: 0.88 g/cm³
- Purity: 50.5% peroxide content (range 49-51%), rest phlegmatizer
- Expiry Date: not provided by the sponsor
- Storage Conditions: < -20 °C

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF on delivery
- Age at study initiation: 8 -9 weeks
- Housing: The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 2017-02-01 To: 2017-03-02

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

12.5%, 25% and 50% (v/v)

No. of animals per dose:

- 5 mice/group
- 5 mice/ pre screen test

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Before the initiation of the pre-screen test, a solubility test was performed to define the vehicle and the maximum concentration, which is technically applicable to the animals. The maximum technically applicable concentration of the test item was found to be 100%. The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in AOO (Acetone, Sigma-Aldrich, lot no. STBG2167V, expiry date: 17/08/2021 and lot no. SZBF3480V, expiry date: 12/2021; olive oil highly refined, Sigma Aldrich, lot no. BLBQ4885V, expiry date: 07/03/2017).

- Irritation:
In order to determine the highest tolerated and not excessively irritant test concentration a pre-screen test was performed, which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation. The mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination. Both ears were observed for erythema and scored according to Table 1 (see "Any other information on materials and method"). Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%. Animals no. 1 and no. 2 were treated with a test item concentration of 100% (undiluted). Animals no. 3 and no. 4 were treated with a test item concentration of 50% (diluted with AOO) and one further animal was treated with 100% AOO and served as negative control.

- Systemic toxicity:
During this period also all clinical signs were recorded. Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).

- Ear thickness measurements:
Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured using a Vernier digital caliper.

- Erythema scores:
See Table 1 in "Any other information on results". For the results of the pre-screen tests please refer to the box "Any other information on results".

MAIN STUDY
- Controls
AOO was used as vehicle and served as negative control. Due to animal welfare the negative control was shared. Positive controls (1% phenylenediamine dissolved in AOO) are performed periodically. The recent results are included in the result section.

- Other Materials
3H-methyl thymidine (TRK 300, 20 Ci/mmol; PerkinElmer, lot no. 201610E), diluted to a working concentration of 80 µCi/mL. Phosphate buffered saline (PBS), BSL Munich, lot no. 26.10.16, expiry date: 26/10/2017. Trichloroacetic acid (TCA), Sigma-Aldrich, lot no. BLBN7603V, expiry date: 27/08/2017.

- Preparation of the Test Item
The preparations were made immediately prior to each dosing.

- Preparation of the Animals
The animals were randomly selected using the validated departmental computerised system E WorkBook (version 9.4.0, ID Business Solutions Ltd.). Identification was ensured by cage number and individual marking (tail).

- Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

- Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

- Test Regime
-> Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.
-> Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.
-> Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4 °C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
-> Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
- Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted. EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3= c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a, b) and one below (c, d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated. A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).
On the basis of the test results, the test substance may be classified into one of the following categories in conformity with the criteria given in Commission Regulation (EU) No 286/2011 as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, sixth revised edition, 2015:
Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser
a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or
b) if there are positive results from an appropriate animal test.
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value ≤ 2%
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value > 2%
WARNING, exclamation mark. May cause an allergic skin reaction.

Positive control substance(s):

other: 1% phenylenediamine in AOO

Results and discussion

Positive control results:

The positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system (see Table 2 in box "Any other information on results").

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

mean of five animals

Value:

16.3

Variability:

SD = 4.3

Test group / Remarks:

12.5 % (v/v)

Results 2

Key result

Parameter:

SI

Remarks:

mean of five animals

Value:

21.2

Variability:

SD = 3.6

Test group / Remarks:

25 % (v/v)

Results 3

Key result

Parameter:

SI

Remarks:

mean of five animals

Value:

19.2

Variability:

SD = 2.7

Test group / Remarks:

50 % (v/v)

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Please see Table 3 in box "Any other information on results"

EC3 CALCULATION
The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were above 3.

CLINICAL OBSERVATIONS:
Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal. The animals treated with the test item concentrations showed local findings and effects which are not considered to be signs of excessive irritation.

BODY WEIGHTS
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Any other information on results incl. tables

Results of the pre-screen tests

Solubility tests:

The maximum technically applicable concentration of the test item was found to be 100%. The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in AOO.

Irritation and toxicity test:

One of the animals treated with the undiluted test item showed slightly reduced spontaneous activity on day 6 which is considered a sign of systemic toxicity. One of the animals treated with the undiluted test item showed signs of excessive irritation (increased ear thickness > 25%) on day 6 at the right application site, the other animal at the left application site. All other clinical findings were considered not to be signs of systemic toxicity or signs of excessive irritation. All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the pre-screen test.

Results of the main study

Table 2: Stimulation Indices obtained for the Positive-Control Group

Test Item	Concentration [%]	Animal Number	Stimulation Index
Negative Control AOO	100	101
		102
		103
		104
		105
		MV	1.0
		SD
Positive Control Phenylenediamine	1	106	7.8
		107	4.7
		108	3.5
		109	5.7
		110	8.7
		MV	6.1
		SD	1.9

SD = standard deviation; MV = mean value

Table 3: Stimulation Indices obtained in the main experiment

Test Item	Concentration [%]	Animal Number	Stimulation Index
Negative Control AOO	100	101
		102
		103
		104
		105
		MV	1.0
		SD
Dibutylperoxicarbonate in AOO	12.5	1	9.9
		2	17.3
		3	17.4
		4	23.0
		5	14.1
		MV	16.3
		SD	4.3
Dibutylperoxicarbonate in AOO	25	6	16.8
		7	22.7
		8	26.6
		9	22.2
		10	17.6
		MV	21.2
		SD	3.6
Dibutylperoxicarbonate in AOO	50	11	21.8
		12	15.1
		13	18.1
		14	18.6
		15	22.5
		MV	19.2
		SD	2.7

SD = standard deviation; MV = mean value

If not noted individually, the results include both lymph nodes of an animal.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In conclusion, in a mouse local lymph node assay, the test item is considered to be a skin sensitizer.

Executive summary:

In a dermal sensitization study conducted according to OECD 429 with Dibutyl peroxydicarbonate (50.5 % peroxide content) dissolved in AOO (4:1 (v/v) acetone/olive oil), young adult female CBA/CaOlaHsd mice (5 per dose group) were tested at concentrations of 12.5% (v/v), 25 % (v/v) and 50 % (v/v) in a local lymph node assay (LLNA). Due to animal welfare reasons the negative control was shared and a periodically performed positive control (1% Phenlyenediamine in AOO) was used. Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal. The animals treated with the test item concentrations showed local findings and effects which are not considered to be signs of excessive irritation. No mortality was observed in any of the animals. Each of the three tested concentrations exceeded the stimulation index of 3 (16.3 (12.5%), 21.2 (25%), 19.2 (50%)). Therefore an EC3 value could not be calculated. In this study, Dibutyl peroxydicarbonate is a dermal sensitizer.