Docosyl stearate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 Aug - 9 Oct 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

non-GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

2010

Deviations:

yes

Remarks:

non-GLP

GLP compliance:

no

Remarks:

The study was conducted to meet the requirement of a regulation (Japan) other than Regulation (EC) No. 1907/2006 and therefore not conducted according to GLP.

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

Docosyl stearate

EC Number:

244-971-0

EC Name:

Docosyl stearate

Cas Number:

22413-03-2

Molecular formula:

C40H80O2

IUPAC Name:

docosyl octadecanoate

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

- Source: Charles River Laboratories Japan, INC., Kanagawa, Japan
- Age at study initiation: 8 weeks (seven days before test day 1)
- Weight at study initiation: 19.7 - 23.3 g (one day before test day 1)
- Housing: 4 animals in a polycarbonate cage (225W, 338D, 140H mm) supplemented with ALPHA-dri (Shepherd Specialty Papers) and CARE FEEAZ (Hamri) as bedding material
- Diet: Radiation-sterilized solid feed CRF-1 (Oriental Yeast), ad libitum
- Water: UV-sterilized tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 50 ± 10
- Ventilation: 15 times/hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

acetone / olive oil (4:1, v/v) used for negative and positive reference items, dimethylformamide used for test item

Concentration:

1, 5, 10, 20 and 40% (Pre-Screen Test)
1, 10 and 40% (Main study)

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS
A preliminary experiment was carried out in 5 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Five concentrations of 1, 5, 10, 20 and 40% dissolved in DMF were examined. Only 1 animal per concentration was used. Systemic toxicity and irritation were not observed. Therefore, the application concentrations of 1, 10 and 40% were chosen.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay: BrdU-ELISA (Alternative endpoints of the murine local lymph node (measurement of BrdU content to indicate an increased number of proliferating cells in the draining auricular lymph nodes).
- Proliferation is measured by comparing the mean proliferation in each test group to the mean proliferation in the vehicle treated control group (VC). The ratio of the mean proliferation in each treated group to that in the concurrent VC group, termed the SI, is determined, and should be ≥ 1.6 before further evaluation of the test substance as a potential skin sensitizer is warranted.

TREATMENT PREPARATION AND ADMINISTRATION
The test item was diluted in dimethylformamide (DMF). The vehicle acetone / olive oil (4:1, v/v) was used as negative reference item. The test item solution was administered to the dorsum of both animals´ ears at an application volume of 25 μL/ear. Three groups of 4 female animals each were examined. The concentrations (1, 10 and 40%) were chosen based on a preliminary experiment. In addition, three further groups of 4 female animals treated with a 25% concentration of positive control, negative control (acetone / olive oil (4:1, v/v)) and vehicle (DMF) were examined.

Sensitization and administration
25 μL of administration solution was applied to both ear auricles of an animal with a micropipette for 3 days.
After 48 hours of final sensitization, BrdU preparation solution was intraperitoneally administered at 0.5 mL / animal.

Observation
Each animal was observed at least once daily from day 1 to day 6 when the auricular lymph nodes were collected. Body weight was measured on the day of animal arrival, of grouping, day 1 and day 6.

Excision and measurement of the auricular lymph node
Animals were euthanized by excessive anesthesia by ethyl ether on day 6. The auricular lymph nodes were excised and two of them were weighed together. The lymph nodes were stored at -80 °C. When the auricular lymph nodes were excised, the periphery of the auricle was observed. Any abnormality that was observed, was recorded.

Measurement of BrdU content
After the auricular lymph nodes were thawed at room temperature, 4 zirconia beads (2 mm in diameter) and 200 μL of phosphate buffered saline were added and homogenized. This cell suspension was filtered and phosphate buffered saline was added by the final volume of 25 mL. 100 μL of this prepared solution was placed in 3 wells of a 96-well microplate. Similarly 100 μL of phosphate buffered saline was placed in 3 wells as a blank and the 96-well microplate centrifuged.
After removing the supernatant, it was dried with a dryer, and BrdU was measured using Cell Proliferation ELISA, BrdU Measurement Kid (Cell Proliferation ELISA, BrdU colorimetric, Roche Diagnostics K.K.).
Absorbance at a measurement wavelength of 370 nm and a reference wavelength of 492 nm was measured with a microplate reader at 2.5-minute intervals for 15 minutes after addition of the chromogenic substrate solution. The data at the time when the measured value of BrdU of the negative control group was between 0.001 and 0.200 was adopted. With respect to the absorbance (OD:370 nm – OD: 492 nm, measured value of BrdU) of each individual, the mean value of 3 wells was taken as the measurement value of each individual.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The decision process regards a result as positive when SI ≥ 1.6. However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result (SI value between 1.6 and 1.9) is declared positive.

Results and discussion

Positive control results:

The positive control was dissolved in acetone / olive oil (4:1, v/v). Positive controls were used to demonstrate appropriate performance of the assay and competence of the laboratory to successfully conduct the assay. An index for the lymph node cell count above 1.6 is considered positive. SI of the positive control is 2.9 ± 0.3 (p ≤0.01). Therefore, the study can be regarded as valid.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 0.9

Test group / Remarks:

40% test item

Results 2

Key result

Parameter:

SI

Value:

ca. 0.9

Test group / Remarks:

10% test item

Results 3

Key result

Parameter:

SI

Value:

ca. 0.9

Test group / Remarks:

1% test item

Results 4

Key result

Parameter:

SI

Value:

ca. 1

Test group / Remarks:

DMF

Results 5

Key result

Parameter:

SI

Value:

ca. 2.9

Test group / Remarks:

Positive control

Results 6

Key result

Parameter:

SI

Value:

ca. 1

Test group / Remarks:

Negative control

Any other information on results incl. tables

Table1: Summary of result

NO.	Item	Observation	Weight (g)		auricular lymph node	BrdU			SI
			Day 1	Day 6	(mg)	Ave.	±	SD
101	NC (AOO)	-	20	20.3	4.2	0.153	±	0.016	1.2
102	NC (AOO)	-	23.2	21.9	4.5	0.142	±	0.007	1.1
103	NC (AOO)	-	24	23	4.2	0.103	±	0.008	0.8
104	NC (AOO)	-	23.6	22.8	4.1	0.103	±	0.003	0.8
201	PC (25%, HCA)	crusta of both ears (Day6)	19.5	18.9	9	0.325	±	0.021	2.6
202	PC (25%, HCA)	crusta of both ears (Day6)	20.9	20.3	9	0.361	±	0.006	2.9
203	PC(25%, HCA)	desquamation of right ear (Day6)	22.8	23.5	10.5	0.351	±	0.041	2.8
204	PC (25%, HCA)	desquamation of both ears (Day6)	21.6	20.8	10.3	0.403	±	0.025	3.2
301	vehicle (DMF)	-	19.6	20.5	5	0.172	±	0.014	1.1
302	vehicle (DMF)	-	20.8	22.9	4.5	0.164	±	0.005	1.0
303	vehicle (DMF)	-	21.3	22.6	4.3	0.146	±	0.007	0.9
304	vehicle (DMF)	-	22.3	24.9	4.0	0.148	±	0.003	0.9
401	1% test item	-	19.7	19.3	4.2	0.145	±	0.005	0.9
402	1% test item	-	21	21.6	4.9	0.148	±	0.01	0.9
403	1% test item	-	21.6	22	4.5	0.127	±	0.003	0.8
404	1% test item	-	22.4	23.4	4.9	0.156	±	0.001	1.0
501	10% test item	-	18.8	19.2	4.2	0.147	±	0.002	0.9
502	10% test item	-	20.6	21.1	4.6	0.176	±	0.003	1.1
503	10% test item	-	23.5	23	4.5	0.133	±	0.016	0.8
504	10% test item	-	23.3	23.4	4.8	0.128	±	0.006	0.8
601	40% test item	-	19.9	20.4	4.9	0.131	±	0.014	0.8
602	40% test item	-	20.5	19.1	5.1	0.162	±	0.006	1.0
603	40% test item	-	21.2	20.5	4.8	0.159	±	0.01	1.0
604	40% test item	-	22.9	22.8	4.4	0.12	±	0.007	0.8

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008