Docosyl stearate

Endpoint summary

Description of key information

In vivo (LLNA): not sensitising

In vivo (LLNA): not sensitising (read-across from CAS 3687-46-5, WoE)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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

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

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.

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.

Key result

Parameter:

SI

Value:

ca. 0.9

Test group / Remarks:

40% test item

Key result

Parameter:

SI

Value:

ca. 0.9

Test group / Remarks:

10% test item

Key result

Parameter:

SI

Value:

ca. 0.9

Test group / Remarks:

1% test item

Key result

Parameter:

SI

Value:

ca. 1

Test group / Remarks:

DMF

Key result

Parameter:

SI

Value:

ca. 2.9

Test group / Remarks:

Positive control

Key result

Parameter:

SI

Value:

ca. 1

Test group / Remarks:

Negative control

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

Interpretation of results:

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

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

weight of evidence

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Control

Remarks on result:

other:

Remarks:

Source: CAS 3687-46-5

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

25%

Remarks on result:

other:

Remarks:

Source: CAS 3687-46-5

Key result

Parameter:

SI

Value:

2

Test group / Remarks:

50%

Remarks on result:

other:

Remarks:

Source: CAS 3687-46-5

Key result

Parameter:

SI

Value:

2.1

Test group / Remarks:

100%

Remarks on result:

other:

Remarks:

Source: CAS 3687-46-5

Interpretation of results:

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

Conclusions:

The available LLNA test on the suitable source substance showed no skin sensitisation effect. Therefore, the target substance docosyl stearate (CAS 22413-03-2) is not expected to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

CAS 22413-03-2

An in vivo skin sensitisation study (Local Lymph Node Assay, LLNA) was performed with docosyl stearate (CAS 22413-03-2) according to OECD 442B (Fujifilm Safety Evaluation, 2015). A preliminary experiment was carried out in 5 female CBA:J mice 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, 4 animals per dose level were used. The test substance formulations, the vehicle (DMF), negative control (acetone / olive oil (4:1, v/v)) or the positive control (25% α-hexyl cinnamic aldehyde in acetone / olive oil (4:1, v/v)) were applied epicutaneously onto the dorsal part of each ear (25 µL/ear) for three consecutive days. All mice were sacrificed three days after the last treatment (on Day 6) and the weight of the lymph nodes was determined. The auricular lymph node, 4 zirconia beads (2 mm in diameter) and 200 μL of phosphate buffered saline were added and homogenized. This cell suspension was filtered and 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 with a measurement wavelength of 370 nm and a reference. Based on the study results, the test substance was not skin sensitising.

 

CAS 3687-46-5

An in vivo skin sensitisation study (Local Lymph Node Assay, LLNA) was performed with decyl oleate (CAS 3687-46-5) according to OECD 429 and under GLP conditions (Notox, 2010b). Five female CBA/J mice per dose were treated with 0, 25, 50 and 100% of the test substance in acetone/olive oil. The test substance formulations or the vehicle were applied epicutaneously onto the dorsal part of each ear (25 µL/ear) for three consecutive days. All mice were sacrificed three days after the last treatment (on Day 6) and the weight of the lymph nodes was determined. The cell proliferation of pooled lymph nodes from individual animals was measured as 3H-methyl thymidine incorporation and determined by beta-scintillation counting. The stimulation indices were 1.0, 1.2, 2.0 and 2.1 for the control, 25, 50 and 100% groups, respectively. The SI slightly increased with the dose level, but the increase was not statistically significant. Positive and vehicle controls were valid. An EC3 value of the test substance could not be calculated, as all stimulation indices were <3. Based on the study results, the test substance was not skin sensitising.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation do not meet the classification criteria according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.