Octyl laurate

Endpoint summary

Description of key information

Skin sensitation (OECD 442B): sensitizing

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Sep - 02 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes

Type of study:

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

Species:

mouse

Strain:

CBA:JN

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 -10 weeks
- Weight at study initiation: 17 - 20 g (at arrival)
- Housing: group-caged (up to 5) during acclimatisation; individual during the study
- Diet: 4 RF 21 (Mucedola S.r.l., Settimo Milanese (MI) Italy), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): approx. 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Remarks:

(AOO)

Concentration:

100, 50 and 25%

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
In the pre-screen test, 25 μL of five different concentrations of the test item (5, 10, 25, 50 and 100% w/w dissolved in AOO) were applied to the dorsum of both ears of all animals, once a day for 3 consecutive days. All animals were observed twice daily for mortality and morbitiy and for clinical signs on Day 1 before and 1 h after dosing on Day 2 to 6 daily (approx. 1 h after daily dosing). Body weights were recorded prior to dosing (Day 1) and on the day of necropsy (Day 6). Furthermore, erythema measurements were performed daily (once before first dosing, before dosing on Days 2 and 3 and daily thereafter), ear thickness was measured on Day 1 (pre-dose), Day 3 (before dosing) and Day 6. After sacrifice, regularly shaped biopsies were obtained from both ears and weighed together. No necropsy was performed on the animals.

- Compound solubility: At a concentration of 50% w/w in acetone/olive oil 4:1 v/v, a good solution was obtained
- Irritation: No irritation was observed
- Systemic toxicity: No signs of toxicity were observed.
- Ear thickness measurements: No relevant increase was induced by treatment (values of Day 6 compared to Day 1). The evaluation of ear punch weight indicated a very slight increase in the animals treated at 100% concentration (26%), when compared to the animals treated with the vehicle. However, this increase was not considered significant, since it was very slight and no other parameter to evaluate irritation was altered.
- Erythema scores: No erythema was observed.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA: BrdU-ELISA method
- Criteria used to consider a positive response: The test item is considered to induce sensitisation when the Stimulation Index (SI) for any single treatment dose group is ≥ 1.6.
It is not required that an increased response is observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION: In the main assay, the test item was used at the higher concentrations that were systemically tolerated and judged not to cause excessive local skin irritation (i.e. erythema grading (score) ≥ 3 at any day of measurement and/or ear thickness ≥ 25% with respect to Day 1 and/or ear punch weight ≥ 25% with reference to the negative control group).
In the main study 25 μL of three different concentrations of the test item (25, 50 and 100% w/w dissolved in AOO) or the negative or positive control were applied to the dorsal surface of each ear once a day for 3 consecutive days. On Day 5, the animals were injected with 0.5 mL/animal of a solution of BrdU at a concentration of 10 mg/mL in physiological saline (0.9% NaCl), using a plastic graded syringe. All animals were observed twice daily for mortality and morbitiy and for clinical signs before dosing commenced and daily up to sacrifice (approximately 1 h after dosing on Days 2, 3 and 5). The animals were weighed at allocation (Day 1) and on sacrifice (Day 6). The animals were sacrificed on Day 6, approximately 24 h after BrdU injection. No necropsy was performed on the sacrificed animals. Shortly after, the auricular lymph nodes were rapidly excised, pooled on individual basis and collected in a solution of 2% BSA-PBS (2% bovine serum albumine (BSA) in phosphate buffered saline (PBS)). Cell suspensions were prepared for the evaluation of proliferation. A single cell suspension of lymph node cells (LNC) was prepared from each mouse by gentle mechanical disaggregation through a 70 μm nylon mesh. The LNC were re-suspended in 2% BSA-PBS. BrdU was measured by ELISA using a commercial kit (Roche Applied Science, batch no.17267000), according to manufacturer instructions. Absorbance (OD) was detected at 450 nm (with reference wavelength: 690 nm).

Positive control substance(s):

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

Statistics:

Differences between each treated group and the concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous, a Modified t test (Cochran and Cox) was applied.

Positive control results:

The positive control substance (29% hexyl cinnamic aldehyde) induced a positive reaction, determined by a SI of 4.54. Thus the study was considered as valid.

Key result

Parameter:

SI

Remarks:

mean of 4 animals

Value:

1.25

Test group / Remarks:

25% test group

Key result

Parameter:

SI

Remarks:

mean of 4 animals

Value:

3.24

Test group / Remarks:

50% test group

Key result

Parameter:

SI

Remarks:

mean of 4 animals

Value:

5.4

Test group / Remarks:

100% test group

Parameter:

SI

Remarks:

mean of 4 animals

Value:

4.54

Test group / Remarks:

positive control group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : Significantly increased lymphoproliferation (SI ≥ 1.6, p < 0.01) was observed for the test item at treatment concentrations of 50 and 100%. At 25% of the test item, a slight but not significant increase in the lymphoproliferation was observed (SI = 1.25).

DETAILS ON STIMULATION INDEX CALCULATION : BrdU labelling index = (OD450–ODblank450) - (OD690 - ODblank690)

The BrdU labelling index was calculated for each mouse and a group mean was subsequently calculated. Results for each treatment group were expressed as the mean Stimulation Index (SI). The SI was derived by dividing the mean BrdU labelling index/mouse within each test item group and the positive control groups by the mean labelling indices for the respective vehicle group.

EC3 CALCULATION : An EC3 value was not calculated.

CLINICAL OBSERVATIONS: Neither mortality nor clinical signs were recorded in animals treated at all dose levels investigated.

BODY WEIGHTS: Changes in body weight observed during the study were within the expected range for this strain and age of animals.

Table 1: Mean Stimulation Indices

Compound	Concentration [%]	BrdU labeling Index/ mouse				BrdU labeling Index/ group		Stimulation index (SI)
		Replicate A	Replicate B	Replicate C	Mean	Mean ± SD	CV%
AOO	-	0.074	0.082	0.086	0.081	0.091 ± 0.014	15.04	1.00
		0.075	0.071	0.087	0.078
		0.099	0.108	0.096	0.101
		0.103	0.106	0.104	0.105
Test substance	25	0.095	0.078	0.089	0.088	0.114 ± 0.020	17.39	1.25
		0.160	0.126	0.118	0.135
		0.125	0.117	0.092	0.112
		0.114	0.139	0.113	0.122
	50	0.263	0.218	0.209	0.230	0.296 ± 0.095	32.11	3.24**
		0.380	0.348	0.337	0.355
		0.375	0.413	0.402	0.397
		0.197	0.215	0.190	0.201
	100	0.404	0.375	0.307	0.362	0.493 ± 0.164	33.29	5.40**
		0.370	0.423	0.387	0.394
		0.510	0.497	0.466	0.491
		0.732	0.753	0.690	0.725
HCA	29	0.459	0.450	0.362	0.424	0.414 ± 0.068	16.32	4.54
		0.279	0.324	0.354	0.319
		0.503	0.465	0.465	0.478
		0.480	0.419	0.409	0.436

CV% = Coefficient of Variation

SD = Standard Deviation

** = Statistically significant increase vs. vehicle control group (p<0.01)

Interpretation of results:

other: CLP/EU GHS Category 1 (H317) according to Regulation (EC) No 1272/2008

Conclusions:

CLP: Skin Sens. 1

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A LLNA ( BrdU-ELISA) was performed with Octyl laurate (CAS 5303 -24 -2) according to OECD Guideline 442B (Croda, 2017). For the pre-screentest, one animal (mouse) per dose (5, 10, 25, 50 and 100% of the test substance) was tested. No signs of toxicity were observed. Neither did the treatment induce relevant increase of ear thickness. The evaluation of ear punch weight indicated a very slight increase in the animals treated at 100% concentration (26%), when compared to animals treated with the vehicle. However, this increase was not considered significant, since it was very slight and no other parameter to evaluate irritation was altered. Thus, 100% was chosen as highest test concentration in the main assay, where 4 animals per group (vehicle control, positive control and 25, 50 and 100% of the test substance) were tested .

The validity of the study could be confirmed by the positive reaction of the positive control substance (29% hexyl cinnamic aldehyde), determined by an increased lymphoproliferation and a SI of 4.54. For the test substance concentrations 25, 50 and 100% SI values of 1.25, 3.24 and 5.4 were obtained indicating a skin sensitising potential of the test substance, which is considered a weak skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation meet the criteria for classification according to Regulation (EC) 1272/2008, and is therefore classified as skin sensitiser Category 1 (H317).