Sorbitan, tridocosanoate

Endpoint summary

Description of key information

Skin sensitisation in vivo (OECD 429, LLNA): not sensitising

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:

13 Nov - 19 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2010

Deviations:

yes

Remarks:

No data on range finding study are given in the report.

GLP compliance:

yes

Type of study:

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

Species:

mouse

Strain:

CBA

Remarks:

/N, SPF

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Central Lab. Animal Inc., Republic of Korea
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks
- Weight at study initiation: 17.9 - 22.2 g (prior to dose range finding study)
- Housing: 2 - 3 animals per cage in polysulfone cages
- Diet: Pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C, Envigo RMS, Inc., U.S.A.), ad libitum
- Water: public tap water, ad libitum (analysis was performed)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 - 24.7
- Humidity (%): 51.6 - 62.7
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

methyl ethyl ketone

Remarks:

(MEK)

Concentration:

2.5, 5 and 10%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TEST:
In the pre-screen test, 25 μL of the test item (0.5, 1, 2.5, 5 and 10% in MEK) were applied to the dorsum of both ears of two animals, once a day for 3 consecutive days. All animals were observed daily for clinical signs. Body weights were recorded prior to dosing and on the day of necropsy. Furthermore, erythema measurements were performed daily, ear thickness was measured on Day 1 (pre-dose) and Day 3 and 6.

- Compound solubility: The test item was soluble in the vehicle at a concentration of up to 10%.
- Systemic toxicity: No signs of toxicity were observed.
No further informations were given on results of the pre-screen test.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: mouse local lymph node assay (LLNA): BrdU-ELISA
- Criteria used to consider a positive response: A substance is regarded as sensitizer in the LLNA test inducing a Stimulation Index (SI) ≥ 1.6.

TREATMENT PREPARATION AND ADMINISTRATION: Based on the negative results in the pre-screen test, the test item was used at 2.5, 5 and 10% concentration in the main study. 25 μL of each dose formulation were applied to the dorsum of both ears of each animal once a day for 3 consecutive days. Observations took place like described above for the pre-screen test. On Day 5, the animals were injected inter-peritoneally with 0.5 mL/animal of a BrdU solution (10 mg/mL in phosphate buffered saline (PBS)). The animals were sacrificed on Day 6, approximately 24 h after BrdU injection. Using a punch for skin biopsy of 6 mm in diameter the inner tissue of the ear was removed about 1 mm from the outside of the ear and tissues of both sides were weighed together. The draining auricular lymph nodes from each mouse ear were excised and processed separately in PBS for each animal. For each mouse, a single-cell suspension of lymph node cells (LNC) was prepared by #70 nylon mesh to generate a single cell suspension. BrdU was measured by ELISA using a commercial kit (Roche Applied Science, batch no. 24890800), according to manufacturer instructions. Absorbance (OD) was detected at 370 nm (with reference wavelength: 492 nm).

Positive control substance(s):

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

Statistics:

Statistical analysis was conducted using a statistical program (version 9.3, SAS Institute Inc., U.S.A.) for the data including body weight, erythema score, ear thickness, ear weight and stimulation index. Bartlett’s test was employed on homogeneity of variance (significance level: 0.05) for body weights, ear thickness, ear weight and stimulation index data. One-way analysis of variance (ANOVA) was employed on homogeneous data. Dunnett’s ttest was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive control group (G5). Since not significant, Kruskal-Wallis test was employed on heterogeneous data and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive control group.
Kruskal-Wallis test for the erythema score was employed on heterogeneous data, and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive control group.

Positive control results:

The positive control substance (25% HCA) induced a positive reaction, determined by a SI of 2.53. Thus, the study was considered as valid.

Key result

Parameter:

SI

Remarks:

mean of 5 animals

Value:

1.13

Test group / Remarks:

2.5% test group

Key result

Parameter:

SI

Remarks:

mean of 5 animals

Value:

0.76

Test group / Remarks:

5% test group

Key result

Parameter:

SI

Remarks:

mean of 5 animals

Value:

0.75

Test group / Remarks:

10% test group

Parameter:

SI

Remarks:

mean of 5 animals

Value:

2.53

Test group / Remarks:

positive control group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : No (significant) increase in lymphoproliferation (SI > 1.6) was observed at all tested concentrations of the test substance.

DETAILS ON STIMULATION INDEX (SI) CALCULATION :

BrdU labelling index = (ABS em – ABS blank em) – (ABS ref – ABS blank ref)

em = Emission wavelength
ref = Reference wavelength

SI = Mean of BrdU labelling index/mouse of test substance/ Mean of BrdU labelling index of negative control


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

BODY WEIGHTS: No significant changes in body weight were observed during the study.

OTHER OBSERVATIONS:
Erythema: No erythema were observed in the treated animals at any dose or reading time point.
Ear thickness: On Day 6, a significant, but not relevant increase was observed in the highest dose group (10%) when compared to the negative control (Mean ± SD: 0.22 ± 0.0 mm vs. 0.21 ± 0.0 mm (NC), respectively). Except of this, no significant differences when compared to the negative control group.
Ear weight: There were no significant differences when compared to the negative control group.

Table 1: Stimulation Indices in mice

Compound	Concentration [%]	BrdU labelling Index (Animal No.)					BrdU labeling Index	Stimulation Index (SI)
		1	2	3	4	5	Mean ± SD	Mean ± SD
Vehicle control (MEK)	0	0.25	0.18	0.15	0.21	0.16	0.19 ± 0.04	1.0 ± 0.21
Test substance	2.5	0.27	0.2	0.17	0.18	0.26	0.21 ± 0.05	1.13 ± 0.24
	5	0.19	0.13	0.03	0.15	0.21	0.14 ± 0.07	0.76 ± 0.37
	10	0.18	0.12	0.13	0.12	0.16	0.14 ± 0.03	0.75 ± 0.15
Negative control (HCA)	25	0.49	0.52	0.45	0.51	0.43	0.48 ± 0.04	2.53 ± 0.20 **

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

MEK = methyl ethyl ketone

HCA = α-Hexylcinnamaldehyde

 

Interpretation of results:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation in vivo

The skin sensitisation potential of sorbitan tridocosanoate was evaluated in a Local Lymph Node Assay (LLNA) that was performed according to OECD guideline 429 and in compliance with GLP (Riken Vitamin, 2018). Based on the results of a pre-test, the main LLNA was performed with test item formulations of 2.5, 5 and 10%

The test item was dissolved in methyl ethyl ketone and 25 µL each were administered on three consecutive days to the dorsal surface of both ears of 5 female CBA/N mice. A similar constituted group of animals received the solvent or the positive control (25%α-hexyl-cinnamaldehyde). Five days after the first topical application of the test item, solvent or positive control, the animals were injected with 0.5 mL BrdU solution. At terminal sacrifice on Day 6, approx. 24 h after BrdU injection, skin biopsies were taken from both ears and the draining auricular lymph nodes were harvested for preparation of single cell suspensions. Cellular proliferation indicated by BrdU incorporation was quantified by ELISA.

No mortality or clinical signs of toxicity were observed and all animals gained weight as expected. On study Day 6, a significant but not relevant increase in ear thickness was observed at the highest concentration tested (10%). There were no further skin reactions observed.

Stimulation indices (SI values), obtained for the test item were 1.13, 0.76 and 0.75 at 2.5, 5 and 10%, respectively, and were below the criteria of a positive response (SI value > 1.6). The positive control gave a significant response (SI value 2.53) at 25%, confirming the validity of the protocol used for this study.

Under the conditions of the test, sorbitan tridocosanoate is not considered to have a skin sensitising potential.

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 criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.