[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jan - 10 Feb 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

An in vivo study was conducted because in vitro/in chemico test methods described under Annex VII, 8.3.1 of Regulation (EC) No. 1907/2006 are not applicable, or the results obtained from those studies are not adequate for classification and risk assessment. For details, please refer to the document attached below.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2010

Deviations:

yes

Remarks:

humidity in animal room out of range for one day

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

adopted in 2003

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC No 640/2012, Part B

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:

2,2-bis[(hexadecanoyloxy)methyl]propane-1,3-diyl dihexadecanoate

Cas Number:

18641-58-2

Molecular formula:

C69H132O8

IUPAC Name:

2,2-bis[(hexadecanoyloxy)methyl]propane-1,3-diyl dihexadecanoate

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Remarks:

inbred

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: approx. 11 weeks
- Weight at study initiation: 20.5 – 26.9 g
- Housing: in groups up to 5/sex in polycarbonate cages (Makrolon MIII type, height 18 cm), with sterilised wooded fibers as bedding. For enrichment, paper and shelters were provided.
- Diet: pelleted rodent diet (SM R/M, Ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 39 – 44
- Air changes (per hr): ≥ 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: 15 Jan - 08 Feb 2021

Study design: in vivo (LLNA)

Vehicle:

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

Remarks:

AcOO

Concentration:

Pre-test: 25% (w/w)
Main study: 5, 10 and 25% (w/w)

No. of animals per dose:

5 females

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at the testing facility. At 50%, the formulation was too dry for application to the ears.
- Irritation: Only very slight irritation of the ears was observed.
- Systemic toxicity: There were no signs of systemic toxicity.
- Ear thickness measurements: No increase in ear thickness of 25% or more at a test item concentration of 25% (w/w) was noted from study Days 1 – 3 and 1 - 6 and thus, the test substance was not considered irritating at these concentrations.
- Erythema scores: At 25% (w/w) the animals showed very slight erythema (score 1) on both ears. The signs were observed on Days 2 and 3 and the animals were free from skin reactions thereafter. In addition, white test item remnants were present on the dorsal surface of the ears of both animals dosed at 25% between Days 1 and 3, which did not hamper scoring of the skin reactions.

MAIN STUDY
Based on the pre-screen test results, test substance concentrations of 5, 10 and 25% (w/w) were chosen for the main study.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay. Cellular proliferation was determined by incorporation of 3H-methyl-thymidine.
- Criteria used to consider a positive response: The test item was considered positive in the LLNA if the incorporation of 3H-methyl-thymidine, indicated by stimulation index, was at least 3-fold when compared to control mice.

TREATMENT PREPARATION AND ADMINISTRATION:
The dorsal surface of both ears was topically treated with 25 µL/ear at concentrations of 5, 10 and 25% w/w. The animals were treated on each ear for 3 consecutive days (Induction Days 1, 2 and 3).
On study Day 6, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine. About 5 hours after injection, the animals were euthanized and the draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The draining lymph nodes were pooled for each experimental group.
Following excision of the nodes, single cell suspensions of pooled lymph node cells (LNC) were prepared in PBS by gentle separation through stainless steel gauze. LNC were washed twice with an excess of PBS by centrifugation and exposed to 5% trichloroacetic acid (TCA) for DNA precipitation overnight in the refrigerator. Afterwards, the precipitates were re-suspended in TCA and transferred to scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR) and the number of radioactive disintegrations per minute (DPM) was assessed.

CLINICAL SIGNS
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Post-dose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing). All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

BODY WEIGHT
Animals were weighed individually on Day 1 (pre-dose) and 6 (prior to necropsy)

Positive control substance(s):

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

Results and discussion

Positive control results:

Reliability checks are performed regularly. A periodic positive control experiment using 5, 10 and 25% solutions of alpha-hexyl-cinnamaldehyde (v/v) in acetone:olive oil (4:1) was performed in Nov 2020. The SI values obtained were 2.1, 3.6 and 9.0 at 5, 10 and 25 %, demonstrating the validity of the test. An EC3 value of 8.0% was established, which fell into the acceptable range of 4.8 - 19.5%.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

5% formulation

Results 2

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

10% formulation

Results 3

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

25% formulation

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION:
The SI of the 5, 10 and 25% treatment group was 1.6, 1.4 and 1.1%, respectively.

EC3 CALCULATION:
The EC3 value could not be calculated, since all SI values were below the threshold value of 3.

CLINICAL OBSERVATIONS:
One animal died during the IV injection with 3H-methyl thymidine on Day 6, which was considered not test item related. In addition, no clinical signs of systemic toxicity were noted in any animal.

BODY WEIGHTS:
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness):
No irritation was observed in any of the animals. White test item remnants were present on the dorsal surface of the ears of all animals dosed at 10% and 25% between Days 1 and 3, which did not hamper scoring of the skin reactions.

Any other information on results incl. tables

Table 2: Relative size of lymph nodes, radioactivity counts (DPM) and stimulation index (SI)

Test item formulation	Animal	Relative size auricular lymph nodes		DPM		SI
		left	right	DPM/animal	mean ± SEM	mean ± SEM
0%	1	n	n	81	193 ± 57	1.0 ± 0.3
	2	n	n	206
	3	-	-	-
	4	n	n	140
	5	n	n	345
5%	6	n	n	400	306 ± 43	1.6 ± 0.2
	7	n	n	301
	8	n	n	378
	9	n	n	152
	10	n	n	298
10%	11	n	n	469	276 ± 53	1.4 ± 0.3
	12	n	n	267
	13	n	n	239
	14	n	n	259
	15	n	n	146
25%	16	n	n	327	214 ± 35	1.1 ± 0.2
	17	n	n	185
	18	n	n	144
	19	n	n	263
	20	n	n	153
DPM: disintegrations per minute; SEM: standard error of the mean; n: normal; -: animal died during IV injection with 3H-methyl thymidine on Day 6, not attributed to treatment with the test item

Table 3: Body weights and skin reactions

Group	Test item (% w/w)	Animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
			Body weight (g)	Erythema^x		Erythema^x		Erythema^x		Erythema^x		Erythema^x		Erythema^x		Body weight (g)
				left	right	left	right	left	right	left	right	left	right	left	right
1	0	1	25.4	0	0	0	0	0	0	0	0	0	0	0	0	25.8
		2	25.9	0	0	0	0	0	0	0	0	0	0	0	0	26.0
		3	26.2	0	0	0	0	0	0	0	0	0	0	0	0	25.6
		4	23.6	0	0	0	0	0	0	0	0	0	0	0	0	24.3
		5	22.6	0	0	0	0	0	0	0	0	0	0	0	0	24.6
2	5	6	22.0	0	0	0	0	0	0	0	0	0	0	0	0	22.4
		7	22.2	0	0	0	0	0	0	0	0	0	0	0	0	23.4
		8	24.6	0	0	0	0	0	0	0	0	0	0	0	0	25.4
		9	24.7	0	0	0	0	0	0	0	0	0	0	0	0	25.0
		10	25.0	0	0	0	0	0	0	0	0	0	0	0	0	24.9
3	10^*	11	26.9	0f	0f	0f	0f	0f	0f	0	0	0	0	0	0	25.7
		12	20.8	0f	0f	0f	0f	0f	0f	0	0	0	0	0	0	22.9
		13	25.4	0f	0f	0f	0f	0f	0f	0	0	0	0	0	0	25.0
		14	25.0	0f	0f	0f	0f	0f	0f	0	0	0	0	0	0	24.3
		15	23.6	0f	0f	0f	0f	0f	0f	0	0	0	0	0	0	22.7
4	25^#	16	24.1	0f	0f	0f	0f	0f	0f	0	0	0	0	0	0	25.3
		17	24.0	0f	0f	0f	0f	0f	0f	0	0	0	0	0	0	23.8
		18	23.1	0f	0f	0f	0f	0f	0f	0	0	0	0	0	0	22.5
		19	20.5	0f	0f	0f	0f	0f	0f	0	0	0	0	0	0	22.2
		20	24.7	0f	0f	0f	0f	0f	0f	0	0	0	0	0	0	24.0
^x: Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear)
^*: Applied using a pipette with the tip cut off.
^#: Applied using a spatula, instead of using a pipette
^f: White staining of test item remnants on the dorsal surface of the ears did not hamper scoring for erythema.

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

Conclusions:

The skin sensitisation potential of the test substance was assessed in a LLNA. Application of the test substance to mice ears at 5, 10 and 25% resulted in a calculated mean SI value of 1.6, 1.4 and 1.1%, respectively. The positive control demonstrated the validity and sensitivity of the test. Thus, the test substance is considered not sensitising according to Regulation (EC) No. 1272/2008.