[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:

24 Apr to 17 Jun 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

no

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:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted in 2012

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA): DA

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, vegetable-oil, polymd., esters with 1,2-hexadecanediol

Cas Number:

68910-99-6

Molecular formula:

Not applicable (i.e., UVCB substance)

IUPAC Name:

Fatty acids, vegetable-oil, polymd., esters with 1,2-hexadecanediol

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

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: SPF
- Age at study initiation: 12-13 weeks old
- Weight at study initiation: 20.9 - 27.0 g
- Housing: group housing; up to 5 animals per cage in polycarbonate cages (Makrolon MIII type; height 18 cm)
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal tap-water in water bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 25 Apr 2018 To: 10 Jun 2018

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Pre-screen test: 25 and 60% (w/w)
Main study: 10, 25 and 60% (w/w)

No. of animals per dose:

5 females/group

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements
- Concentrations tested: 25 and 60%
- Irritation: the highest concentration to be used in the main study should induce well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied
- Systemic toxicity: the highest concentration should cause no systemic toxicity
- Ear thickness measurements: conducted prior to dosing on Days 1 and 3, and on Day 6 with a digital thickness gauge (Kroeplin C110T-K)
- Erythema scores: determined according to numerical scoring system ranging from 0 (no erythema) to 4 (severe erythema)

MAIN STUDY :
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by beta-scintillation
- Criteria used to consider a positive response: SI ≥ 3

TREATMENT PREPARATION AND ADMINISTRATION:
Test item dosing formulations were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.

During the induction phase (days 1 - 3) the dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day; local irritation reactions were assessed.

IN-LIFE DATES: 25 Apr to 10 Jun 2018

EXCISION OF NODES AND TISSUE PROCESSING
On day 6, an injection of 0.250 mL phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). 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 nodes were pooled for each animal in PBS.

A single cell suspension of lymph node cells (LNC) was prepared from each mouse in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS followed by centrifugation at 200 g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

RADIOACTIVITY MEASUREMENTS
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

IN-LIFE PROCEDURES, OBSERVATIONS, MEASUREMENTS
Mortality/Moribundity Checks
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. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations - Postdose Observations
Postdose 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 Weights
Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy).

Irritation
Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded. Erythema and eschar formation were scored according to Draize.

Terminal procedures
No necropsy was performed, since all animals survived until the end of the observation period.

Positive control substance(s):

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

Statistics:

All results presented in the tables are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented. DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

Results and discussion

Positive control results:

A routine six-month reliability test (performed in November 2017; study number 20152821) was conducted to assess the sensitivity of the CBA/J mice at the testing facility to a known sensitizer. The positive control substance hexyl cinnamic aldehyde (25% (v/v) in acetone:olive oil (4+1)) was considered to be a sensitizer under the conditions of the test (SI 3.5).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

2.3

Test group / Remarks:

60%

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The SI values calculated for the test item concentrations 10, 25 and 60% were 1.0, 1.6 and 2.3, respectively.

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

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity were observed in any treatment group or in the control group.

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

MACROSCOPIC EXAMINATION OF THE LYMPH NODES AND SURROUNDING AREA
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Any other information on results incl. tables

Main Study

- Skin Reactions / Irritation

The very slight irritation of the ears as shown by the animals treated at 60% between Days 1 and 3 was considered not to have a toxicologically significant effect on the activity of the nodes. Transparent test item remnants were present on the dorsal surface of the ears of all animals treated at 25 and 60% between Days 1 and 3, which did not hamper scoring of the skin reactions.

Table 1. Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

Group	TS^1 (%)	Animal	Node size^2		DPM^3/ animal	mean DPM ± SEM^4				mean SI ± SEM
			left	right
1	0	1	n	n	223	353	±	56		1.0	±	0.2
		2	n	n	310
		3	n	n	285
		4	n	n	542
		5	n	n	404
2	10	6	n	n	254	366	±	39		1.0	±	0.1
		7	n	n	372
		8	n	n	336
		9	n	n	499
		10	n	n	371
3	25	11	n	n	355	571	±	143		1.6	±	0.4
		12	n	n	385
		13	n	n	567
		14	n	n	977
		15	n	n	2572^5
4	60	16	n	n	466	799	±	125		2.3	±	0.4
		17	n	n	557
		18	n	n	1119
		19	n	n	871
		20	n	n	983

¹  TS = test item (% w/w).

²  Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +, ++ or +++: degree of enlargement, n: considered to be normal).

³    DPM= Disintegrations per minute.

⁴    SEM = Standard Error of the Mean.

⁵    Value not used for interpretation (outlier based on Dixon’s Q-test).

Applicant's summary and conclusion

Interpretation of results:

other:

Remarks:

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

Conclusions:

CLP: not classified