Fatty acids, C18-(unsaturated) dimers and their monoesters and diesters with 2-ethylhexanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-08-29 to 2011-11-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD 429 and EU method B.42 in a GLP cerfified testing facility.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(During the acclimation phase, the relative humidity in the animal room ranged between 45 – 100% for a maximum of 30 minutes and between 45 – 75% instead of 45 – 65% for a few hours.)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Details on test material:

Identity: 400160
Appearance: Colourless, yellowish liquid
Batch No.: KL-11-039
Purity: 100 % (GPC)
Storage: At room temperature
Expiration Date: March 24, 2016

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaCrl

Sex:

female

Details on test animals and environmental conditions:

Animals: Female mice of 9-11 weeks of age and with 18-23 g of body weight were included in the study
Housing: Animals were housed in groups
Lighting: 12 h light/12 h dark
Temperature: 22 ± 2°C
Relative humidity: 45 to 65%
Food: Animals had access to pelleted standard diet, ad libitum
Water: Tap water, ad libitum
Bedding: Granulated soft wood bedding
Acclimatisation: Study animals were acclimated to their housing for 5 d prior to their first day of dosing

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

5, 10, 25% (w/v)

No. of animals per dose:

4 females (nulliparous and non-pregnant)

Details on study design:

On each day of dosing, the test item was prepared at the appropriate concentrations (w/v) by dissolving in acetone:olive oil (4:1 v/v). The application volume of 25 µL was used.
4 groups of 4 female mice were treated on the dorsal surface of both ears once per day for 3 consecutive days as follows:
Group 1: vehicle
Group 2: BYK-400160 (5% w/v)
Group 3: BYK-400160 (10% w/v)
Group 4: BYK-400160 (25% w/v)
On day 6, the mice were injected, i.v., with 20.0 µCi of 3H-Methyl thymidine (3HTdR) per mouse. 5 h later, the mice were euthanized and the draining auricular lymph nodes were removed. The lymph node cells were treated with 5% trichloroacetic acid (TCA) to precipitate the DNA. The resulting pellets were counted in a β-scintillation counter to determine incorporation of 3HTdR.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated for the body weight parameter.

Results and discussion

Positive control results:

The positive control, HCA at 10% and 25% resulted in a stimulation index (SI) of 2.18 and 8.08, respectively. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Vehicle control = 1.00 400160 (5% w/v) = 1.30 400160 (10% w/v) = 1.58 400160 (25% w/v) = 2.00

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM per lymph node: Vehicle control = 226.3 400160 (5% w/v) = 294.2 400160 (10% w/v) = 358.6 400160 (25% w/v) = 452.1

Any other information on results incl. tables

Calculation and Results of Individual Data

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

Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	16	---	---	---	---
---	BG II	15	---	---	---	---
0	1	1826	1811	8	226.3	1.00
5	2	2369	2354	8	294.2	1.30
10	3	2884	2869	8	358.6	1.58
25	4	3632	3617	8	452.1	2.00

BG =  Background (1 mL 5% trichloroacetic acid) in duplicate

1    =  Control Group

2-4 =  Test Group

S.I. =  Stimulation Index

^a)   =  The mean value was taken from the figures BG I and BG II

^b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No signs of systemic toxicity were observed during the study period. On d 3 and 4, the animals treated with a test item concentration of 25% showed an erythema of the ear skin (Score 1). Animals treated with 5 or 10% test item concentration did not show any signs of local skin irritation.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3H-methyl thymidine, was within the range commonly recorded for animals of this strain and age.

 

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

400160 was not a skin sensitiser under the conditions tested.

Executive summary:

The dermal sensitisation of 400160 was tested in the local lymph node assay according to EU method B.42 in a GLP certified testing facility. 3 groups of young adult CBE/CaCrl mice (n=4/group, female) were treated daily with the test item at concentrations of 5, 10 and 25% (w/v) in acetone:olive oil (4:1 v/v) by topical application to the dorsum of each ear for 3 consecutive days. A control group of 4 mice was treated with the vehicle only. 5 days after the first topical application the mice were injected intravenously into a tail vein with 3HTdR. Approximately 5 h after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3HTdR measured in a β-scintillation counter.

As positive control α-hexylcinnamaldehyde in acetone:olive oil (4:1 v/v) was used. No signs of systemic toxicity were observed during the study period. On d 3 and 4, the animals treated with a test item concentration of 25% showed an erythema of the ear skin (Score 1). Animals treated with 5 or 10% test item concentration did not show any signs of local skin irritation. No deaths occurred during the study period.

Stimulation Indices (S.I.) of 1.30, 1.58, and 2.00 were determined with the test item at concentrations of 5, 10, and 25% (w/v) in acetone:olive oil (4:1 v/v), respectively. Although a dose response was observed, the test item 400160 was not a skin sensitizer as none of the Stimulation Indices exceeded the threshold value of 3.