[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:

From 1 December 2006 to 9 January 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP compliant; no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: LLNA

Test material

Test material information

Constituent 1

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: B&K Universal Ltd, Hull, UK
- Age at study initiation: eight to twelve weeks
- Weight at study initiation: 15 to 23 g
- Housing: individually in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

1, 2.5, or 5%

No. of animals per dose:

Five

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 5%
- Irritation: None
- Lymph node proliferation response: Not determined


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The test material will be regarded as a sensitiser if at least on concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values.


TREATMENT PREPARATION AND ADMINISTRATION: The test material was freshly prepared in the vehicle and the mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days. Five days following the first topic application of the test material all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containg 3H-methyl thymidine (3HTdR:80µCi/ml, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.
Five hours later, the draining auricular lymph nodes were excised and processed into PBS. A single cell suspension of lymph node cells was prepared from each mouse by rinsing the cells with 4 ml PBS through a 200-mesh stainless steel gauze into a petri dish. The lymph node cells suspension was transferred to a centrifuge tube and was pelleted at 1400 rpm for ten minutes. The pellet was resuspended in 10 ml PBS and re-pelleted. Cells were precipitated with 3 ml 5% trichloroacetic acid at 4 °C for 18 hours. The precipitates were recovered, resuspended and transferred to 10 ml of scintillation fluid. 3HTdR incorporation was measured by ß-scintillation counting.

Positive control substance(s):

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

Results and discussion

Positive control results:

The positive control, α-hexylcinnamaldehyde, was considered to be a sensitiser.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Stimulation index for 1%, 2.5% and 5% is 0.94, 0.69 and 1.10, respectively. See also table 14.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM for vehicle control, 1%, 2.5% and 5% are 1988.22, 1862.99, 1366.01 and 2183.85, respectively. See also table 14.

Any other information on results incl. tables

Table 14 - Results LLNA

Concentration (%w/w) in acetone/olive oil 4:1	Animal number	dpm/animal	Mean dpm/animal (standard deviation)	Stimulation Index	Result
Vehicle	1-1	1445.35	1988.22 (±530.51)	N/A	N/A
	1-2	2144.98
	1-3	2048.27
	1-4	2763.51
	1-5	1539.00
1	2-1	2025.69	1862.99 (±594.55)	0.94	Negative
	2-2	2504.88
	2-3	1640.39
	2-4	956.94
	2-5	2187.03
2.5	3-1	2797.37	1366.01 (±847.83)	0.69	Negative
	3-2	749.26
	3-3	1167.96
	3-4	1386.95
	3-5	728.50
5	4-1	2160.63	2183.85 (±740.63)	1.10	Negative
	4-2	2553.50
	4-3	929.15
	4-4	2450.67
	4-5	2825.32

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.