[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:

2 March 2009 to 17 March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Signature: 15/10/2007; Date of Inspection: 21/08/2007

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of disodium N-(1-oxooctadecyl)-L-glutamate and disodium N-(1-oxohexadecyl)-L-glutamate

EC Number:

700-014-3

Molecular formula:

See remarks

IUPAC Name:

Reaction mass of disodium N-(1-oxooctadecyl)-L-glutamate and disodium N-(1-oxohexadecyl)-L-glutamate

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-12 weeks old.
- Weight at study initiation: 15-23 grams.
- Housing: Suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C.
- Humidity (%): 30-70%.
- Air changes (per hr): Approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light.

IN-LIFE DATES: From: Day 1 To: Day 6

Study design: in vivo (LLNA)

Vehicle:

other: 1% pluronic L92 in distilled water.

Concentration:

Preliminary Screening Test: 10% w/w
Main test: 2.5%, 5% & 10%

No. of animals per dose:

Preliminary Screening Test: 10% w/w - 1 mouse
Main test: Vehicle control - 4 mice; 2.5% - 4 mice; 5% - 4 mice; 10% - 4 mice

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 1% pluronic L92 in distilled water was chosen as the vehicle as it produced the highest concentration that was suitable for dosing.
- Irritation: REFER BACK TO IRRITATION STUDY.
- Lymph node proliferation response: No signs of systemic toxicity.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay in the Mouse.
- Criteria used to consider a positive response: The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was freshly prepared as a solution in 1% pluronic L92 in distilled water.
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 (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

Positive control substance(s):

other: 2,4-Dinitrobenzenesulfonic acid.

Statistics:

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (stimulation Index).

Results and discussion

Positive control results:

Concentration (% w/w) in 1 % pluronic L92 in distilled water: 10
Stimulation Index: 13.71
Result: Positive

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

Concentration (% w/w) in 1% pluronic L92 in distilled water

Remarks on result:

other: See Table 2.

Results 2

Key result

Parameter:

SI

Value:

1.01

Test group / Remarks:

Concentration (% w/w) in 1% pluronic L92 in distilled water

Remarks on result:

other:

Remarks:

See Table 2

Results 3

Key result

Parameter:

SI

Value:

1.58

Test group / Remarks:

Concentration (% w/w) in 1% pluronic L92 in distilled water

Remarks on result:

other:

Remarks:

See Table 2

Any other information on results incl. tables

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 1.

No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were 10 %, 5 % and 2.5 % w/w in 1 % pluronic L92 in distilled water.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 2.

A stimulation index of less than 3 was recorded for the three concentrations of the test material (10 %, 5 % and 2.5 % w/w in 1 % pluronic L92 in distilled water).

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 3.

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

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 4.

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.

Table 1: Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (% w/w) in 1 % pluronic L92 in distilled water	Animal Number	Body weight (g)		Day
		Day 1	Day 6	1		2		3		4		5		6
				Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
10	S-1	18	18	0	0	0	0	0	0	0	0	0	0	0	0

0 = No signs of systemic toxicity

Table 2: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in 1% pluronic L92 in distilled water	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	2423.64	302.96	na	na
2.5	2181.70	272.71	0.90	Negative
5	2456.92	307.12	1.01	Negative
10	3833.08	479.14	1.58	Negative

dpm =  Disintegrations per minute

a = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

Table 3: Individual Clinical Observations and Mortality Data

Concentration (% w/w) in 1 % pluronic L92 in distilled water	Animal Number	Day
		1		2		3		4		5		6
		Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
Vehicle	1-1	0	0	0	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0	0	0	0
2.5	2-1	0	0	0	0	0	0	0	0	0	0	0	0
	2-2	0	0	0	0	0	0	0	0	0	0	0	0
	2-3	0	0	0	0	0	0	0	0	0	0	0	0
	2-4	0	0	0	0	0	0	0	0	0	0	0	0
5	3-1	0	0	0	0	0	0	0	0	0	0	0	0
	3-2	0	0	0	0	0	0	0	0	0	0	0	0
	3-3	0	0	0	0	0	0	0	0	0	0	0	0
	3-4	0	0	0	0	0	0	0	0	0	0	0	0
10	4-1	0	0	0	0	0	0	0	0	0	0	0	0
	4-2	0	0	0	0	0	0	0	0	0	0	0	0
	4-3	0	0	0	0	0	0	0	0	0	0	0	0
	4-4	0	0	0	0	0	0	0	0	0	0	0	0

0 = No signs of systemic toxicity

Table 4: Individual Bodyweights and Bodyweight Changes

Concentration (% w/w) in 1 % pluronic L92 in distilled water	Animal Number	Body weight (g)		Body weight Change (g)
		Day 1	Day 6
Vehicle	1-1	15	14	-1
	1-2	19	19	0
	1-3	19	19	0
	1-4	18	19	1
2.5	2-1	18	19	1
	2-2	18	19	1
	2-3	20	20	0
	2-4	18	19	1
5	3-1	19	20	1
	3-2	19	19	0
	3-3	18	19	1
	3-4	18	18	0
10	4-1	17	18	1
	4-2	18	19	1
	4-3	18	19	1
	4-4	19	19	0

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Executive summary:

A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following: OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002); and Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10 % w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as a solution in 1 % pluronic L92 in distilled water at concentrations of 10 %,5 % or 2.5 % w/w. A further group of four animals was treated with 1 % pluronic L92 in distilled water alone.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in 1 % pluronic L92 in distilled water	Stimulation Index	Result
2.5	0.90	Negative
5	1.01	Negative
10	1.58	Negative

Under the conditions of the test, exposure to the test material did not cause a threefold or greater increase in 3HTdR incorporation compared to control values and the stimulation index remained < 3 at all concentrations tested. The test material is considered to be not sensitising in accordance with the OECD guideline.