N,N''-(methylenedi-4,1-phenylene)bis[N'-octylurea]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 October, 2003 - 27 October, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2002)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-060-3

EC Name:

-

Cas Number:

122886-55-9

Molecular formula:

C31H48N4O2

IUPAC Name:

3-octyl-1-[4-({4-[(octylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): KY-UN
- Stability under test conditions: not stated
- Storage condition of test material: at room temperature and protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation: 19.2 ± 1.1 g
- Housing: Individually housed in disposable crystal polystyrene cages.
- Diet: Free access to A04 C pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France)
- Water: Free access to tap water (filtered using a 0.22 micron filter)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30 - 70
- Air changes (per hr): approx. 12
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

0, 0.5, 1, 2.5, 5 and 10%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
To assess the irritant potential of the test sustance (through ear thickness measurement), a preliminary test was performed on four animals, as follows: - the test substance was prepared at the concentrations of 1, 2.5, 5 and 10%, - for three consecutive days, the animals received applications of 25 µL of the dosage form preparations to the external surface of both ears (one concentration per ear), - measurement of the ear thickness (using a micrometer) was performed each day before treatment and 24 hours after the last application.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test substance was considered as a skin sensitizer when the SI for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

ANIMAL ASSIGNMENT
Five groups of four animals were treated with one test substance concentration per group. One group of four animals was treated with vehicle and another group of four animals was treated with the positive control substance.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: All dosage form preparations were made freshly on the morning of administration and any unused material was discarded that same day.
Rationale for selecting vehicle: Due to the unsatisfactory solubility of the test substance in the first recommended vehicle (acetone/olive oil (4/1, v/v)), methyl ethyl ketone was chosen among the other proposed vehicles. A homogeneous dosage form preparation was obtained at the maximum concentration of 10%.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Clinical signs, morbidity and mortality: The animals were observed at least once a day during the study.
Body weights: The animals were weighed individually on the first day of the study (day 1) and on the day of sacrifice (day 6).
Ear thickness measurements and recording of local reactions: Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test substance. On days 1, 2 and 3 (before application) as well as on day 6 (after sacrifice), the thickness of the left ear of each animal was measured using a micrometer. Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (coloration, presence of residual test substance) was recorded according to guidelines.

Positive control substance(s):

other: Alpha-hexylcinnamaldehyde (HCA)

Statistics:

Not performed.

Results and discussion

Positive control results:

In the positive control group given HCA at the concentration of 25%, a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 8.15) was noted. The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.86

Test group / Remarks:

0.5%

Remarks on result:

other: 0.5% test group

Results 2

Key result

Parameter:

SI

Value:

0.86

Test group / Remarks:

1%

Remarks on result:

other: 1% test group

Results 3

Key result

Parameter:

SI

Value:

0.53

Test group / Remarks:

2.5%

Remarks on result:

other: 2.5% test group

Results 4

Key result

Parameter:

SI

Value:

1.07

Test group / Remarks:

5%

Remarks on result:

other: 5% test group

Results 5

Key result

Parameter:

SI

Value:

0.76

Test group / Remarks:

10%

Remarks on result:

other: 10% test group

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The radioactivity measurements for the substance concentrations 0.5, 1, 2.5, 5 and 10% were 765.99, 758.46, 466.19, 947.39 and 670.31 respectively.

Any other information on results incl. tables

Results Pre-screen test:

No cutaneous reactions and no noteworthy increase in ear thickness were observed in the treated animals, whatever the concentration.

The highest concentration retained for the main test was therefore the maximal practicable concentration, according to the criteria specified in OECD 429 test guideline.

Other results - main study:

No clinical signs and no mortality were observed during the study.

The body weight changes of the treated animals were similar to that of the control animals.

No cutaneous reactions and no noteworthy increase in ear thickness were observed at any of the tested concentrations.

Applicant's summary and conclusion

Interpretation of results:

other: Not skin sensitising

Remarks:

According to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD 429 test guideline and GLP principles, KY-UN was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to and including 10%.

Executive summary:

KY-UN was tested in a LLNA skin sensitisation study, performed according to OECD 429 test guideline and GLP principles.

No clinical signs and no mortality were observed during the study. The body weight changes of the treated animals were similar to that of the control animals. No cutaneous reactions and no noteworthy increase in ear thickness were observed at any of the tested concentrations. Reliable negative and positive controls were included.

The SI values calculated for the substance concentrations 0.5, 1, 2.5, 5 and 10% were 0.86, 0.86, 0.53, 1.07 and 0.76 respectively.

Based on the results KY-UN does not have to be classified for skin sensitisation according to Regulation (EC) No 1272/2008, as the SI appeared not to be ≥ 3 when tested up to and including 10%.