Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N,N'-Dimethylurea
- Test-substance No.: 05/0835-1
- Analytical purity: 98.1 g/100 g
- Purity test: analytical report No.: 05L00235
- Lot/batch No.: 581251AX10
- Stability under test conditions: The stability of the test substance in the vehicle for the maximum application period was confirmed indirectly by
analysis of the correctness of the concentration. The correctness of the concentration of the test-substance preparations (10%, 30% and 70%) for the first application was confirmed by analysis.
- Homogeneity: The test substance was homogeneous by visual inspection

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 – 7 weeks
- Weight at study initiation: 16.8 g – 19.5 g
- Housing: single
- Diet (e.g. ad libitum): Kliba-Labordiaet (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 8 days before the first test-substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 30 – 70%
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Study design: in vivo (LLNA)

Vehicle:
other: 1% aqueous Pluronic®
Concentration:
10%, 30% and 70%
No. of animals per dose:
6
Details on study design:
The skin sensitizing potential of N,N'-Dimethylurea was assessed using the non-radioactive variant of the Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of the auricular lymph nodes on repeated application of the test substance to the dorsal skin of the ears.

SELECTION OF DOSES/CONCENTRATIONS
- The results of a pretest were considered, which showed moderately increased ear weights and slightly increased lymph node weights after application of a 70% preparation in 1% Pluronic® L 92 in doubly distilled water.

VEHICLE
- 1% Pluronic® L 92 Surfactant in doubly distilled water
- Reason for the vehicle: 1% Pluronic® L 92 Surfactant in doubly distilled water was chosen as the vehicle because good solubility of the preparation was achieved. Pluronic® L 92 was recommended by Ryan C.A. et al. (Food Chem. Toxicol 40, 1719-1725, 2002) as suitable to produce aqueous preparations for use in the Murine Local Lymph Node Assay (LLNA).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Randomization: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.:
Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 – 64“.
- Name of test method: non-radioactive variant of the Murine Local Lymph Node Assay
- Criteria used to consider a positive response:
If at least one concentration tested causes a concentration dependent statistically significant and/or biologically relevant increase in cell count and/or lymph node weight without being accompanied by a statistically significant and/or biologically relevant increase in ear weight, the test substance is considered to be a sensitizer.
- further evaluation: If statistically significant and/or biologically relevant increases in ear weights are running in parallel to the increase in cell count and/or lymph node weight, it cannot be ruled out, that the lymph node response was caused by irritation and not by skin sensitization. Then, for identification of the relevance of the statistical evaluation, a comparison of the results of the present test to appropriate historical control values (cf. section “Historical control data”) may be performed. If one or a combination of the measured parameters change statistical significance, evaluation on basis of the criteria described above may be possible. If the statistical comparison with the historical control does not yield results useful for evaluation, further investigations may be necessary to differentiate between irritation and sensitization response.
If a test substance does not elicit a statistical significant increase in lymph node weight and/or cell count but shows a clear concentration related increase in response, further investigating of the sensitization potential at higher concentrations should be considered.
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: No detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted in the raw data.

TREATMENT PREPARATION AND ADMINISTRATION:
- Form of application: Epicutaneous application of the solution is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 2 x 12.5 μL per ear
The total volume of 25 μL per ear and application was split into two portions in order to prevent run off of the test-substance preparations due to high volume. The second daily application was performed immediately after the first daily application was finished in all animals of the study collective.
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- Mortality was checked twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- Treatment groups:
Control group 1 Vehicle: 1% aqueous Pluronic®
Test group 2 Test substance 10% in 1% aqueous Pluronic®
Test group 3 Test substance 30% in 1% aqueous Pluronic®
Test group 4 Test substance 70% in 1% aqueous Pluronic®

TERMINATION
- Three days after the last application the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the cell count (indicator of cell proliferation) and weight of each animal’s pooled lymph nodes. Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of cell count, lymph node weight and ear weight were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group.
Further statistical analyses were performed according to the following:
- Cell count, lymph node weight and ear weight: WILCOXON - Test

Results and discussion

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Stimulation Index: Cell count Test Group: 1; Treatment: vehicle 1% aqueous Pluronic®; Cell Count [Counts/Lymph Node Pair]: Mean: 10,325,000; S.D. 1,057,023; Stimulation Index: 1.00 Test Group: 2; Treatment: 10% in 1% aqueous Pluronic®; Cell Count [Counts/Lymph Node Pair]: Mean: 7,649,333; S.D. 2,090,677; Stimulation Index: 0.74 Test Group: 3; Treatment: 30% in 1% aqueous Pluronic®; Cell Count [Counts/Lymph Node Pair]: Mean: 9,619,333; S.D. 2,283,940; Stimulation Index: 0.93 Test Group: 4; Treatment: 70% in 1% aqueous Pluronic®; Cell Count [Counts/Lymph Node Pair]: Mean: 10,173,333; S.D. 2,066,731; Stimulation Index: 0.99 Stimulation Index: Lymph node weight Test Group: 1; Treatment: vehicle 1% aqueous Pluronic®; Cell Count [Lymph Node Weight [mg]]: Mean: 5.1; S.D. 0.4; Stimulation Index: 1.00 Test Group: 2; Treatment: 10% in 1% aqueous Pluronic®; Cell Count [Lymph Node Weight [mg]]: Mean: 4.5; S.D. 0.8; Stimulation Index: 0.89 Test Group: 3; Treatment: 30% in 1% aqueous Pluronic®; Cell Count [Lymph Node Weight [mg]]: Mean: 5.4; S.D. 0.5; Stimulation Index: 1.06 Test Group: 4; Treatment: 70% in 1% aqueous Pluronic®; Cell Count [Lymph Node Weight [mg]]: Mean: 5.1; S.D. 0.6; Stimulation Index: 1.00 Stimulation Index: Ear weight Test Group: 1; Treatment: vehicle 1% aqueous Pluronic®; Cell Count [Counts/Lymph Node Pair]: Mean: 29.0; S.D. 0.9; Stimulation Index: 1.00 Test Group: 2; Treatment: 10% in 1% aqueous Pluronic®; Cell Count [Counts/Lymph Node Pair]: Mean: 29.2; S.D. 1.0; Stimulation Index: 1.01 Test Group: 3; Treatment: 30% in 1% aqueous Pluronic®; Cell Count [Counts/Lymph Node Pair]: Mean: 29.9; S.D. 1.0; Stimulation Index: 1.03 Test Group: 4; Treatment: 70% in 1% aqueous Pluronic®; Cell Count [Counts/Lymph Node Pair]: Mean: 31.7; S.D. 1.2; Stimulation Index: 1.09* * statistically significant for the value p ≤ 0.01

Any other information on results incl. tables

LLNA

- No signs of systemic toxicity were noticed.

- The test substance did not induce a statistically significant or biologically relevant response of the auricular lymph nodes when applied as 10%, 30% or 70% preparations in 1% aqueous Pluronic®.

- The statistically significant increase in ear weight at the concentration of 70% demonstrates the achievement of a skin irritation effect concentration.

Thus it is concluded that N,N'-Dimethylurea does not have a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information