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: Well documented and reliable GLP and guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012
Reference Type:
other: "Corrections/additions to the final report"
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
yes
Remarks:
The method of determination of BrdU incorporation by ELISA presented in the OECD guidelines was altered to the method by flow cytometry.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
Please refer to confidential details on test material.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc
- Age at study initiation: 9-10 weeks old (main test)
- Weight at study initiation: 18.1-20.6 g (main test)
- Housing: In groups of not more than 5 animals/cage in polycarbonate cages
- Diet: CRF-1 (pelleted feed), Oriental Yeast Co., Ltd. (Chiba), ad libitum
- Water: Drinking water was provided to the animals ad libitum via glass water bottles fitted with sipper tubes.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22.3-24.2 °C
- Humidity: 49.4-69.0 %
- Photoperiod: 12-hour light-dark cycle

IN-LIFE DATES:
pretest - from: 2011-11-2 To: 2011-11-7
main test - from: 2011-11-10 To: 2011-11-15

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Remarks:
(DMF)
Concentration:
Pre and main test:
10, 25 and 50 % (w/v)
No. of animals per dose:
Main-test:
5 animals per group
Pre-test:
2 animals per group
Details on study design:
PRE-SCREENING TEST
The test item formulations in DMF at concentrations of 10,25 or 50 % (w/v) were adminstered once per day to both ear auricles (25 µL/ear) of each 2 mice/group on Days 1-3. The animals were then observed for a further 3-day period (Days 4-6). Clinical signs including skin irritation (erythema formulation), body weights and auricular thickness were recorded repeatedly during the study period. The auricular thickness was measured using a thickness gauge on Days 1 (pre-dosing), 3 (prior to the third dosing (approx. 48 hours after the first dosing)), and 6. The concentrations in the main test were selected based on the results of the pre-screen test.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
At nearly the same time on Days 1, 2 and 3, the test item dosing formulations were applied to the outside of both ears at a volume of 25 μL/ear using micropipettes.

Dosing with BrdU
The 10 mg/mL PBS solution containing BrdU was administered intraperitoneally to the animals on Day 5 at a dose level of 5 mg/animal (0.5 mL/animal) using sterile disposable 26 G needles.

OBSERVATIONS AND EXAMINATIONS
Clinical observations
From Day 1 to the day of excision of the auricular lymph nodes (Day 6). Twice daily on Day 1-3 and Day 5, and once daily during the other periods.

Body weight
Day 1(pre-dosing), 4 and 6

Excision of the auricular lymph nodes
On Day 6 the animals were euthanized by inhalation of carbon dioxide. The right and left auricular lymph nodes were excised and stored in ice-chilled PBS.

Confirmation of irritation
Prior to excision of the auricular lymph nodes, the treated areas (ears) were observed for erythema or edema. In addition, circular samples were collected from the top of both ears using a punch for biopsy (8 mm in diameter). The samples were weighed in pairs.

Measurement of auricular lymph node weights
Weights of the right and left auricular lymph nodes were measured in pairs. Organ-to-terminal body weight ratios (relative weights) were calculated based on the body weights measured on Day 6.

Preparation of lymphocyte suspensions
On Day 6 the right and left auricular lymph nodes from each animal were smashed and the lymphocyte suspension was filtered using a 70 μm mesh disposable filter. The lymphocyte cells in this suspension were counted using the ADVIA 120 Hematology System.

Determination of BrdU incorporation by flow cytometry
Cell suspensions were prepared using stain buffer and lymphocytes were counted using a hemocytometer. The lymphocytes were fixed and the cell membrane was permeabilized. The lymphocytes were treated with deoxyribonuclease (DNase) to expose BrdU epitopes. The cells were immunofluorescently-stained using fluorochrome conjugated anti-BrdU antibody. DNA in the lymphocytes was stained with 7-amino-actinomycin D (7-AAD). Then, these suspensions were used as samples for determination. The ratios of BrdU-positive lymphocytes were determined/calculated using a flow cytometer.

Calculation of the stimulation index (SI)
The stimulation index (SI) was calculated for each treated group according to the formula below:
SI = Mean number of BrdU-positive cells in the test item treated group / Mean number of BrdU-positive cells in the vehicle control group.

Criteria used to consider a positive response:
When SI > 3, the test item was considered to be positive. Since all SI values for the 10, 25 and 50 % (w/v) test item dosing formulations were ≤ 3, the EC3 concentration at which SI was expected to be 3 was not calculated.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis
For quantitative data, mean group values and standard deviations were calculated and analyzed for homogeneity of variance using Bartlett’s test (5% level of significance). When the variance was homogeneous between the groups, Williams test premised on a monotonic increasing dose-response relationship was conducted. When significant differences were not found by Williams test, mean group values were compared between the vehicle control and each test item treated group using Dunnett’s test. When the variance was heterogeneous based on Bartlett’s test, Shirley-Williams test premised on a monotonic increasing dose-response relationship was conducted. When significant differences were not found by Shirley-Williams test, a mean rank test of the Dunnett type was conducted between the vehicle control and each test item treated group. Dunnett´s test was conducted by two-tailed and Williams and Shirley-Williams tests were conducted by one-tailed. Differences from the vehicle control group were evaluated at the 5% level of significance and presented as p < 0.05 or p < 0.01.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The SI values were 1.1, 1.0 and 1.0 in the 10, 25 and 50 % (w/v) groups, respectively. Increases in the ratio and count of BrdU-positive lymphocyte cells were noted in the positive control group as compared to the vehicle control group. The SI value in the positive control group was 7.4.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
BrdU-positive lymphocytes No statistically significant changes were noted in the lymphocyte count or in the ratio or count of BrdU-positive lymphocyte cells in any treated group as compared to the vehicle control group. Increases in the ratio and count of BrdU-positive lymphocyte cells were noted in 1 animal in the 10 % (w/v) group. However, no changes were noted in any other animal in this group or in any animal in the 25 or 50 % (w/v) group. Therefore, these changes were considered to be incidental changes with no dose-relationship.

Any other information on results incl. tables

Pre-screening test

- Irritation: No skin irritation at the application area, was observed in any animal (2 animals/group) in the 10, 25 or 50 % (w/v) group throughout the observation period.

- Clinical signs: no effect

- Changes in body weight: no effect

- Auricular thickness: no effect

Main test

Clinical observations

No clinical signs, including skin irritation at the application area, were observed in the 10, 25 or 50 % (w/v) group throughout the observation period. In the positive control group, very slight erythema was observed in both ear auricles of all 5 animals at 1 hour post-dosing on Days 2 and 3.

Body weight

No statistically significant body weight changes were noted in any treated group as compared to the vehicle control group. Furthermore, there were no notable body weight changes in any animal in the positive control group.

Irritation of the auricles at excision of the auricular lymph nodes and ear weights

No skin irritation at the application area was observed in any animal in any group. No statistically significant changes were noted in the ear weights between the vehicle control and any treated group. In the positive control group, increased ear weights were noted as compared to the vehicle control group.

Auricular lymph node weights

No statistically significant changes were noted in the absolute or relative weight of the auricular lymph nodes in any treated group as compared to the vehicle control group. In the positive control group, increases in the absolute and relative weights of the auricular lymph nodes were noted as compared to the vehicle control group.

- All dose formulations used in this study were formulated appropriately as evidenced by analytically determined mean concentrations of 10.17, 25.42 and 50.59 % (w/v; n = 2) as compared to the nominal concentrations of 10, 25 and 50 % (w/v), respectively.

Table 1: BrdU-positive lymphocytes and SI values (LLNA-FC method) on day 6

Group

BrdU-positive lymphocytes

mean ± SD (n = 5)
[× 10³/mL]

SI

NC (DMF)

20.42 ± 5.15

1.0

10 % (w/v) test item

21.85 ± 15.91

1.1

25 % (w/v) test item

20.42 ± 3.90

1.0

50 % (w/v) test item

20.35 ± 7.91

1.0

PC (25 % (v/v) HCA

151.16 ± 39.12

7.4

BrdU: Bromodeoxyuridine, DMF: N,N-Dimethylformamide, HCA:α-Hexyl cinnamic aldehyde, LLNA-FC: local lymph node assay – flow cytometry,

n: number of samples NC: negative control, PC: positive control, SD: standard deviation, SI: stimulation index

Applicant's summary and conclusion