Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 10, 2006 to January 17, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed according to OECD Guideline 429 and EU method B.42, with GLP.

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
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Erythromycin A-9 Oxime (E) Hydrochloride.
- Physical state: white powder.
- Lot/batch No.: B5310059
- Storage condition of test material: room temperature.
- Other:
pH: 6.16 (1% solution in deionised water, w/v, determined with a pH-Meter WTW pH340)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelman GmbH, D-33176 Borchen.
- Age at study initiation: About 8 weeks at the first administration.
- Weight at study initiation: 17.7 - 21.9 g.
- Housing: Group caging in Makrolon cages type IV (33 cm x 55 cm bottom area, 20 cm height). Wire meshes lids. Sanitation cages once a week..
- Diet (e.g. ad libitum): Altromin 1324 forte, gamma irradiated with 25 kGy 60Co, ad libitum (Producer: Altromin GmbH, D-32791 Lage) Random samples of feed are analysed for contaminants by Altromin.
- Water (e.g. ad libitum): Tap water offered in Makrolon bottles with stainless steel cannulae ad libitum.
- Acclimation period: 5 days; clipping of hair and washing with antibiotic solution was performed during acclimatisation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Average of 22.0 ºC (continous control and recording)
- Humidity (%): Average of 48.1 % (continous control and recording)
- Photoperiod (hrs dark / hrs light): Artificial light from 6 a.m. to 6 p.m.
- air exchange: Approx. 12/h.

Study design: in vivo (LLNA)

Vehicle:
other: DMSO (w/w)
Concentration:
Group A (low dose): 5 % (w/w) solution of test substance in DMSO.
Group B (MID dose): 10 % (w/w) solution of test substance in DMSO.
Group C (high dose): 26.5 % (w/w) solution of test substance in DMSO.

Group P (positive control): 25% (v/v) solution of hexyl cinnamic aldehyde in acetone:olive oil (4:1, v/v)
Group K (negative control): DMSO
No. of animals per dose:
5 females.
Details on study design:
RANGE FINDING TESTS:
A range finding study was performed with two animals/concentration with following concentrations: 26.5 % and 10 % (26.5 % is the maximal achievable concentration that avoids overt systemic toxicity and excessive local irritation). Animals were treated with 25 µL test substance on the dorsum of each ear on 3 consecutive days. Ear thickness and body weight were measured on Day 1 before the first administration and on Day 4 about 24 hours after the last administration. In animals of both groups test substance remnants at the application sites were observed on Days 3 and 4, which is of no relevance for the results of a skin sensitisation study. None of the animals showed overt systemic toxicity, excessive local skin irritation at the application sites or an important increase in ear thickness in the range finding study. Therefore 26.5 % was chosen as highest test substance concentration and ear thickness measurement was not performed in the main study. As mid concentration 10 % was chosen, since 26.5 % is very near at the proposed 25%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: According to the guidelines the decission process with regard to a positive response includes a stimulation index of equal to or greater than 3, together with consideration of dose-response.

TREATMENT PREPARATION AND ADMINISTRATION: The test substance solutions were prepared freshly on each day of administration by the solution of test substance in DMSO (w/w) using an ultrasonic bath for the highest concentration. No analysis of test substance preparations were made. The test substance was solved in DMSO and was administered to 3 groups of 5 female. Administration was performed epicoutaneously to the dorsal surface of both ears, once a day, on 3 consecutive days. The volume applied was 25 µL. 5 days after the first topical administration, each animal received 20 µCi 3HTdR by slow intravenous administration. Approximately 5 hours after 3HTdR injection all animals were sacrificed and the draining auricular lymph nodes were rapidly excised. The lymph nodes of each group were pooled in PBS. A single cell suspension of lymph node cells was prepared by gentle mechanical disaggregation of the pooled lymph nodes through a 70 µm cell strainer. 3HTdR incorporation was determined with a β-scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Application of 25 % HCA in AOO resulted in an SI of 3.5. This result proves the sensitivity of the strain of animals used anmd the reliability of the experimental technique.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Group A (low dose): 4.1 Group B (mid dose): 6.6 Group C (high dose): 7.9 Group P (positive control): 3.5 Group K (negative control): 1
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Group A (low dose): 36177 dpm Group B (mid dose): 58470 dpm Group C (high dose): 69441 dpm Group P (positive control): 30431 dpm Group K (negative control): 8796 dpm

Any other information on results incl. tables

All animals survived till the end of the study.

No abnormal behaviour or clinical signs were detected during the experiment in the animals.

No local irritations were observed at the application sites of all animals of all test substance groups and both control groups throughout the whole study.

Body masses and body mass gains of all animals were in the range to be expected from animals of the same strain, sex and age. Body weight loss was noted in 2/5 animals in the high dose treatment group.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The Stimulation Index of the tested test substance concentrations were 4.1 (low dose), 6.6 (mid dose) and 7.9 (high dose) (SIs > 3). According to the OECD Guideline 429 and the EU Method B.42, the test substance is regarded as a sensitizer in the LLNA.
Executive summary:

The Local Lymph Node Assay was performed according to OECD Guideline 429 and the B.42 EU Method, to evaluate a possible sensitizing potential of the test substance. The test substance was solved in DMSO and was administered to three groups of 5 female CBA/Ca mice. Administration was performed epicutaneously to the dorsal surface of both ears, once a day on 3 consecutive days. The volume administered was 25 µL/ear. The concentrations used were: 5, 10 and 26.5 % w/w (solution of the test substance in DMSO). Two groups with 5 animals each served as positive and negative controls. Both control substances were administered under identical conditions as the test substances. The SIs of the tested test substance concentrations were 4.1 (low dose), 6.6 (mid dose) and 7.9 (high dose) (SIs > 3). According to the OECD Guideline 429 and the EU Method B.42, the test substance is regarded as a sensitizer in the LLNA.