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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Decycloxirane
- Batch: 10317704
- Purity: 97.3 - 97.4%
- Physical state, appearance: Colorless, clear liquid
- Storage conditions: At room temperature
- Expiry Date: 27 July 2014

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V. The Netherlands
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 18.4 - 22.1 g
- Housing: Makrolon Type II (pre-test) / III (main study), wire mesh top; granulated soft wood bedding
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days prior to start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2, 5, 10% (w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which could be technically used was 100% of the undiluted test item. Test item solution at different concentrations was prepared using acetone/olive oil (4+1, v/v) as vehicle.
- Irritation/systemic toxicity: To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6. At the tested concentrations the animals did not show any signs of systemic toxicity. From day 2 to 6, the animals showed an erythema of the ear skin (score ranged from 1 to 3). Furthermore, the measured ear weight of both animals exceeded the threshold of 25% recommended by OECD 429 in comparison with the historical controls (animal treated with 50%: 39.8%; animal treated with 100%: 52.2%). Therefore, a second pre-test was performed using test item concentrations of 10 and 25%. At these concentrations the animals did not show any signs of systemic toxicity. From day 2 to 6, the animal treated with 25% showed an erythema of the ear skin (score 1). Furthermore, the measured ear weight of animal 2 exceeded the threshold of 25% recommended by OECD 429 (30.6%). The animal treated with 10% did not show any sign of local skin irritation. Thus, the test item in the main study was assayed at 2, 5, and 10%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

MAIN STUDY
- Topical application: Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2, 5, and 10% in acetone/olive oil (4+1, v/v). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
- Administration of 3H-methyl-thymidine: Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 19.7 μCi of 3H-methyl thymidine (equivalent to 78.7 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
- Determination of incorporated 3HTdR: Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.
- Determination of Lymph Node Weight and Cell Count: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CAS®1, Schärfe System). The values obtained were taken down manually.
- Determination of Ear Weights: After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
- Criteria used to consider a positive response: The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index; Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
- An index was calculated for the lymph node weight and –cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle treated group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response and the cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation). A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as a statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test used for detection of possible outliers (performed with Microsoft Excel 2007). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2013. SI of 1.8, 3.2, and 5.8 were determined at 5, 10, and 25% substance, respectively.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: In this study Stimulation Indices (S.I.) of 0.68, 0.92, and 2.92 were determined with the test item at concentrations of 2, 5, and 10% in acetone/olive oil (4+1, v/v), respectively. A dose response was observed.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The Disintergration per minute (DPM) were determined to be 1893.1, 1290,7, 1744.1, and 5530.9 for the vehicle control group, 2%, 5% and 10% test group, respectively

Any other information on results incl. tables

No deaths occurred during the study period.

No signs of systemic toxicity were observed during the study period. On day 3 (1h after the third application), the animals treated with a test item concentration of 10% showed an erythema of the ear skin (score 1). Animals treated with 2 and 5% test item concentration did not show any signs of local skin irritation.

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

 

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant increase in lymph node weights and –cell counts was observed in the high dose group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The index determined for the lymph node cell count was slightly exceeded in the high dose group (index of 1.62).

 

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not reached or exceeded in any of the treated groups.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information