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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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 (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid, melt
Details on test material:
Please refer to the section "confidential details on test material" below.

In vivo test system

Test animals

Species:
mouse
Strain:
other: Pre-test: Mice, CBA/CaOlaHsd; Main Study: Mice, CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Pre-test: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
Main experiment: Charles River UK, Manston Road, Margate, Kent CT9 4LT, United Kingdom
- Age at study initiation: 8 – 9 weeks old (beginning of treatment)
- Weight at study initiation: 21.6 - 17.6 g (main experiment), 21.7 and 20.8 g (pre-test)
- Housing: Group
Cage Type: Makrolon Type II (pre-test)/ III (main study), with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
Bedding: Granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)
- Diet: Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands)
- Water: Tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination.
- Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2°C
- Humidity: 31 – 65% (acclimation period); 45 – 65% (main study)
- Photoperiod (hrs dark / hrs light): Artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Pre-test: 50 % solution in acetone:olive oil (4+1 v/v)
Main experiment: Vehicle Control (acetone:olive oil (4+1 v/v)); 10%, 25%, 50% solution
No. of animals per dose:
Pre-test: 2 animals
Main experiment: 5 animals per dose group
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429.
- Irritation: A pre-test was performed in two animals.
- Lymph node proliferation response: Eventual signs of local irritation were documented and the ear thickness was determined.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Proliferative response of the lymph node cells
- Criteria used to consider a positive response:
(i) incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice (stimulation index)
(ii) data are compatible with a conventional dose response
(iii) cutoff-value for the lymph node cell count index: 1.55 (based on BALB/c mice)
(iv) cutoff-value for the ear weight index: 1.1 (based on BALB/c mice)

TREATMENT PREPARATION AND ADMINISTRATION:
- The test item mixed with acetone:olive oil (4+1 v/v) was added to achieve the required test substance concentration. The different test substance concentrations were prepared individually. The preparations were made freshly before each dosing occasion.
- A concentration control analysis of all three doses (and if applicable a homogeneity analysis of the lowest dose) was performed as a separate study.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated for body weight, ear weight, lymph node weight and lymph node cell count, and for DPM values (group mean DPM ± standard deviation). For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-ANOVA was used as 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). However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Vehicle Control: 1.00 10% solution: 0.87 25% solution: 0.85 50% solution: 1.22
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Mean DPM per animal (2 lymph nodes)*: Vehicle Control: 1173.5 +/- 399.2 10% solution: 1016.1 +/- 443.9 25% solution: 1002.3 +/- 502.4 50% solution: 1428.3 +/- 696.7 *) Mean DPM/animal was determined by dividing the sum of the calculated values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

Any other information on results incl. tables

No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. 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 cutoff-value for the lymph node cell count index of 1.55 (reported for BALB/c mice) for a positive response was not exceeded in any of the tested dose groups. For BALB/c mice, a threshold for the ear weight index of 1.1 was reported for a positive response. None of the indices determined for the test item treated groups exceeded this threshold.

Applicant's summary and conclusion