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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:
2010-07-01 to 2010-07-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to OECD 429 (2010), EU B.42 (Commission directive 2004/73/EC) and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Remarks:
Harlan Laboratories Ltd.
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
(R*,R*)-α,4-dimethyl-α-(4-methyl-3-pentenyl)cyclohex-3-ene-1-methanol
EC Number:
208-205-9
EC Name:
(R*,R*)-α,4-dimethyl-α-(4-methyl-3-pentenyl)cyclohex-3-ene-1-methanol
Cas Number:
515-69-5
Molecular formula:
C15H26O
IUPAC Name:
6-methyl-2-(4-methylcyclohex-3-en-1-yl)hept-5-en-2-ol
Test material form:
other: liquid
Details on test material:
(+/-) alpha-Bisabolol

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., Maasheseweg 87c, 5804 AB Venray; Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: 18.6 – 24.1 g
- Housing: Group housing in Makrolon Type-II cages with standard softwood bedding.
- Diet: Pelleted standard Kliba 3433 mouse maintenance diet, available ad libitum.
- Water: Community tap water from Itingen/Switzerland, available ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30 - 70%
- Air changes: 10 - 15 air changes per hour
- Photoperiod: 12 hour fluorescent light/12 hour dark cycle

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0% (vehicle), 10%, 25% and 50%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by the guideline OECD 429. The highest test item concentration which can be technically used, was a 50% (w/w) solution in acetone/olive oil (4:1 v/v).
- Irritation: Two mice were treated with concentrations of 100% (undiluted) and 50% (w/v) in acetone/olive oil (4:1 v/v) on three consecutive days. In the pre-test clinical signs were recorded within 4 ± 2 hours and 24 ± 4 hours after each application as well as on the day corresponding to the day of preparation of the main experiment. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity. However at the undiluted concentration, a slight erythema was observed at the head of the animal treated with undiluted test item. As a consequence, the sponsor decided not to test the undiluted concentration in the main study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (dpm/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of the test groups relative to that recorded for the control group (Stimulation Index S.I.). Before dpm/lymph node values were determined, the mean scintillation-background dpm was subtracted from test and control raw data.

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by topical application to the dorsal surface of each ear lobe with the test item at concentrations of 50%, 25% or 10% (w/v) in acetone/olive oil (4:1 v/v). The application volume of 25 μL was spread over the entire dorsal surface of each ear lobe (Ø about 8 mm) once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle.
Five days after the first topical application, all mice were administered with 250 μL of phosphate-buffered saline (PBS) containing 80.46 μCi/mL 3HTdR (equal to 20.11 μCi 3HTdR) by intravenous injection via a tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of CO2.
The draining lymph nodes were rapidly excised and pooled in PBS for each experimental group (8 lymph nodes per group). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through a gauze (200 μm mesh size) and transferred in centrifugation vials. After washing twice with approximately 10 mL phosphate buffered saline the lymph node cells were resuspended in approximately 3 mL 5% trichloroacetic acid and incubated at approximately + 5 °C for at least 18 hours for precipitation of macromolecules. The supernatant was removed by centrifugation. The precipitates were then resuspended in 1 mL 5% trichloroacetic acid and transferred to glass scintillation vials with 10 mL of ‘Irga-Safe Plus’ scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5% trichloroacetic acid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
For the body weight mean values standard deviations were calculated.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: In this study S.I. of 1.9, 4.0 and 7.9 were determined with the test item dissolved in acetone/olive oil (4:1 v/v) at concentrations of 10%, 25% and 50% (w/v), respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM: (Control): 1657 Group 1 (10%): 3107 Group 2 (25%): 6482 Group 3 (50%): 12708

Any other information on results incl. tables

In this study S.I. of 1.9, 4.0 and 7.9 were determined with the test item dissolved in acetone/olive oil (4:1 v/v) at concentrations of 10%, 25% and 50% (w/v), respectively. The EC3 value calculated was 17.9%.

Test article concentration %

 

Measurement dpm

dpm-BG

Number of lymph nodes

dpm per lymph nodea)

S.I.

 

BG I

47

-

-

-

-

 

BG II

61

-

-

-

-

 

CG

1657

1603

8

200

-

10

TG 1

3107

3053

8

382

1.9

25

TG 2

6482

6428

8

804

4.0

50

TG 3

12708

12654

8

1582

7.9

BG = Background (1 mL 5% trichloroacetic acid) in duplicates

Control = Control Group

TG = Test Item Group

S.I. = Stimulation Index (Ratio = dpm/lymph node TG / dpm/lymph node Control)

a) = Since the lymph nodes of the animals of a dose group were pooled, dpm/lymph node was determined by dividing the measured value by the number of lymph nodes pooled

Viability / Mortality: No deaths occurred during the study period.

Clinical Signs: Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period.

Body Weights: The body weights of the animals were within the range commonly recorded for animals of the strain and age.

Applicant's summary and conclusion

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