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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:
From 21 February 2011 to 16 March 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; asaqsuate coherence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

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 lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-HEPTANOL
- Physical state: colorless liquid
- Analytical purity: 99.81%
- Lot/batch No.: 1103007
- Expiration date of the lot/batch: 28 October 2011
- Storage conditions of test material: at room temperature in fireproof storage cabinet.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the first day of the treatment period, the animals of the preliminary and main tests were approximately 9 weeks old
- Weight at study initiation: a mean body weight  standard deviation of 19.9 +/- 0.9 g
- Housing: individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained (except for the 5 hours
following the 3H-TdR injections) autoclaved sawdust (SICSA, Alfortville, France).
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered using a 0.22 micron filter) contained in bottles.
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): : 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: From: 23 February 2011 To: 16 March 2011.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10, 25, 50 and 100%.
No. of animals per dose:
4 animals per dose.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The test item was prepared at the chosen concentrations in AOO by successive dilutions. The dosage form preparation
were homogenized by vortex.
The reference item was dissolved in AOO at the concentration of 25% (v/v).
Fresh dosage form preparations were made extemporaneously of the day of each administration and the dosage form preparations were stored at room temperature prior to use.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay (LLNA) based on the design adopted by ICCVAM (Interagency Coordination Committee on
the Validation of Alternative Methods, ICCVAM 1999) and ECETOC (Monograph No. 78 Skin sensitization Testing for the Purpose of Hazard
Identification and Risk Assessment, September 2000), with the addition of the evaluation of local irritation
- Criteria used to consider a positive response: stimulation index of 3.

TREATMENT PREPARATION AND ADMINISTRATION:
The maximum concentration was selected according to the criteria specified in the International Guidelines and on the basis of the data obtained in
the preliminary test:
- the concentrations were selected from the concentration series 100 (for liquids), 50, 25, 10, 5, 2.5, 1, 0.5%, etc.
- the maximum concentration was the highest achievable concentration, whilst avoiding overt systemic toxicity and excessive local irritation.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: DPM per node: Group 1: Vehicle: 104.13 Group 2: Test item at 5%: 149.13 Group 3: Test item at 10%: 119.88 Group 4: Test item at 25%: 176.50 Group 5: Test item at 50%: 442.63 Group 6: Test item at 100%: 372.50
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM per group: Group 1: Vehicle: 833 Group 2: Test item at 5%: 1193 Group 3: Test item at 10%: 959.00 Group 4: Test item at 25%: 1412 Group 5: Test item at 50%: 3541 Group 6: Test item at 100%: 2980

Applicant's summary and conclusion

Interpretation of results:
other: weak sensitizer
Conclusions:
The test item, N-HEPTANOL, induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.
According to the EC3 value obtained, the test item should be considered as a weak sensitizer.
Executive summary:
The objective of this study was to evaluate the potential of the test item, N-HEPTANOL, to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel. This study was conducted in compliance with the principles of Good Laboratory Practice. MethodsA preliminary test was first performed in order to define the concentrations of test item to be used in the main test. In the main test, 28 female CBA/J mice were allocated to seven groups: ·          five treated groups of four animals receiving the test item at the concentration of 5%, 10%, 25%, 50% and 100%in a mixture Acetone/olive oil (4/1; v/v) (vehicle), ·          one negative control group of four animals receiving the vehicle, ·          one positive control group of four animals receiving the reference item, a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in a mixture acetone/olive oil (4/1; v/v). During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6. ResultsFollowing the solubility assay, acetone/olive oil (4/1, v/v) was chosen as vehicle A solution was obtained at the maximum tested concentration of 50%. Consequently, the concentrations selected for the preliminary test were 10%, 25%, 50% and 100%. Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (100%). Clinical signs and mortalityNo treatment-related mortality and no clinical signs were observed during the observation period.Local irritation As all acceptance criteria were met, this experiment was therefore considered valid.  
The EC3value is equal to 38%.
ConclusionThe test item, N-HEPTANOL, induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC3 value obtained, the test item should be considered as a weak sensitizer.