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Administrative data

Description of key information

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, nerol induced positive response.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 August - 20 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline 429 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Preliminary test - Approximately 12 weeks; Main test - Approximately 8 weeks
- Weight at study initiation: Preliminary test - 22.7 g (21.8 - 23.2 g); Main test - 19.7 g (18 - 21.6 g)
- Housing: Animals were housed by groups of two (preliminary test) or four (main test) in polycarbonate cages (Tecniplast 1145T, 435 cm^2) containing autoclaved sawdust (SICSA, Alfortville, France).
- Diet: SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap water (filtered using a 0.22 µm filter), ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary test: 10, 25, 50 and 100 %
Main test: 5, 10, 25, 50 and 100 %
No. of animals per dose:
Preliminary test: 1 dose/ear (10 and 25 % in left and right ears of 2 females, respectively; 50 and 100 % in left and right ears of 2 females, respectively)
Main test: 4 females/dose
Details on study design:
PRELIMINARY TEST:
- Compound solubility: Test item was soluble at the concentration of 50 % in Acetone/Olive Oil (4/1; v/v).
- Irritation: Dryness was observed on Day 6 in right ear of all females treated at 25 and 100 %. No notable increase in ear thickness was observed at any tested concentrations. The highest concentration retained for the main test was therefore 100 %.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT: Animals were allocated to the groups using a manual randomization procedure
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item is considered as a skin sensitizer when the SI for a dose group is ≥ 3 together with consideration of a dose-response relationship. Other relevant criteria such as cellularity, radioactivity levels and ear thickness are also taken into account to evaluate the data.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of control and test item were applied to the dorsal surface of both ears on Days 1, 2 and 3. On Day 6, 250 µL of 0.9 % NaCl containing 20 μCi of 3H-TdR (specific activity of 20 Ci/mmol) was injected into the tail vein of each experimental mouse. Five hours later, all mice were sacrificed by an intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group. A single cell suspension of auricular lymph node cell (ALNC) was prepared by mechanical disaggregation in Petri dishes using the plunger of a syringe. Cell suspensions were washed with 0.9 % NaCl and precipitated with 5 % (w/v) trichloroacetic acid (TCA) at 4 °C. Pellets were re-suspended in 1 mL TCA and 3 mL of Ultima GoldxR scintillation fluid (Packard) was added in order to measure incorporation of 3H-TdR using β-scintillation counting. The results were expressed as disintegrations per minute (dpm) per group and as dpm/node.

Stimulation Index (SI) were calculated according to the following formula:
SI = dpm per node of the treated group / dpm per node of the vehicle control group
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data
Positive control results:
Stimulation index for α-hexylcinnamaldehyde at 25 % v/v was 4.30 and classified as a sensitiser.
Parameter:
SI
Remarks on result:
other: Stimulation index for 5, 10, 25, 50 and 100 % were 1.10, 1.77, 3.16, 5.12 and 2.47, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: - DPM per group for vehicle, 5, 10, 25, 50 and 100 % were 4203, 4639, 7449, 13264, 21516 and 10386, respectively. - DPM per node for vehicle, 5, 10, 25, 50 and 100 % were 525.38, 579.88, 931.13, 1658.00, 2689.50 and 1298.25, respectively.
Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Nerol induced positive response in a LLNA study. Therefore, it is classified as "R43: May cause sensitization by skin contact" according to Directive 67/548/EEC and nerol is classified in category 1 according to CLP Regulation (EC) N° 1272/2008.
Executive summary:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, groups of CBA/J mice (4 females/dose) were topically applied with 25 µL of nerol at concentrations of 5, 10, 25, 50 and 100% to the dorsal surface of both ears for three consecutive days. Vehicle control group of four females received the vehicle (acetone/olive oil (4/1; v/v)). 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 results were expressed as disintegrations per minute (dpm) per group; dpm/node and the obtained values were used to calculate Stimulation Indices (SI). Animals were observed for mortality, clinical signs and body weight during the study. The test concentrations for the main study were determined from a preliminary study (10 and 25% in left and right ears of 2 females, respectively; 50 and 100% in left and right ears of 2 females, respectively).

No mortality and no clinical signs were observed during the observation period. Body weight of the animals was unaffected by the test item treatment.Erythema was observed on day 6 in 2/4 females treated at the concentration of 50 % and in all females treated at 100%. Dryness of ear skin was noted in all females treated at 25, 50 and 100% on day 6. No notable increase in ear thickness was observed at 5, 10, 25 and 50%. A slight increase in ear thickness of 10% was observed in females treated at 100% (mean value = 10.42%). A significant and dose-related lymphoproliferation (SI > 3) was noted at 25 and 50 %. The decrease of the SI noted at the concentration of 100%, even if erythema were noted in all mice at this concentration, may be explained by the test item being applied as supplied and not diluted in the vehicle. Therefore, the availability of test item over the ear skin and its skin penetration may be lowered under these conditions. In absence of slight local irritation at 25 and 50%, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value was equal to 23%. The SI of the positive control (α- hexylcinnamaldehyde) was > 3; this experiment was therefore considered valid.

 

Therefore, nerol induced positive response. Nerol is classified "R43: May cause sensitization by skin contact" according to Directive 67/548/EEC and nerol is clasified in category 1 (sub-category 1B) according to CLP Regulation (EC) N° 1272/2008.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, groups of CBA/J mice (4 females/dose) were topically applied with 25 µL of test item, nerol at concentrations of 5, 10, 25, 50 and 100% to the dorsal surface of both ears for three consecutive days. Vehicle control group of four females received the vehicle (acetone/olive oil (4/1; v/v)). 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 results were expressed as disintegrations per minute (dpm) per group; dpm/node and the obtained values were used to calculate Stimulation Indices (SI). Animals were observed for mortality, clinical signs and body weight during the study. The test concentrations for the main study were determined from a preliminary study (10 and 25% in left and right ears of 2 females, respectively; 50 and 100% in left and right ears of 2 females, respectively).

No mortality and no clinical signs were observed during the observation period. Body weight of the animals was unaffected by the test item treatment. Erythema was observed on day 6 in 2/4 females treated at the concentration of 50% and in all females treated at 100%. Dryness of ear skin was noted in all females treated at 25, 50 and 100% on day 6. No notable increase in ear thickness was observed at 5, 10, 25 and 50%. A slight increase in ear thickness of 10% was observed in females treated at 100% (mean value = 10.42%). A significant and dose-related lymphoproliferation (SI > 3) was noted at 25 and 50%. The decrease of the SI noted at the concentration of 100%, even if erythema were noted in all mice at this concentration, may be explained by the test item being applied as supplied and not diluted in the vehicle. Therefore, the availability of test item over the ear skin and its skin penetration may be lowered under these conditions. In absence of slight local irritation at 25 and 50%, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value was equal to 23%. The SI of the positive control (α- hexylcinnamaldehyde) was > 3; this experiment was therefore considered valid.


Migrated from Short description of key information:
In a LLNA, nerol induced skin sensitisation (EC3=23%).

Justification for selection of skin sensitisation endpoint:
Only one study available for this endpoint

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Nerol induced positive response in a LLNA study (EC3 = 23%). Therefore it is classified as "R43: May cause sensitization by skin contact" according to Directive 67/548/EEC and nerol is classified in category 1B according to CLP Regulation (EC) N° 1272/2008.