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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:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Cymbopogon nardus, ext.
EC Number:
289-753-6
EC Name:
Cymbopogon nardus, ext.
Cas Number:
89998-15-2
IUPAC Name:
Cymbopogon nardus, ext.
Test material form:
liquid
Details on test material:
Source: J. Manheimer, Inc
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
J. Manheimer, Inc

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Interfauna UK, Shaw's Farm, Blackthorne, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17–21 g
- Housing: 4 animals per cage
- Diet (e.g. ad libitum): ad libitum (Porton Combined Diet, pelleted diet; Special Diets Services Ltd.,
Witham, United Kingdom)
- Water (e.g. ad libitum): ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19–25
- Humidity (%): 30–70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: EtOH:DEP 1:3
Concentration:
2.5%, 5%, 10%, 25% or 50% w/v
No. of animals per dose:
4
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: For each concentration of test material, a stimulation index (SI) relative to the concurrent vehicle treated control was calculated. The SI value for each te st material was calculated by dividing the mean dpm at a given dose level by the mean dpm of the vehicle control group. A material was considered a sensitizer if at least one concentration of the test material was observed to have an SI value of 3 or more.

ADMINISTRATION
- Test item: 25 μL of the test substance or vehicle control was applied to the back of the ear in 4 female mice per dose group. Dosing occurred daily for three consecutive days.
- 3H-Methyl Thymidine: on day 6 after the first application, the animals were injected intravenously (tail vein) with 250 μL phosphate buffered saline (PBS) containing 20 μCi of [3H] methyl thymidine.

TERMINATION
Five hours later, the mice were euthanized and the draining auricular lymph nodes were excised and pooled for each experimental group. Suspensions of the lymph node cells were prepared (200-mesh stainless steel gauze). The cell suspensions were washed thrice in PBS and precipitated overnight at 4 °C with 5% w/v trichloroacetic acid (TCA). The samples were pelleted by centrifugation, and resuspended in 1 ml of TCA. The incorporation of 3HTdR was measured by by scintillation counting
and expressed as disintegrations per minute (dpm) per lymph node for each experimental group.
Positive control substance(s):
not specified
Statistics:
The EC3 value was taken as a measure of relative sensitization potential for each material. Using two data points on the dose response curve, one immediately above and one below the SI value of three, the EC3 value was calculated utilizing the following equation presented by Basketter et al. (1999): EC3 = c+[(3-d)/(b-a)*(a-c) where the data points lying directly above and below the SI value of 3 on the dose–response curve have the coordinates (a,b) and (c,d), respectively.

Results and discussion

Positive control results:
Not specified

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
> 50
Variability:
not specified
Test group / Remarks:
tested up to 50%
Parameter:
SI
Value:
1.4
Test group / Remarks:
2.5% test substance
Parameter:
SI
Value:
5
Test group / Remarks:
5.0% test substance
Parameter:
SI
Value:
1.2
Test group / Remarks:
10% test substance
Parameter:
SI
Value:
1.2
Test group / Remarks:
25% test substance
Parameter:
SI
Value:
2.7
Test group / Remarks:
50% test substance
Cellular proliferation data / Observations:
Mean DPM at 0 (vehicle), 2.5, 5, 10, 25, and 50% were 925, 1300, 819, 1074, 1127 and 2528 respectively.

DETAILS ON STIMULATION INDEX CALCULATION
The SI value for each test material was calculated by dividing the mean dpm at a given dose level by the mean dpm of the vehicle control group.

EC3 CALCULATION
The EC3 value, or estimated concentration of test material required to elicit an SI of 3 or more, was derived from the dose–response data by linear extrapolation.

Applicant's summary and conclusion

Interpretation of results:
other: not sensitising
Remarks:
in accordance with Annex I of the CLP Regulation (1272/2008/EC).
Conclusions:
Under the conditions of this study, the Citronella Nardus Oil was not considered to be a sensitiser based on a derived EC3 value of >50%.
Executive summary:

The skin sensitisation potential of citronella nardus oil has been tested according to the OECD TG 429 (Local Lymph Node Assay) guideline. An amount of 25 μL of the test substance (at 2.5, 5, 10, 25 and 50%) or vehicle control was applied to the back of the ear in 4 female mice per dose group. Dosing occurred daily for three consecutive days. On day 6 after the first application, the animals were injected intravenously (tail vein) with 250 μL phosphate buffered saline (PBS) containing 20 μCi of [3H] methyl thymidine. Five hours later, the mice were euthanized and the draining auricular lymph nodes were excised and pooled for each experimental group. Suspensions of the lymph node cells were prepared (200-mesh stainless steel gauze). The cell suspensions were washed three times in PBS and precipitated overnight at 4 °C with 5% w/v trichloroacetic acid (TCA). The samples were pelleted by centrifugation, and resuspended in 1 ml of TCA. The incorporation of 3HTdR was measured by scintillation counting and expressed as disintegrations per minute (dpm) per lymph node for each experimental group. Stimulation Indices of 1300, 819, 1074, 1127, 2528 were observed at concentrations of 2.5,

5, 10, 25 and 50% respectively. The EC3 was calculated to be >50%. Based on these results the substance is not considered to be a skin sensitiser.

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