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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:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
OECD guideline 429 study with deviations: no data on physicochemical properties of test substance; no data on individual weights, housing conditions of test animals; no information on time course of onset and signs of toxicity

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Lack of data on test substance; no data on individual weights, housing conditions of test animals
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
(R)-p-mentha-1,8-diene
EC Number:
227-813-5
EC Name:
(R)-p-mentha-1,8-diene
Cas Number:
5989-27-5
Molecular formula:
C10H16
IUPAC Name:
4-isopropenyl-1-methylcyclohexene
Details on test material:
- Name of test material (as cited in study report): R(+)-limonene
- Source: Fluka Chemicals, Gillingham, UK
- Analytical purity: 99%

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Seralab, Oxon, UK
- Age at study initiation: 8-12 weeks
- Housing: four/cage on flushing metal racks
- Diet: SDS PCD pelleted diet; Special Diets Services, Witham, UK; ad libitum
- Water: ad libitum

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0 (vehicle), 25, 50 and 100% v/v
No. of animals per dose:
Four females
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay (Kimber and Basketter, 1992)
- Criteria used to consider a positive response: Stimulation index (SI) ≥ 3 (Kimber and Basketter, 1992).

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of mice received 25 µL of various concentrations of limonene in vehicle, or vehicle alone, on the dorsum of both ears daily for 3 consecutive days. Five days following the initiation of treatment, all mice were injected intravenously with 250 µL of 20 µCi 3H-TdR in PBS. Five hours later draining auricular lymph nodes were excised and a single cell suspension prepared. The incorporation of 3H-TdR was measured by β-scintillation counter (dpm/node and stimulation index [SI]) (Kimber and Basketter, 1992).

The estimated concentration of chemical required to induce a stimulation index of 3 relative to concurrent vehicle-treated controls (EC3 value) was derived by linear interpolation by method as explained by Basketter et al. (1999).

The EC3 value was calculated by interpolating between two points on the SI axis, one immediately above, and the other immediately below, the SI value of three. The vehicle-treated control value (SI=1) cannot be used for the latter. Where the data points falling immediately above and below the SI value of three have the co-ordinates (a,b) and (c,d), respectively, then the EC3 value may be calculated using the following equation (Basketter et al., 1999): EC3=c + [3−d) / (b−d)] × (a−c).
Statistics:
None

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
0 % v/v
Key result
Parameter:
SI
Value:
1.84
Test group / Remarks:
25 % v/v
Key result
Parameter:
SI
Value:
2.44
Test group / Remarks:
50 % v/v
Key result
Parameter:
SI
Value:
3.95
Test group / Remarks:
100 % v/v
Key result
Parameter:
EC3
Value:
68.5
Remarks on result:
other: EC3= estimated concentration to induce a Stimulation index of 3

Any other information on results incl. tables

Table 1: Local lymph node assay responses to limonene

Exposure Concentration (% v/v)

dpm/node

Stimulation index (SI)

EC3 (%)

0

476

1

68.5

25

877

1.84

50

1164

2.44

100

1882

3.95

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The calculated EC3-value for d-limonene was found to be 68.5%. Under these test conditions, d-limonene was found to be sensitising and should be clasified as skin sensitiser 1B according to the criteria of the CLP Regulation (EC) N° (1272-2008).
Executive summary:

In a skin sensitization study conducted following a method similar to OECD guidelines 429, four groups of CBA/Ca strain female mice (4/concentration) were exposed topically on the dorsum of both ears to 25 µL of vehicle (acetone/olive oil, 4:1 v/v), 25, 50 and 100% v/v of d-limonene daily for 3 consecutive days. Five days following initiation of exposure all mice were injected 250 µL of phosphate buffered saline (PBS) containing 20 µCi of [3H] methyl thymidine (3H-TdR). The animals were then sacrificed after 5 hours and suspension of lymph node cells was prepared from auricular lymph nodes. The incorporation of 3H-TdR was measured by β-scintillation counting as disintegrations per minute (dpm) per node for each experimental group. A Stimulation index of 3 or greater was considered to be indicative of a potential to cause contact sensitization.

D-limonene at exposure concentration of 0 (vehicle), 25, 50 and 100% v/v resulted in 476, 877, 1164 and 1882 dpm/node and stimulation index of 1, 1.84, 2.44 and 3.95, respectively.

The calculated EC3-value for d-limonene was found to be 68.5%. Under these test conditions, d-limonene was found to be sensitising and should be clasified as skin sensitiser according to the CLP Regulation (EC) N° (1272-2008).