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Description of key information

d-Limonene was found to be a skin sensitiser in two LLNA studies conducted according to OECD Guideline 429.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 07 to 18, 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted in compliance with OECD guideline 429 with minor deviations: no certificate of analysis of the test substance
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:
yes
Remarks:
no certificate of analysis of the test substance
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17.7-20 g
- Housing: Housed in a group of 4 mice (maximum) per cage
- Diet (e.g. ad libitum): Diet (RM1) (Special Diet Services Limited, Witham, UK), ad libitum
- Water (e.g. ad libitum): Mains water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70%
- Air changes (per hour): 15/hour (minimum)
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light
Vehicle:
other: ethanol/diethyl phthalate (3: 1 v/v)
Concentration:
0, 10, 25, 50, 75 or 100% v/v in ethanol/diethyl phthalate (3: 1 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
- Criteria used to consider a positive response: One or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of test material at concentrations of 0 (vehicle control), 10, 25, 50, 75 or 100% v/v in ethanol/diethyl phthalate (3: 1 v/v) were applied to the dorsal surface of each ear on Days 1, 2 and 3. On Day 6, 250 μL phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine (specific gravity: 2.0 Ci/mmol) was injected into the tail vein of each experimental mouse. Five hours later, all mice were killed by inhalation of halothane vapour followed by cervical dislocation and the draining auricular lymph node of each ear was excised into PBS. A single cell suspension of lymph node cells (LNC) was prepared by gentle separation through 200-mesh stainless steel gauze. LNC were washed thrice with 10 mL of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4 °C. Pellets were re-suspended in 1 mL TCA and transferred to scintillation vials containing 10 mL of scintillation fluid (Optiphase) for 3H-counting. The number of radioactive disintegrations per minute (DPM) was measured using a Packard Tri-Carb Liquid Scintillation Counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data
Positive control results:
Mean DPM
- Vehicle (acetone/olive oil 4:1 v/v) = 2263
- Test material (5 % w/v) = 5496
- Test material (10 % w/v) = 7571
- Test material (25 % w/v) = 24752

Stimulation Index
- Test material (5 % w/v) = 2.4
- Test material (10 % w/v) = 3.3
- Test material (25 % w/v) = 10.9
Key result
Parameter:
SI
Value:
ca. 1.3
Test group / Remarks:
Test material (10% v/v)
Key result
Parameter:
SI
Value:
ca. 3.4
Test group / Remarks:
Test material (25% v/v)
Key result
Parameter:
SI
Value:
ca. 4
Test group / Remarks:
Test material (50% v/v)
Key result
Parameter:
SI
Value:
ca. 8.8
Test group / Remarks:
Test material (75% v/v)
Key result
Parameter:
SI
Value:
ca. 6.5
Test group / Remarks:
Test material (100% v/v)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: - Vehicle = 2511 - Test material (10% v/v) = 3319 - Test material (25% v/v) = 8554 - Test material (50% v/v) = 9916 - Test material (75% v/v) = 22063 - Test material (100% v/v) = 16259

- Dermal reactions: Application of the test substance did not result in an increase in visual levels of irritancy to the skin on or around the ear area for the duration of the study.

Table 1 - Skin sensitisation potential of d-limonene in ethanol/diethyl phthalate (3:1 v/v)

 

Concentration of

test substance

(% v/v)

Number of lymph nodes

assayed     

Disintegrations

per min (dpm)

dpm

per lymph node

Test:control

ratio

0 (vehicle only) 

 8 

 2511 

 314 

Not applicable

 10 

 8 

 3319 

 415 

 1.3 

 25 

 8 

 8554 

 1069 

 3.4 

 50 

 8 

 9916 

 1240 

 4.0 

 75 

 8 

 22063 

 2758 

 8.8 

 100 

 8 

 16259 

 2032 

 6.5 

 EC3 

 Estimated to be 22% (5500 µg/cm2) 

 

Table 2 - Skin sensitisation potential of the positive control substance (hexylcinnamaldehyde)

 

Concentration of hexylcinnamaldehyde (% w/v)

Number of lymph nodes assayed     

Disintegrations per min (dpm)

dpm per lymph node

Test:control ratio

0 (vehicle only) 

 8 

 2263 

 283 

Not applicable

 5 

 8 

 5496 

 687 

 2.4 

 10 

 8 

 7571 

 946 

 3.3 

 25 

 8 

 24752 

 3094 

 10.9 

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
d-Limonene is classified as ‘R43 May cause sensitisation by skin contact’, according to Directive 67/548/EEC and in ‘Category 1’ according to CLP Regulation (EC) No 1272/2008.
Executive summary:

In a local lymph node assay performed in CBA/Ca strain mice according to OECD Guideline 429 and in compliance with GLP, 25 µL of d-limonene were applied to groups of mice (4 females/dose) at concentrations of 0 (vehicle control), 10, 25, 50, 75 or 100% v/v in ethanol/diethyl phthalate (3: 1 v/v) to the dorsal surface of each ear for three consecutive days. On Day 6, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and measured for radioactivity expressed as number of disintegrations per minute (DPM). Historic data of hexylcinnamaldehyde (5, 10 and 25 % w/v) in acetone/olive oil (4:1 v/v) was used as the data for positive control group.

 

Mean DPM for 0, 10, 25, 50, 75 or 100% d-limonene were observed to be 2511, 3319, 8554, 9916, 22063 or 16259 dpm, respectively. Stimulation index for 10, 25, 50, 75 or 100% d-limonene were calculated to be 1.3, 3.4, 4.0, 8.8 or 6.5, respectively. The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was 22% v/v (5500 µg/cm2). No increase in visual levels of irritancy to the ear skin was observed during the study.

 

Therefore, d-limonene is classified as ‘R43 May cause sensitisation by skin contact’, according to Directive 67/548/EEC and in ‘Category 1’ according to CLP Regulation (EC) No 1272/2008.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting 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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
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)
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

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
Positive control results:
Not applicable
Parameter:
SI
Remarks on result:
other: 1, 1.84, 2.44 and 3.95 at 0, 25, 50 and 100% v/v, respectively
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 476, 877, 1164 and 1882 at 0, 25, 50 and 100% v/v, respectively

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

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The calculated EC3-value for d-limonene was found to be 68.5%. Therefore, d-limonene was found to be sensitising and should be clasified as skin sensitiser according to Directive 67/548/EEC and CLP Regulation (EC) No 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 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%. Therefore, d-limonene was found to be sensitising and should be clasified as skin sensitiser according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

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

A study was identified as a key study (Betts, 2004). This study was performed according to OECD Guideline 429 and in compliance with GLP. This LLNA assay gave positive results, as SI for 10, 25, 50, 75 or 100% d-limonene were calculated to be 1.3, 3.4, 4.0, 8.8 or 6.5, respectively. The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was 22% v/v (5500 µg/cm²). No increase in visual levels of irritancy to the ear skin was observed during the study.

Moreover, this result was confirmed by another supporting study (Warbrick, 2001). In this OECD Guideline 429 GLP study, d-limonene at exposure concentration of 0 (vehicle), 25, 50 and 100% v/v resulted in 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% (17125 µg/cm²).

The mean EC3 of these two studies is 11312 µg/cm². This value is close to reported LLNA weighed mean EC3 values from five LLNA studies (=10075 µg/cm²). Moreover, these animal data are consistent with the NOEL (highest concentration tested with no sensitisation in human) found in humans, based on Human Repeated Insult Patch Test (10000 µg/cm²) (see section 7.10.4).  

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

d-Limonene was found to be sensitising and should be classified as skin sensitiser according to the criteria of Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.