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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:
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
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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

Results and discussion

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

In vivo (LLNA)

Resultsopen allclose all
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

Any other information on results incl. tables

- 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 

Applicant's summary and conclusion

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.