Tangor, Murcote, ext.

Skin sensitisation

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:

other: see 'Remark'

Remarks:

GLP study conducted in compliance with OECD guideline 429 with minor deviations: no certificate of analysis of the test substance.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted in compliance with OECD guideline 429 with minor deviations: no certificate of analysis of the test substance. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.

Justification for type of information:

Rationale & experimental data
For 5 oils studies are available, showing that these oils were not sensitising. This in contrast to the major constituent D-limonene, which is classified with R43. For mandarin oil and lime oil, no studies are available. The lime oil is lowest in D-limonene content, however, it also consists of a number of other constituents that are classified R43. In all oils, the content of constituents classified with R43 is >1%, which is the threshold mentioned for classification and labelling in the Dangerous Preparations Directive and CLP. Therefore, the results obtained in the studies with the citrus NCSs overrule the available studies of these NCSs. The result of D-limonene is read across to the other NCSs and the whole group is considered classified as R43 or H317.

Conclusion and dose descriptor
For 5 oils studies are available, showing that these oils were not sensitising. However, D-limonene, being classified as R43, is a known skin sensitizer. As D-limonene is present in all NCSs of this group at levels higher than the 1% threshold, this result is read across to the other NCSs and the whole group is considered classified as R43 or H317.

Classification & Labelling
Based on the available information the NCSs in this category are to be considered a skin-sensitising substance and should be classified as R43 or H317.

Reason / purpose for cross-reference:

read-across source

No. of animals per dose:

ale

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

Parameter:

SI

Remarks on result:

other: - Test material (10% v/v) = 1.3 - Test material (25% v/v) = 3.4 - Test material (50% v/v) = 4.0 - Test material (75% v/v) = 8.8 - Test material (100% v/v) = 6.5

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:

Under the test conditions, d-limonene is classified as ‘R43 May cause sensitisation by skin contact’, according to the criteria of Annex VI to the Directive 67/548/EEC and ‘Category 1’ according to the CLP Regulation (EC) N° (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, groups of mice (4 females/dose) were applied with 25 µL of d-limonene 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.

 

Under the test conditions, d-limonene is classified as ‘R43 May cause sensitisation by skin contact’, according to the criteria of Annex VI to the Directive 67/548/EEC and ‘Category 1’ according to the CLP Regulation (EC) N° (1272-2008).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

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

Test material information

Constituent 1

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:

(4R)-1-methyl-4-(prop-1-en-2-yl)cyclohexene

Constituent 2

Reference substance name:

Dipentene

EC Number:

205-341-0

EC Name:

Dipentene

Cas Number:

138-86-3

Molecular formula:

C10H16

IUPAC Name:

4-isopropenyl-1-methylcyclohexene

Details on test material:

- Name of test material (as cited in study report): d-Limonene
- Source: Millenium Speciality Chemicals
- Physical state: Colourless liquid
- Analytical purity: 99.7%
- Lot/batch No.: Not specified
- Storage condition of test material: Ambient temperature in the dark

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)

Results

Results 1

Parameter:

SI

Remarks on result:

other: - Test material (10% v/v) = 1.3 - Test material (25% v/v) = 3.4 - Test material (50% v/v) = 4.0 - Test material (75% v/v) = 8.8 - Test material (100% v/v) = 6.5

Results 2

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

Conclusions:

Under the test conditions, d-limonene is classified as Skin Sensitiser Category 1 according to the CLP Regulation (EC) N° (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, groups of mice (4 females/dose) were applied with 25 µL of d-limonene 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.

 

Under the test conditions, d-limonene is classified as Skin Sensitiser Category 1 according to the CLP Regulation (EC) N° (1272-2008).