Orange, sweet, ext.

Endpoint summary

Description of key information

Read across based on d-limonene content: sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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. 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.

Reason / purpose for cross-reference:

reference to same study

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)

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:

1.3

Test group / Remarks:

Test material (10% v/v)

Key result

Parameter:

SI

Value:

3.4

Test group / Remarks:

25% (v/v)

Key result

Parameter:

SI

Value:

4

Test group / Remarks:

50% (v/v)

Key result

Parameter:

SI

Value:

8.8

Test group / Remarks:

75% (v/v)

Key result

Parameter:

SI

Value:

6.5

Test group / Remarks:

100% (v/v)

Cellular proliferation data / Observations:

Disintegrations per minute (DPM)
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:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

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

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See attached justification

Reason / purpose for cross-reference:

read-across source

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:

1.3

Test group / Remarks:

Test material (10% v/v)

Key result

Parameter:

SI

Value:

3.4

Test group / Remarks:

25% (v/v)

Key result

Parameter:

SI

Value:

4

Test group / Remarks:

50% (v/v)

Key result

Parameter:

SI

Value:

8.8

Test group / Remarks:

75% (v/v)

Key result

Parameter:

SI

Value:

6.5

Test group / Remarks:

100% (v/v)

Cellular proliferation data / Observations:

Disintegrations per minute (DPM)
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:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The key study is a local lymph node assay performed with major constituent limonene which was read across to orange oil. The study was performed in CBA/Ca strain mice according to OECD guideline 429 and in compliance with GLP.

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).

 

In a supporting study, a human patch test was performed in which human volunteers were exposed to orange oil repeatedly during three weeks (study summary included in 7.10.4). Skin reactions were noted in 4% of the subjects at 24 hr, no reaction was noted at any other reading (48 hr, 72 hr) or in the control group. It can be concluded that orange oil does not have sin sensitising properties. However, as the major constituent of

orange oil is limonene, which is classified for skin sensitisation, orange oil will be classified for skin sensitisation.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the skin sensitising properties of the major constituent of orange oil, d-limonene, orange oil has to be labelled as skin sensitiser in accordance with the criteria outlined in Annex I of 1272/2008/EC (CLP/EU-GHS)