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EC number: 907-808-0 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Density
- Particle size distribution (Granulometry)
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- Endpoint summary
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
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- Endocrine disrupter testing in aquatic vertebrates – in vivo
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- Toxicological Summary
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Endpoint summary
Administrative data
Description of key information
REACTION MASS OF 1,4-CINEOLE, 1,8-CINEOLE, DL-LIMONENE AND TERPINOLENE is classified as skin sensitizer because 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
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Justification for type of information:
- D-Limonene is one of the main constituent of REACTION MASS OF 1,4-CINEOLE, 1,8-CINEOLE, DL-LIMONENE AND TERPINOLENE so data on D-limonene can be extrapolated to the substance multiconstituent.
- Reason / purpose for cross-reference:
- read-across source
- 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
- Interpretation of results:
- Category 1B (indication of skin sensitising potential) based on GHS criteria
- 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:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Justification for type of information:
- D-Limonene is one of the main constituent of REACTION MASS OF 1,4-CINEOLE, 1,8-CINEOLE, DL-LIMONENE AND TERPINOLENE so data on D-limonene can be extrapolated to the substance multiconstituent.
- 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:
- 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
- Interpretation of results:
- Category 1B (indication of skin sensitising potential) based on GHS criteria
- 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.
Referenceopen allclose all
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 |
- 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 |
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 category 1B according to the criteria of Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.
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