Registration Dossier

Administrative data

Description of key information

In a GLP study conducted in compliance with OECD guideline 429 with d-limonene in ethanol/diethyl phthalate, an EC3 value of 22% was obtained.

In an study conducted similarly to OECD guideline 429 with limonene in acetone/olive oil, an EC3 value of 68.5% was obtained.

In an study conducted similarly to OECD guideline 429 with beta-phellandrene in acetone/olive oil, an EC3 value of 5.6% was obtained.

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:
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 constituents of multiconstituent substance REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE. Therefore, data on d-limonene can be used for extrapolation to REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE. See read-across justification document in section 13.
Reason / purpose:
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
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:
d-Limonene is classified as ‘R43 May cause sensitisation by skin contact’, according toDirective 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, 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 the 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% of 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:
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 constituents of multiconstituent substance REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE. Therefore, data on d-limonene can be used for extrapolation to REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE. See read-across justification document in section 13.
Reason / purpose:
read-across source
Positive control results:
Not applicable
Key result
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 d-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:
Beta-phellandrene is one of the main constituents of multiconstituent substance REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE. Therefore, data on beta-phellandrene can be used for extrapolation to REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE. See read-across justification document in section 13.
Reason / purpose:
read-across source
Principles of method if other than guideline:
Not applicable
Positive control results:
EC3 values of cinnamic alcohol and cinnamic aldehyde were 1.5 M (20.1%) and 0.10 M (1.3%), respectively.
Key result
Parameter:
EC3
Remarks:
in % w/v
Value:
5.6
Cellular proliferation data / Observations:
Refer Table 7.4.1/1

Table 7.4.1/1: LLNA responses

 

Test concentration (% w/v)

[3H]thymidine incorporation (dpm/lymph node)

SI

EC3 value

Classification

% w/v

M

0

478

5.6

0.41

Moderate

1

540

1.1

10

2283

4.8

20

10964

23

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test material is classified as skin sensitiser, sub-category 1B, according to the GHS and CLP Regulation (EC) No 1272/2008.
Executive summary:

In a local lymph node assay, groups of mice (4/dose) were exposed to the test material at concentrations of 0 (vehicle control), 1, 10 or 20% w/v. Results were expressed as mean dpm/lymph node for each experimental group and as stimulation index (SI), i.e., test group/control group ratio. EC3 value (the estimated concentration required to induce an SI of 3) was calculated by linear interpolation.

At 0, 1, 10 and 20% w/v of the test material, the mean [3H]thymidine incorporation (dpm/lymph node) were noted to be 478, 540, 2283 and 10964, respectively. Stimulation index at 1, 10 and 20% w/v of the test material was determined to be 1.1, 4.8 and 23, respectively, and the EC3 was calculated to be 5.6% w/v (0.41 M). The EC3 values of positive controls cinnamic alcohol and cinnamic aldehyde were reported to be 1.5 (20.1%) and 0.10 M (1.3%), respectively.

Therefore, the test material is classified as skin sensitiser, sub-category 1B, according to the GHS and CLP Regulation (EC) No 1272/2008.

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

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 the dorsal surface of each ear of 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) 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 radioactivity was measured 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.

In a skin sensitization study conducted following a method similar to OECD Guideline 429, four groups of CBA/Ca strain female mice(4 females/dose) 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 d-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%.

In a local lymph node assay, groups of mice (4/dose) were exposed to beta-phellandrene at concentrations of 0 (vehicle control), 1, 10 or 20% w/v. Results were expressed as mean dpm/lymph node for each experimental group and as stimulation index (SI), i.e., test group/control group ratio. EC3 value (the estimated concentration required to induce an SI of 3) was calculated by linear interpolation. At 0, 1, 10 and 20% w/v of the test material, the mean [3H]thymidine incorporation (dpm/lymph node) were noted to be 478, 540, 2283 and 10964, respectively. Stimulation index at 1, 10 and 20% w/v of the test material was determined to be 1.1, 4.8 and 23, respectively, and the EC3 was calculated to be 5.6% w/v (0.41 M). The EC3 values of positive controls cinnamic alcohol and cinnamic aldehyde were reported to be 1.5 (20.1%) and 0.10 M (1.3%), respectively.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

d-Limonene and beta-phellandrene were found to be skin sensitisers and should be classified as skin sensitisers category 1B according to the criteria of CLP Regulation (EC) No 1272/2008. As the registered substance contains more than 1% w/w of d-limonene and beta-phellandrene, which are the main constituents of the registered substance, it should also be classified as skin sensitiser category 1B according to the criteria of classification for mixtures of CLP Regulation (EC) No 1272/2008.