Registration Dossier

Administrative data

Description of key information

Two studies are avialble on d-limonene, one main impurity of the registered substance.

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.

Key value for chemical safety assessment

Skin sensitisation

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%.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

d-Limonene was found to be a skin sensitiser 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- and l-limonene, which are the main impurities 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.