Hazardous substance assessment – D-Limonene

Identification

Chemical name:

D-Limonene

CAS #:

5989-27-5

Chemical composition:

C₁₀H₁₆

Synonyms:

(+)-(4R)-Limonene; (4R)-(+)-Limonene; Cyclohexene, 1-methyl-4-(1-methylethenyl)-, (R); (R)-(+)-1-Methyl-4-(1-methylethenyl)cyclohexene; (+)-4-Isopropenyl-1-methylcyclohexene; (R)-p-Mentha-1,8-diene.

UN #:

2052

Pictogram(s):

Figure 1 - Text description

The symbol within the pictogram is a flame with a line underneath it. This symbol indicates that hazardous products with this pictogram can ignite easily and burn rapidly if they are not stored and handled properly.

Figure 2 - Text description

The symbol within the pictogram is a black silhouette of a person's head and chest with a white star shape spreading out from the center of the chest. This symbol indicates that hazardous products with this pictogram can cause certain health effects for example:

• carcinogenicity
• specific target organ effects following single or repeated exposure
• reproductive toxicity

Figure 3 - Text description

The symbol within the pictogram is an exclamation mark. This symbol indicates that hazardous products with this pictogram can cause certain health effects for example:

• skin irritation
• eye irritation
• skin sensitization

WHMIS classification

Health hazards:

Skin Irritation – Category 2

Skin Sensitizer – Category 1B

Aspiration Hazard – Category 1

Physical hazards:

Flammable Liquids – Category 3

Health hazards

Acute Toxicity (Oral):

Does not meet criteria

Median lethal dose (LD₅₀) (rat, male) = 4,400 mg/kg^{Footnote 1}

In a standard acute oral toxicity study, Wistar rats (10 per sex per dose) were given a single oral dose of D-limonene at 1,500, 1,900, 2,500, 3,300, 4,300, 5,600, 7,300, 9,400, 12,200, or 15,900 mg/kg. The LD₅₀ was 4,400 mg/kg in male rats and 5,200 mg/kg in female rats (based on study summary^{Footnote 1}).

The available data do not meet the classification criteria for Acute Toxicity (Oral).

Acute Toxicity (Dermal):

Does not meet criteria

LD₅₀ (rabbit) > 5,000 mg/kg^{Footnote 1}

In an acute dermal toxicity study, New Zealand White rabbits (10 per group) were administered D-limonene at 5,000 mg/kg on clipped abraded abdominal skin using an occlusive patch for 24 hours (based on study summary^{Footnote 1}). No deaths occurred throughout the 7-day study. Normal body weight gain was observed in all animals. At necropsy, macroscopic examination of main organs showed no abnormalities.

The available data do not meet the classification criteria for Acute Toxicity (Dermal).

Acute Toxicity (Inhalation – Gases):

Not applicable

D-Limonene is not a gas. The classification criteria for Acute Toxicity (Inhalation – Gases) do not apply to this substance.

Acute Toxicity (Inhalation – Vapours):

No data available

Acute Toxicity (Inhalation – Dusts and Mists):

No data available

Skin Corrosion / Irritation:

Category 2

Dermal exposure to undiluted D-limonene in 1 human subject caused burning, itching, aching and a purpuric rash. No information on reversibility was provided (allergic reaction cannot be precluded)^{Footnote 2}. In a human patch test study, there was evidence of sensory effects and urticarial responses on removal of the patches and irritation persisted for up to 72 hours in many volunteers. The authors concluded that D-limonene was a skin irritant in humans^{Footnote 3}.

In a primary dermal irritation study in animals performed according to the Organisation for Economic Co-operation and Development Test Guideline (OECD TG) 404 and in compliance with good laboratory practice (GLP), the intact skin of the flank of 3 albino rabbits was exposed to 0.5 mL of undiluted D-limonene under a semi-occlusive patch for 4 hours^{Footnote 4}. Mean individual scores at 24, 48 and 72 hours were, respectively, 2/4, 2/4 and 2/4 for erythema and 1.33/4, 2/4 and 1.33/4 for edema. Signs of erythema and edema, as well as desquamation of the skin surface, were observed in all animals on day 7. In another 4-rabbit study using undiluted D-limonene in compliance with OECD TG 404 and GLP, the mean individual scores for 24, 48 and 72 hours for the respective rabbits for erythema were 2, 2, 1.7 and 2. The mean individual scores for the respective rabbits for edema were 1.3, 0.7, 1.3 and 2^{Footnote 5}. In all of the above animal studies, erythema and edema scores do not meet classification criteria.

The available human data meet the classification criteria for Skin Irritation – Category 2.

Serious Eye Damage / Eye Irritation:

Does not meet criteria

In an eye irritation study conducted according to OECD TG 405 and in compliance with GLP, 3 male New Zealand White rabbits were exposed to 0.1 mL of undiluted D-limonene in one of the eyes while the other eye served as a control (based on study summary^{Footnote 4}). The eyes were examined for irritation scores at 1 hour and 1, 2, 3, 4 and 7 days after dosing. Instillation of D-limonene resulted in slight-to-moderate redness of conjunctivae associated with moderate chemosis in all treated animals after 1 hour of instillation. The irritation completely resolved within 7 days. Mean individual 24-, 48- and 72-hour scores for the 3 animals were all 0 for corneal opacity and iritis; 0.3, 1 and 1.3 for conjunctival redness; and 1, 0.3 and 1 for chemosis.

The available data do not meet the classification criteria for Serious Eye Damage / Eye Irritation.

Respiratory Sensitization:

No data available

Skin Sensitization:

Category 1B

D-Limonene has been shown to cause skin sensitization in humans^{Footnote 6Footnote 7}. In one study, 7 dermatology patients were exposed to 1% solutions of melaleuca oil or 1% solutions of the constituents comprising this oil. Of the 7 subjects, 6 showed positive sensitization reactions to D-limonene^{Footnote 7}. In a case report, a 53-year-old scientist presented with patchy dermatitis on his fingers and hands. Patch testing was negative; however, an allergic reaction was noted after exposure to the diluted solvent and DL-limonene (used as an analogue in this study)^{Footnote 6}. In another human patch test study, D-limonene caused strong reactions for all types of patches within 10 to 15 minutes of exposure. The study authors concluded that the test substance was a skin sensitizer in humans^{Footnote 3}. In a Kligman Maximization test, 25 human volunteers were exposed to D-limonene on a skin site pre-treated for 24 hours with 5% aqueous sodium laurel sulfate (SLS). The test substance exposure lasted 48 hours under occlusive conditions. Following a 10-day rest period, a new skin site was washed with 10% SLS for 1 hour after which there was a challenge with an 8% solution of the test substance. A sensitization reaction with D-limonene was not observed^{Footnote 3}.

In a local lymph node assay performed in CBA/Ca mice according to OECD TG 429 and in compliance with GLP, D-limonene was applied to groups of mice (4 females per dose) at a concentration of 0, 10, 25, 50, 75 or 100% v/v in ethanol/diethyl phthalate (3:1 v/v) (based on study summary^{Footnote 4}). The test substance was applied to the dorsal surface of each ear for 3 consecutive days. Stimulation indices for 10, 25, 50, 75 and 100% D-limonene were 1.3, 3.4, 4.0, 8.8 and 6.5, respectively. The estimated concentration giving rise to a 3-fold increase in lymphocyte proliferation (EC3) was 22% v/v (5,500 µg/cm2). No increase in visual levels of irritancy to the ear skin was observed during the study. The results meet the classification criteria for Skin Sensitizer - Category 1B, as per HPR 8.4.1(4). D-Limonene also tested positive in the guinea pig maximization test (GPMT)^{Footnote 8}. Intradermal induction was initiated on day 0 with an injection of 0.1 mL of 5% D-limonene, 0.1 mL of 5% D-limonene with Freund's complete adjuvant (FCA), or 0.1 mL of FCA alone. Each injection was repeated twice. Positive results for sensitization were obtained; however, the number of animals reacting was not provided in the study.

The available data meet the classification criteria for Skin Sensitizer – Category 1B [HPR 8.4.1(4)].

Germ Cell Mutagenicity:

Does not meet criteria

In vivo: Negative results were reported in a mammalian spot test. Mouse embryos were treated in utero (intraperitoneal administration) with 215 mg/kg D-limonene on days 10 and 11 post conception^{Footnote 9}. No genotoxic effects were noted. In a mammalian comet assay (not GLP compliant, guideline not specified), D-limonene tested negative for genotoxicity (based on study summary^{Footnote 4}).

In vitro: D-Limonene tested negative in an Ames test in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and TA 1538 with and without metabolic activation^{Footnote 9Footnote 10Footnote 3}. It also tested negative in a mouse lymphoma forward mutation assay in L5178Y mouse lymphoma cells, with and without metabolic activation^{Footnote 9}. Negative results were obtained in sister chromatid exchange and chromosomal aberration assays in Chinese hamster ovary cells, with and without metabolic activation^{Footnote 9Footnote 10}.

The available data do not meet the classification criteria for Germ Cell Mutagenicity.

Carcinogenicity:

Does not meet criteria

D-Limonene has been reviewed by the International Agency for Research on Cancer (IARC) and has been classified as Group 3, "Not classifiable as to its carcinogenicity to humans"^{Footnote 11Footnote 12}. D-Limonene has not been reviewed by the American Conference of Governmental Industrial Hygienists (ACGIH).

In a 2-year National Toxicology Program (NTP) carcinogenicity study performed similarly to OECD TG 451 and in compliance with GLP, D-limonene was administered through gavage to groups of 50 B6C3F1 mice per sex per dose at a dose of 0, 250, or 500 mg/kg/day (in males) or 0, 500, or 1,000 mg/kg/day (in females) for 103 weeks (5 days per week)^{Footnote 10}. There was no evidence of carcinogenic activity in any of the animals. In another 2-year study, D-limonene was administered to rats at doses up to 150 mg/kg/day in males or up to 600 mg/kg/day in females by gavage^{Footnote 10}. There was no evidence of carcinogenic activity of D-limonene in female rats. There was, however, increased incidences of tubular cell hyperplasia, adenomas and adenocarcinomas of the kidney in male rats. The mechanism of nephron-carcinogenicity was shown to be rat-specific, mediated by alpha2μ-globulin and was not relevant to humans.

The available data do not meet the classification criteria for Carcinogenicity.

Reproductive Toxicity:

Does not meet criteria

In a developmental/teratogenicity study (guideline not specified), 4 groups of 20 Wistar female rats were orally administered 0, 591, or 2,869 mg/kg D-limonene on days 9-15 of gestation^{Footnote 9}. At 2,869 mg/kg/day, increased maternal mortality and weight loss were reported. Fetal effects at this dose included delayed ossification of the metacarpal bone and proximal phalanx and decreased body weight. Fetal effects were considered secondary to maternal toxicity (due to the severity of the maternal effects). No effects were observed at the lower dose. Similar results were reported in a study with mice^{Footnote 9}. In a rabbit study (guideline not specified), female rabbits were orally administered 0, 250, 500, or 1,000 mg/kg/day D-limonene on days 6-18 of gestation^{Footnote 9}. Increased maternal mortality was noted at 1,000 mg/kg/day. Significant decreases in maternal body weight gain and food consumption were observed at the two highest doses. No treatment-related effects were reported for the offspring. In a study that also examined sub-chronic toxicity and was performed similarly to OECD TG 408 and was also in compliance with GLP, D-limonene was administered through gavage to groups of 10 B6C3F1 mice per sex per dose at a dose of 0, 125, 250, 500, 1,000, or 2,000 mg/kg/day for 13 weeks (5 days per week) (based on study summary^{Footnote 4}). No effects on reproductive organs were observed at either the macroscopic or microscopic level.

The available data do not meet the classification criteria for Reproductive Toxicity.

Specific Target Organ Toxicity – Single Exposure:

Does not meet criteria

Oral Route of Exposure: No human data are available. In an acute rat oral toxicity study, mortality was observed close to the oral LD₅₀ value (4,400 mg/kg in males)^{Footnote 1}. No clinical signs were reported. In another study, that involved male rats and was non-guideline, the oral LD₅₀ was reported to be > 5,000 mg/kg. Animals experienced lethargy but no deaths or clinical signs were reported.

Dermal Route of Exposure: No human data are available. In a dermal acute toxicity animal study, no animals died and an LD₅₀ value of > 5,000 mg/kg was reported (based on study summary^{Footnote 4}). No signs of toxicity were reported.

Inhalation Route of Exposure: No human data are available. In a non-guideline study, mice were exposed to 570 mg/L of D-limonene aerosol for 1 minute (based on study summary^{Footnote 4}). No deaths occurred and only a slight respiratory depression was noted.

The available data do not meet the classification criteria for Specific Target Organ Toxicity – Single Exposure.

Specific Target Organ Toxicity – Repeated Exposure:

Does not meet criteria

Oral Route of Exposure: In a 16-day sub-acute toxicity study performed similarly to OECD TG 407 and in compliance with GLP, D-limonene was administered through gavage to groups of 5 F344/N rats per sex per dose mixed in corn oil at a dose of 0, 413, 825, 1,650, 3,300, or 6,600 mg/kg/day for 16 days (5 days per week)^{Footnote 10}. No treatment-related clinical signs were observed in rats that received doses of 1,650 mg/kg/day or lower. Mortality and reduced body weight gain were observed at the 2 highest doses.

In a 13-week study, doses of D-limonene were administered orally to rats (150 to 2,400 mg/kg/day) and mice (125 to 2,000 mg/kg/day)^{Footnote 10}. Only decreased activity and weight loss were noted in the high-dose groups. No weight gain depression was observed in any of the female groups. All animals survived.

In a 2-year study in rats and mice, D-limonene was administered by gavage to groups of 50 male rats at a dose of 0, 75, or 150 mg/kg/day, of 50 female rats at 0, 300, or 600 mg/kg/day, of 50 male mice at 0, 250, or 500 mg/kg/day and of 50 female mice at 0, 500, or 1,000 mg/kg/day. Deaths occurred in high-dose female rats and low-dose male mice as well as reduced body weight gain in high-dose female mice. Nephropathy was observed in the kidneys of male rats. No other significant clinical effects were reported^{Footnote 10}.

Dermal Route of Exposure: No data available

Inhalation Route of Exposure: Volunteers were administered 10, 225, or 450 mg/m3 of D-limonene in an exposure chamber on 3 different occasions for a 2-hour period each. No signs of respiratory irritation or central nervous system effects were reported. A significant decrease in the vital capacity of volunteers was noted at the highest dose but was considered to be not functionally significant^{Footnote 13}.

The available data do not meet the classification criteria for Specific Target Organ Toxicity – Repeated Exposure.

Aspiration Hazard:

Category 1

No human data are available.

D-Limonene is a liquid hydrocarbon. The dynamic viscosity is 0.897 mPa·s (0.846 centipoise) at 25 °C^{Footnote 4}. The relative density is 0.844^{Footnote 4}. The kinematic viscosity at 25 °C is calculated to be 1.063 cSt (0.897/0.844) or 1.063 mm²/s. It should be noted that the viscosity of D-Limonene was derived from a read-across with L-limonene, an enantiomer having the same physico-chemical properties.

The available data meet the classification criteria for Aspiration Hazard – Category 1 [HPR 8.10.1].

Biohazardous Infectious Materials:

Not applicable

D-Limonene is not a microorganism, protein or nucleic acid.

Physical hazards

Explosives:

Not evaluated*

* Explosives are excluded from the HPAand its regulations. Explosives are regulated under the Explosives Act. For more information, visit Natural Resources Canada.

Flammable Gases:

Not applicable

D-Limonene is not a gas. The classification criteria for Flammable Gases do not apply to this substance.

(Flammable) Aerosols:

Not evaluated

Classification of a hazardous product in the Flammable Aerosols or Aerosols hazard class is product dependent.

Oxidizing Gases:

Not applicable

D-Limonene is not a gas. The classification criteria for Oxidizing Gases do not apply to this substance.

Gases Under Pressure:

Not applicable

D-Limonene is not a gas. The classification criteria for Gases Under Pressure do not apply to this substance.

Flammable Liquids:

Category 3

D-Limonene has a flash point of 45 °C (closed cup)^{Footnote 4}.

The available data meet the classification criteria for Flammable Liquids – Category 3 [HPR 7.6.1(2)].

Flammable Solids:

Not applicable

D-Limonene is not a solid. The classification criteria for Flammable Solids do not apply to this substance.

Self-reactive Substances and Mixtures:

Does not meet criteria

D-Limonene has a boiling point of 177.6 °C^{Footnote 4}. Self-reactive Substances and Mixtures must have a self-accelerating decomposition temperature (SADT) of ≤75 °C to meet the minimum classification in this hazard class.

The available data do not meet the classification criteria for Self-reactive Substances and Mixtures.

Pyrophoric Liquids:

Does not meet criteria

D-Limonene has an auto-ignition temperature of 237 °C^{Footnote 4}. Pyrophoric liquids react at room temperature.

The available data do not meet the classification criteria for Pyrophoric Liquids.

Pyrophoric Solids:

Not applicable

D-Limonene is not a solid. The classification criteria for Pyrophoric Solids do not apply to this substance.

Self-heating Substances and Mixtures:

Does not meet criteria

D-Limonene has an auto-ignition temperature of 237 °C^{Footnote 4}, which is well above the temperature at which spontaneous ignition would need to occur for classification.

The available data do not meet the classification criteria for Self-heating Substances and Mixtures.

Substances and Mixtures Which, in Contact with Water, Emit Flammable Gases:

Not applicable

D-Limonene has a chemical structure that does not contain metals or metalloids and is, therefore, excluded from classification [HPR 7.12.1(1)].

Oxidizing Liquids:

Not applicable

D-Limonene does not contain oxygen, fluorine or chlorine and is therefore excluded from classification (HPR 7.13.1(1)(a)).

Oxidizing Solids:

Not applicable

D-Limonene is not a solid. The classification criteria for Oxidizing Solids do not apply to this substance.

Organic Peroxides:

Not applicable

D-Limonene is not an organic peroxide. The classification criteria for Organic Peroxides do not apply to this substance.

Corrosive to Metals:

Does not meet criteria

D-Limonene is used as a solvent in degreasing metals prior to industrial painting^{Footnote 14}. There is no evidence that D-limonene is corrosive to metals.

The available data do not meet the classification criteria for Corrosive to Metals.

Combustible Dusts:

Not applicable

D-Limonene is not a solid. The classification criteria for Combustible Dusts do not apply to this substance.

Simple Asphyxiants:

Not applicable

D-Limonene is not a gas. The classification criteria for Simple Asphyxiants do not apply to this substance.

Pyrophoric Gases:

Not applicable

D-Limonene is not a gas. The classification criteria for Pyrophoric Gases do not apply to this substance.

Chemicals Under Pressure:

Not evaluated

Classification of a hazardous product in the Chemicals Under Pressure hazard class is product dependent.

Regulatory and other information

Regulatory information:

Hazardous substance assessments are prepared by Health Canada as educational and information resources. Under the HPA, suppliers of hazardous products must, upon the sale or importation of a hazardous product, provide a safety data sheet and label that meet the requirements set out in the HPR.

Other information:

The information and classifications contained in these hazardous substance assessments are based on publicly available sources, such as peer-reviewed literature or reports by international bodies. New information, including proprietary information, could have an impact on the classification of substances or hazardous products containing them. It is the responsibility of the supplier to ensure the accuracy, sufficiency and reliability of their hazardous product classifications.

Last updated:

2022

Prepared by:

Workplace Hazardous Materials Bureau, Health Canada