Chemical and Physical Properties

α-Pinene is a bicyclic monoterpene emitted from plant matter and exists as a colorless, oily liquid with a strong odor.¹ α-Pinene is present in conifer trees and turpentine as a mixture of (+) and (−) enantiomers, which can differ in ratio according to species, source tissue (e.g., needles, xylem), and age.^2-5 There is enantiospecificity to the odor of α-pinene, with (+)-α-pinene producing a slightly minty odor and (−)-α-pinene producing a pine scent.⁶ α-Pinene has a density of 0.859 at 20°C relative to water at 4°C, a boiling point of 155° to 156°C at 760 mm mercury, and a refractive index for sodium light of 1.466 at 20°C.⁷ α-Pinene is relatively hydrophobic with a measured solubility in water of 18 μg/mL at 20°C and a calculated logP value of 4.37.⁸

Monoterpenes such as α-pinene belong to the terpenoid class of chemicals, which are based on isoprene units and represent the largest group of naturally occurring compounds with over 22,000 terpenoids identified.⁹ Structurally related monoterpenes include d-limonene and ∆³-carene.

α-Pinene is a volatile organic compound that can react with nitric oxide to form ozone in the troposphere.¹⁰ Major pathways of removal and transformation of α-pinene from the atmosphere include reactions with hydroxyl radical, nitrate radical, or ozone. Products formed from these reactions are pinonaldehyde, acetone, formaldehyde, formic acid, and hydroxyl radical, among others.¹⁰

Production, Use, and Human Exposure

α-Pinene, produced by pine trees and various other plants, is the main component of turpentine. Although α-pinene is ubiquitous due to its volatilization from pine trees, there are two potential pathways that lead to more significant α-pinene exposure in humans: 1) processing, use, or storage of softwoods or their by-products (e.g., turpentine), and 2) use of personal care products, cleaning products, or air fresheners containing α-pinene as a fragrance component. Measured concentrations of α-pinene occupy a wide range from tens of µg/m³ to hundreds of mg/m³. The current permissible exposure limit and recommended airborne exposure limit for α-pinene is 100 ppm (as turpentine)¹¹ and the threshold limit value is 20 ppm averaged over an 8-hour workshift.¹²

Several studies have measured the levels of α-pinene or combined terpenes in the lumber industry. Demers et al.¹³ measured α-pinene concentrations in Canadian softwood lumber mills using personal passive sampling devices and reported a geometric mean of 0.1 mg/m³ (geometric SD = 3.8 mg/m³; approximately 0.018 ± 0.68 ppm). New Zealand plywood workers were exposed to α-pinene concentrations of 0.5 to 2.4 mg/m³ (approximately 0.090 to 0.43 ppm).¹⁴ Significantly higher α-pinene levels were detected in Finnish sawmills, in the range of 57 to 152 mg/m³ (approximately 10 to 27 ppm).¹⁵ Personal exposure to the monoterpenes α-pinene, β-pinene, and ∆³-carene ranged from 10 to 214 mg/m³ (approximately 1.8 to 38 ppm) in Swedish joinery shops¹⁶ and 0.64 to 28 mg/m³ (approximately 0.11 to 5.0 ppm) in Swedish wood pellet manufacturing facilities.¹⁷ The highest levels of terpenes reported were in Swedish lumber mills and ranged from 100 to 500 mg/m³ (approximately 18 to 90 ppm), with an average of 254 mg/m³ (approximately 46 ppm).¹⁸ Concentrations of α-pinene ranging from 0.078 to 0.333 mg/m³ (approximately 0.014 to 0.060 ppm) were measured in the blower exhaust from a composting facility.¹⁹

α-Pinene is used as a fragrance component in perfumes, air fresheners, personal care products, and household cleaners.²⁰ Rastogi et al.²¹ detected α-pinene in 39% of 59 occupational and domestic products tested with a mean concentration of 41.3 ± 51.5 ppm. A maximum concentration of α-pinene measured in a small chamber following application of floor wax containing the chemical as a major component was 0.0683 mg/m³ (approximately 0.012 ppm).²² α-Pinene is also a common contaminant detected in indoor air samples. An exposure study measuring a number of volatile organic compounds in homes detected α-pinene in 100% of samples collected from three cities in Michigan in summer and winter, with a median concentration of 3.16 μg/m³ (approximately 0.00057 ppm) and a maximum of 139.20 μg/m³ (approximately 0.025 ppm).^23a

Absorption, Distribution, Metabolism, Excretion, and Toxicokinetics

In general, data from human exposure to α-pinene have demonstrated that it is rapidly absorbed after inhalation exposure, accumulates in the fat compartment, is metabolized primarily by hydroxylation and glucuronidation, and is excreted by the kidneys.^24,25

The uptake, distribution, and elimination of α-pinene was investigated in healthy male volunteers following a 2-hour inhalation exposure to 10, 225, and 450 mg/m³ (approximately 1.8, 40, and 81 ppm) (+)-α-pinene or 450 mg/m³ (−)-α-pinene.²⁴ Significant differences were not found between the enantiomers in uptake, distribution, or excretion. α-Pinene exhibited a high degree of uptake from the lungs, averaging 59% for the two higher concentrations, with approximately 8% of the parent compound eliminated in exhaled air. Less than 0.001% of the total uptake was eliminated unchanged in the urine. Saturation of metabolism was not observed, as evidenced by a linear increase in arterial blood concentration with increasing exposure concentration. The clearance value for α-pinene indicated that it was readily metabolized. A tri-phasic elimination curve was observed with half-lives for each phase of elimination equal to 4.8 and 5.6, 38 and 40, and 695 and 555 minutes, respectively, for (+)- and (−)-α-pinene. The long half-life associated with the third phase of elimination indicates a high affinity of α-pinene for poorly perfused tissue (i.e., adipose tissue).

Animal metabolism studies in the rabbit and brushtail possum support the metabolic pathway identified in the human studies and have identified many of the same α-pinene metabolites, with the verbenols representing the major metabolites and myrtenol and myrtenic acid in lesser amounts.^26,27 Metabolism of inhaled α-pinene occurs mainly via hydroxylation and glucuronidation followed by renal elimination.²⁸ The major metabolites cis- and trans-verbenol, representing approximately 1% to 4% of the total uptake, were identified in the urine of experimentally exposed individuals. These urinary metabolites, in addition to trace amounts of the metabolite myrtenol, were also identified in mill workers exposed occupationally to α-pinene, β-pinene, and ∆³-carene.²⁹

In contrast to the profile of absorption, distribution, metabolism, and elimination following inhalation exposure, a case study describing the fate of monoterpenes following an intentional ingestion of pine oil (57% α-pinene, 8% β-pinene, 26% ∆³-carene, 6% limonene, and 3% other) found poor uptake of the monoterpenes from the gastrointestinal tract, slow metabolism, and renal excretion of metabolites.³⁰ The major metabolites identified for α-pinene, borneol and bornylacetate, also differed from those identified following inhalation.

Toxicity

In humans, reports of toxicity resulting from α-pinene alone or terpene mixtures containing α-pinene indicate potential respiratory and skin irritation. Johard et al.³¹ assessed the effects of short-term inhalation exposure to a terpene mixture (α-pinene, β-pinene, and ∆³-carene) on bronchioalveolar lavage fluid from eight healthy volunteers and found that macrophage and mast cell counts increased following exposure to 450 mg/m³. Irritation of the eyes, nose, and throat was observed in volunteers exposed to 450 mg/m³ α-pinene.²⁴

Animal studies designed to address the general toxicity, reproductive toxicity, or developmental toxicity of α-pinene were not found in the literature. α-Pinene elicited sensory irritation (stimulation of specific nerve endings in the nasolaryngeal region leading to characteristic “braking” during exhalation and a corresponding decrease in respiratory frequency) in a mouse bioassay with a concentration that reduces respiratory rate by 50% (RD₅₀) of 1,053 to 1,107 ppm for the more active D-α-pinene enantiomer.³² α-Pinene was positive in an acute dermal irritation assay and negative in a guinea pig maximization test, indicating that it is a skin irritant but not a sensitizer.³³

Carcinogenicity

Very little carcinogenicity data are available for α-pinene. Two epidemiological studies have examined turpentine or terpene exposure in occupational settings and cancer outcomes. In a case-control study of Finnish woodworkers, a weak association [odds ratio (OR) = 1.33; 95% confidence interval (CI): 0.78, 2.27 for any exposure to terpenes lasting over one month] was found between respiratory cancer and exposure to terpenes (primarily α-pinene and ∆³-carene) and other heating products of pine and spruce.³⁴ Another case-control study found an association between paternal exposure to turpentine and neuroblastoma in offspring (OR = 1.9; CI: 1.0, 3.6 to 10.4; CI: 2.4, 44.8 depending on methods for exposure categorization).³⁵ Chronic toxicity studies with α-pinene were not found in the literature.

Genetic Toxicity

Genotoxicity studies of α-pinene indicated that it was not positive in bacterial mutagenicity assays but demonstrated clastogenic and aneugenic effects in one in vitro mammalian cell study.

α-Pinene was not mutagenic in several bacterial mutagenicity assays. α-Pinene, tested at a single concentration of 3 μM, was negative in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 with or without Arochlor 1254-induced rat liver S9 metabolic activation enzymes, and α-pinene tested over concentrations that ranged up to 3 μM was negative in S. typhimurium strains TA98 and TA100 with or without 3-methylcholanthrene-induced rat liver S9 mix.³⁶ Additionally, α-pinene was negative in S. typhimurium strains TA98 and TA100 when tested at concentrations ranging from 10 to 500 μg/plate with or without S9 mix³⁷ and enantiomers of α-pinene were negative in S. typhimurium strains TA97a, TA98, TA100, and TA1535 at concentrations ranging from 100 to 5,000 μg/plate with or without S9 mix.³⁸

Two in vitro genotoxicity studies of α-pinene were performed using mammalian cells. α-Pinene did not induce DNA damage as assessed by the comet assay in human lung A549 cells in a system that allowed exposure to α-pinene by air (concentrations ranged from 1 to 1,800 mg/m³).³⁹ However, α-pinene was clastogenic and aneugenic in V79-C13 Chinese hamster cells exposed in cell culture medium.⁴⁰ Clastogenic activity was evidenced by induction of DNA damage assessed by the comet assay, significant increases in micronucleated cells, and induction of chromosomal breakage assessed by metaphase analysis. With regard to the mechanism of DNA damage, α-pinene generated significant increases in reactive oxygen species as measured by a fluorescence assay. Furthermore, a significant number of the micronuclei observed in the V79-C13 cells stained positive for the presence of kinetochores, the number of chromosomes in metaphase spreads deviated from the modal number (decreased with increasing concentrations of α-pinene), and a significant increase in metaphase spreads showing endoreduplication was noted, suggesting that α-pinene has aneugenic activity. Immunofluorescent detection of tubulin and counterstaining for chromatin showed that the mitotic spindle was disrupted in cells exposed to α-pinene. Although concentrations of α-pinene that ranged from 40 to 50 μM induced very high levels of apoptosis, the clastogenic and aneugenic effects of α-pinene were observed at concentrations ranging from 25 to 35 μM that were accompanied by low levels of apoptosis.

Study Rationale

Originally, turpentine was nominated by the International Union of the United Auto Workers for comprehensive toxicity studies due to widespread human exposure and a lack of data characterizing the chronic effects associated with turpentine. The National Toxicology Program proceeded with testing the main component of turpentine, α-pinene, which has a more diverse and widespread exposure profile. In addition to being the main component in turpentine, α-pinene is also used as a fragrance and flavoring ingredient. Exposure to α-pinene occurs through the use of personal care and cleaning products, as well as occupationally, in lumber processing and building activities. Furthermore, the toxicity data available for α-pinene are inadequate for assessing potential human health effects. This Toxicity Study Report summarizes the results of 2-week and 3-month inhalation toxicity studies with α-pinene in F344/N rats and B6C3F1/N mice.

Footnotes

a

An error was identified in the NTP Toxicity Report on α-pinene (TOX 81). The Production, Use, and Human Exposure text and one reference were updated to accurately reflect indoor exposure levels reported in a study conducted in homes in Michigan. [24 August 2020].