Toxicity studies on western juniper oil (Juniperus occidentalis) and Port-Orford-cedar oil (Chamaecyparis lawsoniana) extracts utilizing Local Lymph Node and Acute Dermal Irritation Assays
A. Morrie Craig1†, Joseph J. Karchesy2, Linda L. Blythe1, Maria del Pilar González-Hernández3, Laurence R. Swan4
1- Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331;
2- Department of Wood Science and Engineering, College of Forestry, Oregon State University, Corvallis, OR 97331;
3- Department of Crop Production, University of Santiago de Compostela, Lugo-España, Spain;
4USDA Forest Service, Klamath Falls, OR 97601
†Corresponding author: A. Morrie Craig
Department of Biomedical Sciences College of Veterinary Medicine, 208 Dryden Hall, 450 SW 30th Street Oregon State University, Corvallis, OR 97331, phone: (541) 737-3036, fax: (541) 737-2730 e-mail: email@example.com
Abstract The essential oil extracts of western juniper oil (Juniperus occidentalis) and Port-Orford-cedar oil (Chamaecyparis lawsoniana) were evaluated for possible dermal toxic effects on mice and rabbits. Mice were tested for their response to both extracts utilizing a Local Lymph Node Assay. Western juniper oil extract at 0.5% and 5% concentrations did not show a Stimulation Index (SI) greater than normal (3.0); however, a 50% concentration did show a positive response at 3.3. Port-Orford-cedar oil extract did not show a positive response at concentrations of 0.5%, 5%, and 50%. A primary dermal irritation study using rabbits had a Primary Irritation Index (PII) of 3.3 with 100% Port-Orford-cedar oil extract. This was reduced to a PII of 0.625 when diluted 1:1 with olive oil. Undiluted western juniper oil extract had a PII score of 2.7. While a 5.0% solution had a PII score of 0.3, a 0.5% solution of western juniper oil was a non-irritant. It would appear that animals bedded on wood shavings have contact with essential oils at concentrations far less than the 2% maximum by weight obtained by steam distillation extraction. These concentrations did not elicit a hypersensitivity response.
Key Words: western juniper (Juniperus occidentalis), Port-Orford-cedar Chamaecyparis lawsoniana), acute toxicity, shavings, essential oil extracts, horses, dogs, laboratory animals
Sawmills produce a large amount of waste. It is not uncommon for 40% to 50% of a log by weight to become processing residue even for commodity products, such as dimensional lumber. An even higher percentage of waste is generated by the limited number of manufacturers who process logs into high-value added products, such as millwork (e.g., flooring and paneling) and sporting equipment (e.g., wooden arrow shafts). Manufacturing residues of many aromatic cedars, such as western juniper (Juniperus occidentalis) and Port-Orford-cedar (Chamaecyparis lawsoniana), can be distilled for their essential oils, thereby extracting value from what traditionally has been discarded or burned, and improving processing economics.
An added impetus to investigate valued-added uses for western juniper is that this species, similar to other Juniperus spp. in the Western United States, has greatly increased in acreage and density over the last century, causing loss of site productivity, decrease in forage, loss of wildlife habitat, and overall decrease in biodiversity (Gedney et al., 1999; Miller and Wigand, 1994; Miller and Rose, 1995; Miller et al., 2000). The costs of western juniper removal to improve rangeland and watershed conditions are high compared to the value of the land. Given this situation, many landowners and land managers are highly interested in investigating potential markets for eastern juniper products to partially defray costs of management (Swan, 2001).
Besides traditional markets for aromatic cedar essential oils, such as fragrances, there has been increased interest in the pharmaceutical properties of these oils. Oil extracts of Alaska cedar (Chamaecyparis nootkatensis) and western juniper have antimicrobial activity that may provide some protection against opportunistic anaerobic bacterial infections when animals are bedded on Port-Orford-cedar or western juniper shavings (Johnston et al., 2001). Nootkatin, a major component of Alaskan cedar (Chamaecyparis nootkatensis) heartwood, has been shown to have antifungal properties (Rennerfelt and Nacht, 1955). Antioxidative effects have been demonstrated in-vitro with extracts from thyme, juniper (Juniperus sp.), and oregano (Takacsova et al., 1995). Similarly, in a rat model, dietary juniper berry oil (Juniperus sp.) reduced hepatic reperfusion injuries through its 5,11,14-eicosatrienoic acid, a poly-unsaturated fatty acid similar to that found in fish oils (Jones et al., 1998). Chavali et al. (1998) also found consumption of juniper oil (Juniperus chinensis beans) in the diet increased the production of tumor necrosis factor-alpha and decreased levels of dienoic eicosanoids, interlukeukin-6 and -10 in mice subjected to an intraperitoneal lethal dose of endotoxin. Butani et al. (2003) found an amelioration of tacrolimus-induced nephrotoxicity in rats when their diets were supplemented with juniper oil (Juniperus sp.) and suggested its possible use to reduce chronic allograft nephropathy in humans. Acaricidal effects of extracts from Alaska cedar (Chamaecyparis nootkatensis) and eastern-red-cedar (Juniperus virginiana L.) woods may be useful in controlling ticks that cause Lyme=s disease in humans and animals (Panella et al., 1997) while Juniperus procera extracts contain anti-termite compounds (Kinyanjui et al., 2000).
A pharmacological screening of different Juniperus oxycedrus L. extracts found low acute toxicity and significant anti-inflammatory and analgesic activity as well as inhibition of rat paw edema induced by carrageenin (Moreno et al., 1998). Juniperus communis L. Aberries@ have been found to have a variety of pharmacodynamic effects including diuretic, carminative, antiseptic, abortive, and anti-diabetic activity (de Medina et al., 1993) while antitumor activities were found with a crude extract of Juniperus chinensis leaves (Ali et al., 1996). Most recently, Juniperus communis wood was tested for its use as an implant material in rabbits with concurrent toxicity studies on both oral and intravenous administrations. It was found that the low concentrations of oil that would be released were tolerated without any detrimental effects (Gross and Ezerietis, 2003).
Toxicity differs between the aromatic cedar species (Hausen, 1981; Mitchell and Roosk, 1979; Ohman 1984; Woods and Calnan, 1976). Most literature focuses on western-red-cedar wood (Thuja plicata) as an allergen in occupational asthma (Horne et al., 2000, Lin et al., 1996, Noertjojo et al., 1996). Few studies exist on Port-Orford-cedar (Chamaecyparis lawsoniana) or juniper wood (Juniperus communis) or their extracts (Gross and Ezerietis, 2003; Meding et al., 1996). Oral gavage of common juniper needles (Juniperus communis) caused abortion in late term pregnanciessimilar to pine needle induced abortion (Gardner et al., 1998). In a study of multiple juniper species extracts used in fragrance and biological additives in cosmetic formulations, there was little toxicity of the oil or tar in animals. Irritant effects on skin were not found with the oils, but there was some evidence of sensitization to the tar (Final report in the safety assessment of Juniperus communis extract, Juniperus oxycedrus extract, Juniperus oxycedrus tar, Juniperus phoenicea extract, and Juniperus virginiana extract, 2001). A juniper (Juniperus sp.) oil-based phytomedicine was tested for nephrotoxicity in Sprague-Dawley rats by oral administration of varying doses and all were found to be non-toxic (Schilcher and Leuschner, 1997). Commercial products of Port-Orford-cedar oil for use in pet care products to repel fleas and ticks are availablea. In two pilot studies at Oregon State University, no toxicity was found in dogs and horses bedded for six and one half months and for eight months respectively on western juniper shavings (Blythe et al., 2001).
The need for additional toxicity studies was identified by the wood products industry because unresolved questions were being raised about the use of western juniper, Port-Orford-cedar, and other aromatic cedar products for horse, dog, and laboratory animal bedding, as well as for fragrance products and topical applications for humans. This study was specifically undertaken to define potential toxicity of the essential oils in western juniper and Port-Orford-cedar. The hypothesis tested was that the application of the oil extracts to the dermis at levels found in shavings would not cause inflammation or skin pathology. Essential oils from western juniper and Port-Orford-cedar were tested for their capacity to induce a hypersensitivity response in mice as measured by the proliferation of lymphocytes in the local draining lymph nodes and in a primary dermal irritation study in rabbits.
2. Materials and Methods
2.1 Extraction and Analyses
Steam distilled essential oils were prepared from western juniper heartwood shavings from live trees harvested in Eastern Oregon and Port-Orford-cedar wood shavings from standing dead and down logs collected in Coos County, Oregon using protocols previously described (Tucker et al., 2000; Adams, 1987) (oil extract obtained from western juniper supplied from Karchesy Laboratory, College of Forestry, Oregon State University, and Port-Orford-cedar from Rose City Archerya). The extracted oils were then analyzed by GC/MS as described by Tucker (2000) and Adams (1987) to determine and reaffirm presence of the major chemical components. Mass spectra were recorded with 5970 Mass Selective detector (MSD)b coupled to a Hewlett Packard (HP) 5890 GCc using a DB-5 column (100m) for western juniper oil and a HP 50m X 0.2mm fused silica column coated with 0.33 um FFAP (crosslinked) for Port-Orford-cedar oil. The GC was operated under the following conditions: injector temperature at 250o C, oven temperature programmed to 60 o C and held for one minute and progressively to 115 o C at 2.5 C/min, 210 o C at 1 C/min and held for 30 minutes; the injection size was 1 ml split 1:10. The MSD ei was operated under the following conditions: electron impact source 70eV, 250 o C. Identification of peaks was made by retention indices and library searches of the GC/MS instrument library supplemented with searches of the National Institute of Standards and Technology (NIST)d, and Wileye libraries. The components of both oils are reported in Figure 1 and Table 1 respectively.
The Local Lymph Node Assay to test an oil’s capacity to induce a hypersensitivity response as measured by the proliferation of lymphocytes in the local draining lymph nodes was conducted in mice for both western juniper oil and Port-Orford-cedar oil extracts. Two groups of twenty-five nine week old female CBA/J mice from Jackson Laboratoriesf were selected for either the western juniper or Port Orford extract study. The number used is the minimum number recommended (NIH Publication No. 99-4494, 1999). Only mice considered suitable for use were placed on the study. Prior to treatment initiation, all mice were weighted. The weight ranges were from 19 to 24 grams. The mice were assigned to treatment groups using a computer-generated randomization method based on body weight. Mice were given identification numbers and identified by tail marks. Mice were housed (grouped five per cage) in compliance with the National Research Council “Guide for the Care and Use of Laboratory Animals”. Calvertg is a USDA Registered and fully Accredited AAALAC Facility. The animal room environment was controlled (target conditions: temperature 18 to 26oC, relative humidity 30 to 70%, 12 hours artificial light and 12 hours dark). Temperatures and relative humidity were monitored daily. All animals had access to Certified Rodent Diet #7012C (Harlan Tekladh) or equivalent ad libitum, unless otherwise specified. The lot numbers and specifications of each lot of all animals used are archived at Calvertg.
The rabbit is a standard species used in dermal irritation studies and is acceptable to regulatory agencies. The number of animals used in this study was the minimum number necessary to properly perform this type of study (Gad, 1994). Six male and six female twenty week and twenty four week old New Zealand White rabbits (HM:(NZW)fBR) from Covancei were used for testing each oil extract. Prior to testing, each rabbit was assessed as to their general health and acclimated/quarantined for a minimum of five days. Rabbits were placed on the study based upon sex, body weight, and apparent good health. All rabbits were housed individually and identified by ear tag numbers. The housing environment was the same as described above for the mice. All rabbits had access to Teklad Certified Rabbit Dieth ad libitum. Water was provided to the animals in all studies ad libitum. Periodic analyses of the water are performed and the results are archived at Calvertg. There are no known contaminants in the diet or water which at the levels detected would be expected to interfere with the purpose, conduct or outcome of the study.
2.3 Local Lymph Node Assay
The mice were weighed on Days 1 and 6. Groups of five mice were treated with 25µl on the dorsal surface of each of the ears once per day for three days with either the vehicle, olive oil, or the test article, western juniper or Port-Orford-cedar extracts, at concentrations of 0.5, 5 and 50%, or the positive control, 0.1% dinitrochlorobenzene (DNCB) in dimethyl sulfoxide (DMSO). On Day 6, the mice were injected with 20 µCi of 3H-thymidine. Five hours later, the mice were euthanized with CO2 and the draining auricular lymph nodes were removed. The lymph node cells were precipitated with 5% trichloroacetic acid (TCA) and the pellets counted in a ß-scintillation counter to determine incorporation of the 3H-thymidine. The mean decays per minute (DPM) for each group was determined. Increases in 3H-thymidine incorporation relative to vehicle-treated control were derived for each group and recorded as Stimulation Indices (SI). The criterion for a positive response is that one or more concentrations of a test article elicit a three-fold or greater increase in isotope incorporation relative to the vehicle control.
2.4 Acute Dermal Irritation Assay
Within 24 hours before the test, the fur was removed from the dorsal area of the trunk of each rabbit, being careful to avoid abrading the skin. In the first set of experiments utilizing the twenty week old rabbits, an undiluted Port-Orford-cedar extract or western juniper extract was administered once (0.5 ml/site) on the clipped skin of two rabbits. The extract was applied to a small area of skin and covered with a gauze patch. The patch was held in contact with the skin with a sheet of rubber dam. The trunk of the animal was wrapped with an elastic bandage dressing which was held in place with non-irritating tape for the duration of the exposure period. Access by the animal to the patch and resultant ingestion/inhalation of the test article was prevented.
At the end of the four hour exposure period, residual extract was removed using gauze and water without altering the existing response or the integrity of the epidermis. Each site was unwrapped and scored according to a technique described by Draize (1959). The scoring system examined the skin for the presence of erythema and edema. The former was graded as 0 for no erythema, with erythema scores of 1 for very slight, 2 for well defined, 3 for moderate to severe, and 4 for severe to eschar formation. Edema was scored in a similar manner with 0 indicating none, 1 very slight, 2 slight, 3 moderate, and 4 severe. A score for each animal was determined using the immediate, 24, 48, and 72 hour observations for calculations and dividing by four. The Primary Irritation Index (PII) is the sum of the scores for all of the animal scores that is divided by six. The PII is considered slight if less than 2, moderate if between 2 and 5, or severe if greater than 5. Due to moderate to severe erythema and slight edema recorded in the first two rabbits administered the Port-Orford-cedar extract, the extract was diluted with olive oil (1:1) and applied in a similar manner to the remaining four rabbits while the western juniper concentration remained undiluted. In a second set of experiments, using three female and three male twenty-four week old rabbits. Four intact skin sites per animal received either 5.0% or 0.5% concentrations of western juniper extract or Port-Orford-cedar extract in olive oil. The application and observation times were identical to those described above. Body weights were recorded at the beginning and termination of the study. All animals were euthanized by barbiturate overdose following experimental termination.
2.5 Statistical Analysis
Evaluation of equality of means of the data from the local lymph node study was made by a one way analysis of variance using the F distribution to assess statistical significance using Systat (version 9.01)j. If statistically significant differences between the means were found, a Dunnett’s test was used to determine the degree of significance from control means. The design of the acute dermal irritation study is such that statistical analysis was not necessary.
3.0 Results and Discussion
Figure 1 and Table 1 illustrate the major components of western juniper oil and Port-Orford-cedar oil extracts. The analyses of the components indicated that they were identical to those that had been isolated previously (Adams, 1987; Tucker et al., 2000). The concentration of extracted oil on a dry weight basis from the western juniper shavings was 1.68% (Adams, 1987) while the Port-Orford-cedar oil was 1.88% (Dr. D. Walker, Essex Laboratory, personal communication, December 29, 2003). The most severe dermal response of the primary dermal irritation study occurred in the initial two rabbits tested with undiluted Port-Orford-cedar extract. The PII score in these rabbits was 3.3. However, when this extract was diluted (1:1) with olive oil, the PII score dropped to 0.625. In the second set of dilution experiments, extracts of Port-Orford-cedar oil at 5% and 0.5% had PII scores of 1.1 and 0.3 respectively. At the 0.5% concentration of Port-Orford-cedar oil extract, the 0.3 score represented only one rabbit which showed a 1 in erythema (barely perceptible). All the other five rabbits scored 0. With the 5.0% concentration, one animal out of the six total showed very slight edema and erythema. Another rabbit showed no edema and very slight erythema. The four remaining rabbits had 0 for a score in both categories. By the end of the experiment, all six rabbits scored 0. Undiluted western juniper oil had a PII score of 2.7 indicating moderate irritation. However, at 5% concentration, western juniper extract had only very slight erythema (barely perceptible) and no edema. No signs of skin irritation were seen with the 0.5% dilution. Thus, at 0.5% concentration, western juniper oil was found to be a non-irritant. Finally, no changes in weight were noted nor were there any toxic clinical effects from any of the substances tested. The results of the Local Lymph Node Assay in the mice are seen in Table 2. Based on data from this study, Port-Orford-cedar oil at concentrations of 0.5, 5 and 50% did not induce a hypersensitivity response and therefore is not considered to be a sensitizer. Only western juniper oil extract at 50% concentration showed a positive response of 3.33 SI with 3.0 or greater representing a positive response and indicating a potential sensitizer. Lesser concentrations of 0.5% and 5% did not show a positive stimulation response. A recent review examined the use of relevant skin sensitization test methods. Three primary objectives of this review were to evaluate which methods best determined a) relative potency, b) the threshold dose necessary for induction of skin sensitization, and c) risk assessment. It was determined that for de novo investigations, the Local Lymph Node Assay is the recommended method for assessment of the influence of a new formulation on skin sensitizing potency. Utilizing this assay, neither western juniper nor Port-Orford-cedar oil extracts showed a positive response at levels to which animals would be commonly exposed on bedding made from shavings of these species. This conclusion is based on the fact that the percentage of western juniper or Port-Orford-cedar oil extracted by steam distillation is less than 2% by dry weight. Two pilot studies appear to support the interpretation that exposure to low concentrations of oil, such as in animal bedding made of western juniper or Port-Orford-cedar shavings, will not elicit a hypersensitivity response. These studies were conducted at Oregon State University with animals housed for greater than six months on western juniper shavings. Twelve healthy adult horses of mixed breeds were bedded on western juniper shavings for a minimum of twelve hours per day and for twenty-four hours a day during inclement weather. Baseline photographs were taken of the legs and ventral abdomen immediately prior to and at the end of the study. Blood samples were taken at the same times and analyzed for complete blood counts and for the following chemical concentrations: blood urea nitrogen, creatinine, creatine kinase, asparatate amino transferase, gamma glutamyl transferase, total bile acids, total protein, albumin, and bilirubin. The horses were examined daily for any possible foot, limb, or abdominal lesions. During and at the end of the study, there was no evidence of any skin lesions or any other clinical or biochemical abnormalities. In a parallel study, eight dogs, primarily Labrador Retrievers, were housed for 198 days on similar western juniper shavings. Physical examinations and blood analyses were identical to those evaluated in the horses. None of the parameters from any of the dogs had a statistically significant change and there were no signs of dermal hypersensitivity or abnormalities.
In summary, this study shows that low concentrations of oil extracts from either western juniper or Port-Orford-cedar had no toxic effects. Further, they did not elicit a hypersensitivity reaction nor a primary skin irritation at the low concentrations to which animals bedded on these materials would be exposed.
This project was supported by funds from the Oregon State University Agricultural Experiment Station. The authors wish to thank Calvert Preclinical Services, Inc. for performing the Primary Dermal Irritation Study and the Local Lymph Node Assay. Also, the authors wish to acknowledge the contributions of Dr. Joan Chapdelaine of Calvert Preclinical Services, Dr. Jennifer Duringer and Ms. Zelda Zimmerman.
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Table 1. Relative percentage of major components of Port-Orford-cedar shavings extracted by steam-distillation and GC/MS analyses.
alpha-pinene 6.53 ± 0.02
limonene 2.69 ± 1.07
fenchone 4.67 ± 0.18
camphor 5.94 ± 1.05
alpha-fenchol 5.51 ± 1.06
alpha-terpineol 14.33 ± 5.80
alpha-muurolene 4.23 ± 1.56
delta-cadinene 8.17 ± 1.75
tau-cadinol 3.42 ± 1.18
alpha-cadinol 5.30 ± 1.77
Table 2. Local Lymph Node Assay results for juniper oil (Juniperus occidentalis) and Port Orford oil (Chamaecyparis lawsoniana) extract toxicity study.
A. Results from Juniper oil
Group Treatment Dose DPM1 (mean ± sem) SI2 (Test/control Ratio) Results 1 Vehicle – 1131 ± 166 – – 2 Juniper oil 0.5% 1555 ± 181 0.83 – 3 Juniper oil 5% 935 ± 238 1.37 – 4 Juniper oil 50% 3767 ± 519 3.33 + 5 DNCB3 0.1% 18310 ± 2068*** 16.19 + B. Results from Port-Orford-cedar oil Group Treatment Dose DPM1 (mean ± sem) SI2 (Test/control Ratio) Results 1 Vehicle – 1469 ± 148 – – 2 Port-Orford-cedar oil 0.5% 803 ± 255 0.55 – 3 Port-Orford-cedar oil 5% 1379 ± 447 0.94 – 4 Port-Orford-cedar oil 50% 2579 ± 584 1.76 – 5 DNCB3 0.1% 19286 ± 3134*** 13.13 +
1DPM = decays per minute; 2SI = stimulation index; 3DNCB = dinitrochlorobenzene
Test/control ratio of 3.0 or greater represents a positive result
***Statistically significant difference compared to the vehicle control group (P < 0.001)
Figure 1. GC/MS chromatogram of the oil extract from western juniper (Juniperus occidentalis) heartwood and the percentage composition of the major components on a dry weight basis. Cedrol (a) was 38.9%; thujopsene (b) was 18.9%; alpha-cedrene (c) was 8.8% and beta-cedrene (d) was 2.6%. The relative percent of yield of components (sum x percent) on a dry matter basis was 1.68%.