Virginia cedarwood oil (hereafter referred to as cedarwood oil) is extracted from Juniperus virginiana trees by steam distillation and contains cedrol, cedrene, cedrenol, cedral, cuperene, thujopsene, and widdrol as primary components. Cedarwood oil is used as a fragrance in cosmetic products, as a pesticide, and as a source material for production of other fragrance materials with cedarwood odors. Cedarwood oil was nominated for toxicity testing by the National Cancer Institute based on widespread and potentially increasing human exposure to the substance, and a lack of toxicology data. The dermal route of administration was selected for these studies because it is the most common route of exposure in humans due to its frequent use as a pesticide and as a fragrance in household products and cosmetics. Male and female F344/N rats and B6C3F1/N mice were administered cedarwood oil dermally for 3 months. Genetic toxicology studies were conducted in Salmonella typhimurium and mouse peripheral blood erythrocytes.
Groups of 10 male and 10 female rats and mice received no treatment (untreated control) or were administered cedarwood oil in 95% aqueous ethanol dermally at concentrations of 0% (vehicle control), 6.25%, 12.5%, 25%, 50%, or 100% (neat) 5 days per week for 14 weeks. Formulations were administered at a volume of 0.5 mL/kg body weight (rats) or 2.0 mL/kg (mice), which resulted in the mice receiving higher doses of cedarwood oil than rats in respect to the equivalent dose in mg/kg. Dose ranges for mice and rats were estimated as 125 to 2,000 mg cedarwood oil/kg body weight and 31.25 to 500 mg/kg, respectively. Statistical analyses of effects in dosed groups (except 100%) were conducted with comparisons to the appropriate vehicle control group; groups dosed with 100% cedarwood oil were compared to the appropriate untreated control group.
With the exceptions of two males in the 100% group, all rats survived to the end of the study. Final mean body weights and body weight gains of 50% and 100% males and females were significantly less than those of their respective control groups, and the mean body weight gain of male rats in the 25% group was significantly less than that of the vehicle control group. Treatment-related clinical observations included irritation, thickening, and ulceration of the skin at the site of application in most males in the 25% or greater groups and most females in the 12.5% or greater groups.
In rats, relative liver weights of males in the 50% and 100% groups, and absolute and relative liver weights of females in the 100% group were significantly greater than those in the respective control groups. The absolute kidney weight of 100% males and relative kidney weights of males in the 50% and 100% groups were significantly greater than those in the respective control groups. The absolute thymus weights of 25% and 100% males and the absolute and relative thymus weights of 100% females were significantly less than those in the respective control groups.
Compared to those in the respective control groups, the incidences of several nonneoplastic lesions of the skin at the site of application were significantly increased in dosed groups of male and female rats. The incidences of epidermal hyperplasia were significantly increased in 12.5% or greater males and all dosed groups of females. The incidences of epidermal hyperkeratosis and sebaceous gland hyperplasia were significantly increased in 25% or greater males and 12.5% or greater females. The incidences of epidermal ulcer were significantly increased in the 50% and 100% groups of males and females. The incidences of chronic active inflammation were significantly increased in 12.5% or greater males and females. The incidences of dermal fibrosis and hair follicle hyperplasia were significantly increased in 25% or greater males and females. Hematology effects included increased leukon (white blood cell and differential counts) in 100% males and 50% or greater females and treatment-related decreases in erythron (hematocrit, hemoglobin, and erythrocyte counts) in 100% males and females. In the kidney of rats, the incidences of renal tubule degeneration were significantly increased in 50% and 100% males compared to those in the respective control groups. In addition, the incidences of renal tubule granular casts and hyaline droplet accumulation were significantly increased in males in the 25% or greater groups compared to those in the respective control groups; the severity of these lesions increased with increasing dose. In the bone marrow of rats, the incidences of hyperplasia were significantly increased in 100% males and females compared to those in the untreated control groups.
Due to the severities of skin lesions at the site of application, all male and female mice in the 100% groups, one male and one female each in the 50% groups, and one male in the 12.5% group were euthanized during weeks 10, 11, and 12, respectively; all other male and female mice survived to the end of the study. The final mean body weights of 25% and 50% males and 12.5% or greater females and the mean body weight gains of 12.5% or greater males and females were significantly less than those of the vehicle control groups. Test article-related clinical observations included irritation, thickening, and ulceration of the skin at the site of application in most dosed mice.
In mice, the absolute liver weights of 50% males and females and relative liver weights of all dosed groups of males and females were significantly greater than those of the vehicle control groups. The absolute kidney weight of 50% females and relative kidney weights of 12.5% or greater females were significantly greater than those of the vehicle control groups. Absolute thymus weights of 25% and 50% males and 12.5% or greater females were significantly less than those of the vehicle controls.
The incidences of epidermal hyperplasia, hyperkeratosis, and ulcer and chronic active inflammation, dermal fibrosis, hair follicle hyperplasia, and sebaceous gland hyperplasia were significantly increased in 12.5% or greater groups of male and female mice compared to those in the respective control groups. In addition, compared to the occurrences in the vehicle control groups, the incidences of epidermal hyperplasia and hyperkeratosis and chronic active inflammation, hair follicle hyperplasia, and sebaceous gland hyperplasia were significantly increased in 6.25% males, and the incidences of epidermal hyperplasia, chronic active inflammation, and sebaceous gland hyperplasia were significantly increased in 6.25% females. Hematology effects included increased leukon (white blood cells and differential counts) in 25% or greater females and treatment-related decreases in erythron (hematocrit, hemoglobin, and erythrocyte counts) in 12.5% or greater males and 25% and 50% females. In the liver, the incidences of hepatocyte glycogen depletion were significantly increased in males and females in the 12.5% or greater groups compared to those in the respective control groups. In the thymus, the incidences of atrophy were significantly increased in 25% or greater males compared to those in the respective controls. In the kidney, the incidence of nephropathy was significantly increased in 100% males compared to that in the untreated controls.
Cedarwood oil was not mutagenic in S. typhimurium strains TA98, TA100, or TA102 with or without exogenous metabolic activation. No increase in micronucleated erythrocytes was seen in blood samples obtained from male B6C3F1/N mice treated with cedarwood oil for 3 months via dermal application; a small increase in micronucleated erythrocytes, judged to be equivocal, was seen in female B6C3F1/N mice, however. No significant alterations in the percentage of polychromatic erythrocytes (reticulocytes) were seen in male or female mice, suggesting that dermally-applied cedarwood oil did not induce bone marrow toxicity.
Under the conditions of the 3-month dermal studies with Virginia cedarwood oil, there were treatment-related lesions in male and female rats and mice. Skin (at the site of application) and kidney were the major targets from administration of cedarwood oil in both rats and mice. Additionally, the liver and the thymus were considered secondary targets of cedarwood oil administration as a result of the skin effects at the site of application in both rats and mice. The most sensitive measures of cedarwood oil administration in each species and sex were: increased incidences of skin (site of application) lesions in male [lowest-observed-effect-level (LOEL) = 12.5%; approximately equivalent to 62.5 mg/kg] and female (LOEL = 6.25%; approximately equivalent to 31.25 mg/kg) rats and increased incidences of skin (site of application) lesions (LOEL = 6.25%; approximately equivalent to 124 mg/kg) in male and female mice.
National Toxicology Program (NTP). 2016. NTP technical report on the toxicity studies of cedarwood oil (Virginia) (CASRN 8000-27-9) administered dermally to F344/N rats and B6C3F1/N mice. Research Triangle Park, NC: National Toxicology Program. Toxicity Report 86. https://doi.org/10.22427/NTP-TOX-86