Evaluation of the Developmental Toxicity of Linalool in Rats

Abstract

The developmental toxicity of linalool, a widely used fragrance ingredient, was evaluated in presumed pregnant Sprague-Dawley rats (25/group). Oral dosages of 0, 250, 500, or 1000 mg/kg/day linalool were administered by gavage on gestational days 7 to 17. The presence of spermatozoa and/or a copulatory plug in situ was designated as gestational day 0. Rats were observed for viability, clinical signs, body weights, and feed consumption. Caesarean sectioning and necropsy occurred on gestational day 21. Uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. Numbers of corpora lutea were also recorded. Fetuses were weighed and examined for gender, gross external changes, and soft tissue or skeletal alterations. There were no maternal deaths, clinical signs, or gross lesions that were considered related to linalool. During the dosage period, mean relative feed consumption was significantly reduced by 7% and mean body weight gains were reduced by 11% at 1000 mg/kg/day. During the postdosage period, feed consumption values at 1000 mg/kg/day were significantly higher than vehicle control values, which corresponded to the increase in body weight gains during this period. Caesarean section and litter parameters, as well as fetal alterations, were not affected by linalool at any of the three dosages tested. On the basis of these data, the maternal no observed adverse effect level (NOAEL) of linalool is 500 mg/kg/day, whereas the developmental NOAEL is ≥ 1000 mg/kg/day. It is concluded that linalool is not a developmental toxicant in rats at maternal doses of up to 1000 mg/kg/day.

Linalool (Figure 1), also known as 3,7-dimethyl-1,6-octadien-3-ol, is a colorless, fragrant liquid distilled from the oils of rosewood, bergamot, coriander seeds, or other plants and trees used in perfume manufacture (Arctander 1969). Many commercial applications exist, the majority of which are based on its pleasant scent (floral, with a touch of spiciness). The worldwide use of linalool is greater than 1000 metric tons per annum (IFRA 2004). More than 95% of synthetic linalool is used for its fragrance and odorant qualities in cosmetics, soaps, perfumes, household cleaners, waxes, and care products, whereas only approximately 1% is added to food and beverages for aroma and flavoring (ICCA 2002).

Human exposure to linalool is widespread, not only from ingredients of formulated food and beverages, cosmetics, and household products, but also from the natural constituent of fruits and spices. Oral exposure to linalool from formulated food products was estimated at up to 72 μg/kg/day for Europe and the USA; adding linalool from natural sources may possibly double this, resulting in an estimated maximal daily intake of 140 μg/kg/day (ICCA 2002). This maximum corresponds to approximately one quarter of the upper limit of the acceptable daily intake (ADI). There is enterohepatic recirculation, but linalool is excreted relatively rapidly by pulmonary and urinary pathways, and there is no tendency for bioaccumulation (ICCA 2002). The maximum daily human skin exposure from the use of multiple cosmetic products containing linalool has been calculated to be 0.3 mg/kg for a 60-kg high-end user of these products (IFRA 2001). How these data are obtained and exposure is determined is reported in Cadby, Troy, and Vey (2002).

The general toxicological characteristics of linalool are well documented (ICCA 2002; Letizia et al. 2003). Bickers et al. (2003) have summarized the significance of the toxicologic and dermatologic effects of linalool and related esters when used specifically as fragrance ingredients. A summary of pertinent findings included in two extensive general reviews (ICCA 2002; Letizia et al. 2003) indicate that linalool has an acute oral mammalian LD₅₀ of around 3.0 g/kg, whereas the acute dermal toxicity (one rabbit study) was calculated to be 5.61 g/kg (Letizia et al. 2003). Linalool is not genotoxic and not a sensitizer in humans, but it is irritating to the skin and eyes, based on rabbit studies when applied undiluted. A 32% solution in acetone is also a mild dermal irritant in humans; lower concentrations produced no irritation (Letizia et al. 2003). However, oxidized linalool has the potential to induce sensitization (Skold et al. 2002, 2004).

Subchronic studies have been performed. In a 28-day oral study, rats were gavaged once daily with 0, 160, 400, or 1000 mg/kg/day coriander oil (a natural source containing 72.9% linalool) in a 1% methylcellulose vehicle. A no observed effect level (NOEL) of 160 mg/kg/day (equivalent to 117 mg/kg linalool) for males and less than 160 mg/kg/day for females was determined (Letizia et al. 2003; RIFM 1990). In a 90-day dermal study, linalool was applied topically to Sprague-Dawley rats at doses of 250, 1000, or 4000 mg/kg/day. Dermal erythema was noted at all dose levels. At 250 mg/kg/day, there were no systemic changes observed (Letizia et al. 2003; RIFM 1980). Linalool is not considered to have a carcinogenic potential, based on an intraperitoneal carcinogenicity and an oral feed cocarcinogenicity study (ICCA 2002; Russin et al. 1989; Stoner et al. 1973).

Coriander oil has been investigated for reproductive and developmental toxicity. Female rats were gavaged daily with 0, 250, 500, or 1000 mg/kg/day of coriander oil (72.9% linalool) in a corn oil vehicle, beginning 7 days prior to mating and continuing through day 4 of lactation (RIFM 1989). The maternal NOEL of coriander oil was determined to be less than 250 mg/kg/day (equivalent to 183.3 mg linalool/kg/day) and the NOEL for the offspring was determined to be 500 mg/kg/day (364.5 mg linalool/kg/day) (ICCA 2002; Letizia et al. 2003; RIFM 1989). Despite the available toxicology profile of linalool, the previously conducted reproductive study was not considered to be up to modern standards because it was performed using a nonspecific test article of essential oil of coriander (with 72.9% linalool and 22.3% other identified terpenoids), old Food and Drug Administration (FDA) guidelines (1966), and a dosing period of 40 days (which may have resulted in metabolic enzyme induction).

The present study was designed and initiated to accurately evaluate the effects of linalool on International Conference on Harmonisation (ICH) Harmonized Tripartite Guideline stages C and D of the reproductive process. Requirements of the FDA (FDA 1994) were used as the basis for study design. All procedures were conducted in compliance with Good Laboratory Practice (GLP) regulations of the FDA (FDA 1987), the Japanese Ministry of Health and Welfare (MHW 1997), and the Organization for Economic Cooperation and Development (OECD 1998).

MATERIALS AND METHODS

Materials

Linalool, a clear, colorless liquid (lot number 5LM301) with an overall purity of 99.5%, was supplied by Millennium Specialty Chemicals (Jacksonville, FL). Corn oil (lot number 015K0115; Sigma-Aldrich, St. Louis, MO) was the vehicle and control article. Both substances were stored at room temperature and protected from light. Dosing formulations were prepared weekly from bulk materials. Samples from each concentration of the dosing suspensions (first and last days of treatment) were analyzed for linalool content by Charles River Laboratories Pre-clinical Services (Worcester, MA).

Animals

Crl:CD(SD) IGS BR VAF/Plus rats (Charles River Laboratories, Raleigh, NC) were used in the study. On the day of study assignment, female rats weighed 207 to 253 g. The rats were assigned to individual housing (stainless steel, wire-bottomed cages) on the basis of computer-generated random units, except during the mating period when each pair of male and female rats was housed in the male rat’s cage. Healthy, mated female rats were then assigned to four dosage groups, 25 rats/group, using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on the day on which sperm was found in the vaginal smear or a copulatory plug was found in the vagina. The presence of spermatozoa or a copulatory plug was designated as gestational day 0 (GD 0).

All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources 1996). The study room was independently supplied with at least ten changes per hour of 100% fresh air passed through 99.97% HEPA filters. Environmental controls were set to maintain temperatures of 64°F to 79°F, a relative humidity of 30% to 70% and a 12:12-h light-dark lighting cycle. Certified Rodent Diet 5002 (PMI Nutrition International, St. Louis, MO) and reverse-osmosis deionized water were provided ad libitum to the rats.

Methods

Dosages for the present study were selected on the basis of a range-finding study in which linalool in a corn oil vehicle was administered daily by gavage to presumed pregnant rats at dosages of 0, 125, 250, 500, or 1000 mg/kg/day on GDs 7 to 17. An 8% decrease in feed consumption at 1000 mg/kg/day during the dosage period was the only adverse effect noted in the dams. There were no untoward changes noted in caesarean sectioning or litter parameters, and no fetal external alterations were found at any dosage level. Based on these data, dosages of 0, 250, 500, and 1000 mg/kg/day linalool were selected for the definitive developmental toxicity study in rats.

Linalool in a corn oil vehicle was administered by gavage to four groups of pregnant rats on GDs 7 to 17 at dosages of 0, 250, 500, or 1000 mg/kg/day. The dosage volume was 10 ml/kg, adjusted daily according to individual body weights recorded directly before gavage and administered at approximately the same time each day.

Animals were observed twice daily for viability and examined for clinical signs, abortions, and premature deliveries before dosage administration and approximately one hour later. Body weights were recorded prior to the start of the study and daily during the dosage and postdosage periods. Feed consumption was recorded on GDs 0, 7, 10, 12, 15, 18, and 21. On GD 21, all rats were euthanized by inhalation of carbon dioxide. Following caesarean sectioning, a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. Uteri of apparently nonpregnant rats were examined while pressed between glass plates to confirm the absence of implantation sites. Uteri from pregnant rats were excised and examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. The number of corpora lutea in each ovary was recorded.

Fetuses were removed from the uterus, weighed, and examined for gender and gross external alterations. Live fetuses then were euthanized by an intraperitoneal injection of pentobarbital before undergoing further examination. Approximately half of the fetuses in each litter were fixed in Bouin’s solution and examined for soft tissue alterations, using a variation of Wilson’s sectioning technique (Staples 1974). The remaining fetuses in each litter were eviscerated, cleared, stained with alizarin red S (Staples and Schnell 1964), and examined for skeletal alterations.

Data generated during the course of study were recorded either by hand or using the Argus Automated Data Collection and Management System and the Vivarium Temperature and Relative Humidity Monitoring System. All data were tabulated, summarized, and/or statistically analyzed using the above systems in conjunction with Microsoft Excel (Microsoft Office 97/2000XP), Quattro Pro 8, and/or The SAS System (version 6.12). Clinical observation and other proportion data were analyzed using the variance test for homogeneity of the binomial distribution (Snedecor and Cochran 1967a). Continuous data were analyzed using Bartlett’s test of homogeneity of variances (Sokal and Rohlf 1969a) and the analysis of variance (Snedecor and Cochran 1967b), when appropriate. Dunnett’s test (Dunnett 1955) was used to identify statistical significance of differences among individual groups. If the analysis of variance was not appropriate, the Kruskal-Wallis test (Sokal and Rohlf 1969b) or Dunn’s method of multiple comparisons (Dunn 1964) was used to identify the statistical significance of differences among the individual groups. If there were greater than 75% ties, Fisher’s exact test (Siegel 1956) was used to analyze the data.

RESULTS

Results of concentration analyses from the start and end of study revealed that all prepared formulations were within the acceptable limits of ±15%. All homogeneity results were within the acceptable range of ≤2% relative standard deviation (RSD).

No maternal mortality occurred during the study. There were no clinical signs of toxicity. All abnormal necropsy observations on GD 21 were considered unrelated to linalool because they occurred in single animals and were unrelated to dose.

No statistically significant differences occurred in mean body weights at necropsy or body weight gains at dosages as high as 1000 mg/kg/day (Table 1). However, compared to vehicle control group values, mean body weight gains were reduced by 11% in the 1000 mg/kg/day dosage group during the dosing period, whereas a compensatory 7% increase occurred during the post-dosage period (GDs 18 to 21). Mean maternal relative and absolute feed consumption values paralleled the body weight gains, with a statistically significant reduction at some time points in the 1000 mg/kg/day dosage group (Table 1).

Pregnancy occurred in 22 (88%), 23 (92%), 20 (80%), and 22 (84%) rats in the four respective dosage groups. One dam in the 250 mg/kg/day dosage group delivered early on the day of scheduled sacrifice; as a result, caesarean-sectioning observations on GD 21 were based on 22, 22, 20, and 22 deliveries. The early delivery was considered unrelated to linalool because this incident was not dose dependent, and no adverse clinical signs or abnormal feed consumption or body weight values were observed prior to delivery. There were no gross lesions at necropsy, and the litter for this dam consisted of 12 live-delivered pups and one early in utero resorption. All pups appeared normal and no gross external, soft tissue, or skeletal alterations were noted.

No caesarean-sectioning or litter parameters were affected by dosages of linalool as high as 1000 mg/kg/day (Table 2). The litter averages for corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, percent resorbed conceptuses, percent live fetuses, and fetal body weights were comparable among the four dosage groups and did not significantly differ. There were four dead fetuses in the 0 (vehicle) mg/kg/day dosage group; no other dead fetuses were observed. No dam had a litter that consisted of all resorbed conceptuses. All placentae appeared normal (Table 2).

Fetal evaluations were based on the 277, 293, 254, and 271 live GD 21 caesarean-delivered fetuses in 22, 22, 20, and 22 litters in the 0, 250, 500, and 1000 mg/kg/day dosage groups, respectively. Each of these fetuses was examined for gross external alterations, whereas 135, 141, 122, and 127 were examined for soft tissue alterations, 146, 152, 132, and 144 were examined for skeletal alterations, and 143, 152, 132, and 144 were examined for ossification site averages. The numbers of fetal alterations observed in each dosage group are summarized in Table 3. Fetal alterations are defined as either malformations (irreversible changes that occur at low incidences with this species and strain) or variations (common findings in this species and strain and reversible delays or accelerations in development).

Gross external alterations included: unilateral cleft palate and whole-body edema in one fetus at 1000 mg/kg/day and a shortened trunk in a fetus at 250 mg/kg/day. Soft tissue changes included only one malformation, microphthalmia (one eye) in one control fetus, which also had a slight dilation of the renal pelvis (a variation that also occurred in another control fetus and in one fetus at 500 mg/kg/day). Umbilical artery variations were present in 0, 2, 2, and 1 fetus from the 0, 250, 500, and 1000 mg/kg dosage groups, respectively.

Skeletal malformations included a cleft palate, which was incompletely ossified, as were the right premaxilla and maxilla, in the edematous previously mentioned fetus at 1000 mg/kg/day. This fetus also had multiple skeletal alterations, including two incompletely ossified sternebral centra; irregular or fused arches in multiple vertebrae; missing thoracic vertebrae; unattached or fused, close-set ribs; and irregular or missing lumbar vertebrae. Fused arches in one or more thoracic vertebrae and a lack of ossification in two sternal centra and in the centrum of the third thoracic vertebra were also observed in one fetus at 500 mg/kg/day, whereas multiple rib and vertebral alterations were noted in the fetus with the shortened body at 250 mg/kg/day. Several minor skeletal variations occurred in a few other control or treated fetuses, but the incidences were not dosage or treatment related.

The noted gross external, soft tissue, or skeletal fetal alterations (malformations or variations) were not considered to be caused by linalool because these changes were not dosage dependent, did not significantly differ from vehicle control group values, and the incidences were within the ranges observed historically at the Testing Facility. In 101 studies (1685 litters; 24,434 fetuses) conducted at the Testing Facility during a 2-year period just prior to when the study was conducted, cleft palate occurred in a total of three control group fetuses, one in each of three different studies (litter incidence = 0.18%; range per study = 0 to 1 [0% to 4.3%]; fetal incidence = 0.01%; range per study = 0 to 1 [0% to 0.3%]). Whole body edema occurred in a total of three control group fetuses, one in each of three different studies (litter incidence = 0.18%; range per study = 0 to 1 [0% to 4.3%]; fetal incidence = 0.01%; range per study = 0 to 1 [0% to 0.3%]). All ossification averages were comparable to vehicle control group values and did not significantly differ among the groups.

Based on the above data, the maternal no observed adverse effect level (NOAEL) of linalool is 500 mg/kg/day due to non-significant reductions in body weight gain and significant reductions in feed consumption. However, in the postdosage period (GDs 18 to 21), these effects of linalool were reversed. The developmental NOAEL is greater than or equal to 1000 mg/kg/day.

DISCUSSION

The primary purpose of this study was to determine if daily exposure to high systemic dosages of linalool during the formative stages of pregnancy (GDs 7 to 18 for the rat) produced any potential adverse effects in pregnant rats or in the developing embryo-fetus, and to determine the maternal and developmental NOAELs. This objective is most easily and accurately achieved by gavage administration (although most linalool use is dermal) because it precludes the dermal irritation observed in rats (Letizia et al. 2003; RIFM 1980) and rabbits (Letizia et al. 2003; RIFM 1984, 1985, 1986), which can cause undue maternal stress and uncertain dermal absorption. The dosing period of GDs 7 to17 was selected because the rat embryo is most susceptible to developmental changes early in gestation (GDs 8 to 11), whereas late in gestation (GDs 17 to 21) growth is the predominant feature (Wilson 1965).

Coriander oil, a natural source containing 72.9% linalool, has also been investigated for reproductive and developmental toxicity (Letizia et al. 2003; RIFM 1989). In the reproductive and developmental toxicity screening test, female rats were gavaged daily with 0, 250, 500, or 1000 mg/kg/day of coriander oil (72.9% linalool) in a corn oil vehicle, with daily dosing beginning 7 days prior to a 7-day cohabitation period with male rats and continuing until day 4 of lactation (RIFM 1989). There was no maternal mortality, but effects on the pregnant dams included excess salivation in all dosage groups, and urine-stained abdominal fur and ataxia and/or decreased motor function at the 1000 mg/kg/day dosage level. Effects on bodyweight and feed consumption were observed at all dosage levels. Reproductive performance of the female rats was not adversely affected by treatment with coriander oil. At the 1000 mg/kg/day dosage level, decreased live litter size, indicating in utero deaths, and a statistically significant increase in pup mortality was observed. The maternal NOEL of coriander oil was determined to be less than 250 mg/kg/day (equivalent to 183.3 mg linalool/kg/day). The NOEL for the offspring was determined to be 500 mg/kg/day (365 mg linalool/kg/day) because a marked decrease in live litter size, indications of in utero deaths, and a statistically significant increase in pup mortality on day 1 was observed at 1000 mg/kg/day. Coriander oil was determined to be not hazardous to the reproductive performance of female rats or development of their offspring because the adverse effects observed occurred at maternally toxic dosages (ICCA 2002; Letizia et al. 2003; RIFM 1989).

The adverse effects noted in the coriander oil study (RIFM 1989) were not observed in the present investigation, which used synthetically produced linalool. Most probably one or more of the minor components of coriander oil may have produced the untoward effects, although the exact cause has not been determined.

Results from the present definitive developmental toxicity study demonstrated that the maternal NOAEL for linalool is 500 mg/kg/day and the developmental NOAEL is equal to or greater than 1000 mg/kg/day. Human skin exposure to linalool has been calculated using multiple cosmetic products containing linalool. The determinant factors for fragrance exposure are quantities of cosmetic used, frequency of use, and concentration of the fragrance material in these products (Ford et al. 2000). Using these factors, total maximum daily exposure on the skin has been determined to be 0.3 mg/kg for high-end users of these products. Based on these calculations and the data from the present investigation, a safety factor of greater than 1500 exists between maximum daily human dermal exposure and possible adverse fetal exposure. It is concluded that linalool is not a developmental toxicant in rats under the conditions tested.