Evaluation of the Developmental Toxicity of Dihydromyrcenol in Rats

Abstract

Dihydromyrcenol, a widely used fragrance ingredient, was evaluated for developmental toxicity in pregnant Sprague-Dawley rats (25/group). Oral dosages of 0, 250, 500, or 1000 mg/kg/d in corn oil were administered on gestational days 7 to 17. Rats were observed for viability, clinical signs, body weights, and feed consumption. Caesarean sectioning and necropsy occurred on gestational day 21. Fetuses were weighed and examined for sex, gross external changes, and soft tissue or skeletal alterations. All rats survived until scheduled termination. No clinical signs were attributed to dihydromyrcenol. There were no gross tissue changes at necropsy. The 1000-mg/kg/d dosage group had reduced mean maternal body weight gains of 5% compared with controls, whereas absolute and relative feed consumption were significantly reduced during the dosage period. This threshold systemic maternal toxicity was associated with threshold developmental toxicity in the 1000-mg/kg/d dosage group. Fetal effects included a minimal ∼3% reduction in fetal body weight; reversible variations in ossification, including retarded ossification of the metatarsal bones in the hindpaws; and an increase in supernumerary thoracic ribs with associated increases or decreases in thoracic and lumbar vertebrae, respectively. Based on these data, maternal and developmental no observable effect levels of 500 mg/kg/d and maternal and developmental no observable adverse effect levels of 1000 mg/kg/d were established for dihydromyrcenol. It was concluded that dihydromyrcenol is not a selective developmental toxicant in rats under the conditions of this study and that a margin of safety of 25 000 exists between reversible developmental delays in rats and the estimated daily human exposure level of 0.02 mg/kg/d.

Dihydromyrcenol is a colorless liquid that is a mixture of approximately 44.2% 2,6- dimethyl-7-octen-2-ol and 54.8% 2,6- dimethyl-7-octen-2-yl formate. The molecular structures are presented in Figure 1.

Dihydromyrcenol has a strong fresh-lime cologne odor, which makes it useful for use as a fragrance in soaps, shampoos, antiperspirants and lotions, and detergents and cleaning agents. Its worldwide volume of use is greater than 1000 metric tons per annum. ¹ It is not found in nature. The maximum human skin exposure from multiple cosmetic products containing dihydromyrcenol has been calculated to be 0.3 mg/kg/d. ² Determination of systemic exposure for fragrance materials is based on the quantities of cosmetic used, the frequency of use, the concentration of the fragrance material in these products, and 100% skin permeation. ³ Cadby et al ⁴ reported how these data are obtained and how exposure is determined in the absence of skin absorption data. For a more accurate estimation, the in vitro human skin absorption of dihydromyrcenol was measured under both in-use (unoccluded) and occluded conditions. Under the most severe conditions, occluded in a 70:30 ethanol/water vehicle, only 5.7% of dihydromyrcenol was absorbed. ⁵ As such, correcting for skin absorption, the maximum daily exposure on the skin is 0.02 mg/kg/d dihydromyrcenol for high-end users.

The general toxicological characteristics of dihydromyrcenol have been evaluated. ^6,7 Briefly, the oral LD₅₀ in rats was 3.0 g/kg ⁸ and 4.1 g/kg. ⁹ By the dermal route, the rabbit LD₅₀ exceeded 5 g/kg. ⁹ A 90- day oral subchronic toxicity study was conducted in rats at dosages of 0, 10, 50, 500, or 1000 mg/kg/d dihydromyrcenol in corn oil. Statistically significant reductions in body weight gain were observed in both sexes at 1000 mg/kg/d and in 500-mg/kg/d males. Hematological and blood chemistry effects were observed in both sexes at 500 and 1000 mg/kg/d and in males treated with 50 mg/kg/d. Statistically significant increases in liver and kidney weights were noted in both sexes in the 500- and 1000-mg/kg/d groups and males in the 50-mg/kg/d dose group. A statistically significant greater incidence and/or severity of groups of basophilic tubules and/or globular accumulations of eosinophilic material were observed in males in all dose groups. Statistically significant tubular necrosis was also observed in males in the 500- and 1000-mg/kg/d dose groups. Thus, for systemic toxicity, the no observable adverse effect level (NOAEL) for male rats was established at 10 mg/kg/d, whereas the NOAEL in female rats was set at 50 mg/kg/d. ¹⁰ Dihydromyrcenol was not mutagenic in a reverse-mutation (Ames) assay. ¹¹

A lack of developmental or reproductive toxicity studies on dihydromyrcenol in the scientific literature prompted the Research Institute for Fragrance Materials to contract Charles River Laboratories Preclinical Services to conduct a developmental toxicity study to evaluate the continued safe use of this substance in fragrances. The study was designed to meet requirements of the Food and Drug Administration ¹² for the evaluation of International Conference on Harmonization (ICH) Harmonized Tripartite Guideline stages C and D of the reproductive process and to determine the maternal and developmental NOAELs in Sprague-Dawley rats. All procedures were conducted in compliance with the good laboratory practice (GLP) regulations of the Food and Drug Administration, ¹³ the Japanese Ministry of Health and Welfare, ¹⁴ and the Organization for Economic Cooperation and Development. ¹⁵

Materials and Methods

Materials

Dihydromyrcenol is a clear, colorless liquid that is a mixture of approximately 44.2% 2,6-dimethyl-7- octen-2-ol (CAS #18479-58-8, C₁₀H₂₀O; molecular weight 156.27) and 54.8% 2,6-dimethyl-7-octen- 2-yl formate (CAS #25279-09-8, C₁₁H₂₀O₂; molecular weight 184.79). Dihydromyrcenol (lot number 4103311) was supplied by International Flavors and Fragrances, Inc (Union Beach, New Jersey). Corn oil (Sigma-Aldrich, Inc, St. Louis, Missouri) was the vehicle and control article. Both dihydromyrcenol and corn oil were stored at room temperature and protected from light. Dosing formulations were prepared weekly from bulk materials and stirred continuously during use.

Samples from each dosing suspension concentration (first and last days of treatment) were analyzed for dihydromyrcenol content by Charles River Laboratories Preclinical Services. Results of all dihydromyrcenol concentration and homogeneity analyses were within ±15% of calculated concentrations and ≤5% relative standard deviation, respectively. The concentrations of the formulations used were found to be stable at room temperature for 10 days.

Animals

Crl:CD (SD) male and female rats approximately 62 days of age on arrival (Charles River Laboratories, Inc, Raleigh, North Carolina) were used in the study. After a short acclimatization period, the rats were assigned to individual housing on the basis of computer-generated random units, except during the 5-day mating period, when each pair of male and female rats was housed in the male rat’s cage. The healthy, mated female rats, weighing 218 to 244 g, were assigned to 4 dosage groups, 25 rats/group, using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on the day when sperm was found in the vaginal smear or a copulatory plug was found in the vagina. The presence of spermatozoa and/or a copulatory plug in situ 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. ¹⁶ The study room was independently supplied with at least 10 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 with relative humidity of 30% to 70%; a 12:12-hour light-dark lighting cycle was used. Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) and reverse osmosis deionized water (with chlorine added to the processed water as a bacteriostat) were provided ad libitum to the rats.

Methods

Dosages of dihydromyrcenol were selected on the basis of a range-finding study, in which 0 (corn oil), 125, 250, 500, or 1000 mg/kg/d was gavaged daily to 8 rats/group on GDs 7 to 17. No mortality or clinical signs were produced at dosages as high as 1000 mg/kg/d, but body weight gains were reduced by 10.0% and absolute and relative feed consumption values were reduced by 19.5% and 17.5%, respectively, in the 1000-mg/kg/d dosage group during the dosage period. No caesarean sectioning or litter parameters were affected by dosages of dihydromyrcenol as high as 1000 mg/kg/d, and no treatment-related fetal gross external alterations (malformations or variations) occurred. Based on these data, dosages of 0, 250, 500, or 1000 mg/kg/d were selected for the definitive developmental toxicity study.

Dihydromyrcenol in a corn oil vehicle was administered orally via gavage to 4 groups of presumed pregnant rats on GDs 7 to 17 at dosages of 0 (corn oil), 250, 500, or 1000 mg/kg/d. The dosage volume of 10 mL/kg was adjusted daily according to individual body weights recorded directly before gavage and was administered at approximately the same time each day.

Animals were observed for viability and examined for abnormal clinical signs twice daily: before dosage administration and approximately 1 hour later. Body weights were recorded prior to the start of the study, on GD 0, 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 and caesarean sectioned, and 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 resorptions. The number of corpora lutea in each ovary also 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. ^17,18 The remaining fetuses in each litter were eviscerated, cleared, stained with alizarin red S, ¹⁹ and examined for skeletal alterations.

Data generated during the course of study were recorded either by hand or by 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 97/2000/XP), Quattro Pro 8, and/or SAS(Version 6.12).

Clinical observations and other proportional data were analyzed by using the variance test for homogeneity of the binomial distribution. ²⁰ Continuous data were analyzed by using Bartlett’s test of homogeneity of variances ²¹ and the analysis of variance. ²⁰ Dunnett’s test ²² was used to identify statistical significance of individual groups. If the analysis of variance was not appropriate, the Kruskal-Wallis test ²¹ or Dunn’s method of multiple comparisons ²³ was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s exact test ²⁴ was used to analyze the data.

Results

All rats survived until scheduled sacrifice on GD 21. Clinical signs included minor incidences of sparse hair coat or localized alopecia, excess salivation, urine-stained abdominal fur, ungroomed coat, rales, ptosis, chromodacryorrhea (colored tears), and soft or liquid feces (Table 1). These observations were all considered unrelated to dihydromyrcenol because the incidences were observed in vehicle control rats, were not dosage dependent, or were transient and did not persist. No gross lesions were noted at necropsy.

Both maternal absolute (g/d) and relative (g/kg/d) feed consumption values were significantly reduced in the 1000-mg/kg/d group for the entire dosage period compared with vehicle controls (see Table 2 for absolute feed data). Reduced feed consumption was most prominent during GDs 7 to 10, which correlated with the negative weight gains during the initial days of the dosage period (Table 2). Body weight gains in the high-dose group for the entire dosage period were reduced by 5% when compared with controls, an observation that was not statistically significant but was considered evidence of a threshold for maternal toxicity. Dosages of dihydromyrcenol as high as 500 mg/kg/d had no adverse effects on feed consumption or body weight gains.

Pregnancy occurred in 22 to 25 rats per dosage group. Caesarean-sectioning observations on GD 21 were based on 22, 24, 24, and 25 pregnant rats with 1 or more live fetuses in the 0-, 250-, 500-, and 1000-mg/kg/d dosage groups, respectively (Table 3). There were no dead fetuses.

Body weights for combined male and female fetuses were reduced approximately 3% in the 1000- mg/kg/d dosage group compared with concurrent vehicle controls. The reduction in female fetuses was statistically significant (P ≤.05). Although the values were within historical control values for the testing facility (Table 3), the reduction correlated with reduced maternal body weight gain and feed consumption that occurred at 1000 mg/kg/d early in the dosage period. No other caesarean sectioning or litter parameters were affected by dosages of dihydromyrcenol as high as 1000 mg/kg/d.

Fetal morphological evaluations were based on 295, 311, 350, and 358 live GD21 caesarean-delivered fetuses in the 0-, 250-, 500-, and 1000-mg/kg/d dosage groups, respectively. Each of these fetuses was examined for gross external alterations. Of the respective fetuses, 140, 147, 167, and 174 were examined for soft tissue alterations, and 155, 164, 183, and 184 were examined for skeletal alterations and fetal ossification site averages. Fetal alterations were defined as (1) malformations (irreversible changes that occur at low incidences in this species and strain) or (2) variations (common findings in this species and strain and reversible delays or accelerations in development). Litter averages were calculated for specific fetal ossification sites as part of the evaluation of the degree of fetal ossification.

No gross external, soft tissue, or skeletal malformations were attributable to dosages of dihydromyrcenol as high as 1000 mg/kg/d. Significant increases in fetal variations attributable to the test article were observed only at the 1000-mg/kg/d dosage level. These changes consisted of reversible minor variations, including (1) a threshold but statistically significant increase in supernumerary ribs, along with associated significant increases and decreases in the respective numbers of thoracic and lumbar vertebrae, and (2) a small but statistically significant retardation in ossification of the metatarsal bones in the hindpaws, evident as a reduction in the mean number of ossified metatarsal bones (Table 4). The incidence of all fetal alterations is summarized in Table 5.

Discussion

The primary purpose of the study was to (1) determine whether daily systemic exposure to dihydromyrcenol during the formative stages of pregnancy could produce potential adverse effects in pregnant rats or in the developing embryo-fetus and (2) determine the maternal and developmental no observable effect levels (NOELs) and/or NOAELs.

Most present dihydromyrcenol use in fragrances is topical, but the above objectives are most easily and accurately achieved by gavage administration. Systemic exposure is often limited or hard to interpret with topical application due to confounding factors such as licking of application sites, irritation, or occlusion, all of which can affect skin absorption, skin metabolism, and skin macromolecules. ²⁵

Results from the present study indicate that at 1000 mg/kg/d, a dosage level defined as a limit dosage by ICH guidelines, ¹² dihydromyrcenol produced threshold levels of maternal and developmental toxicity. The significant reductions in maternal feed consumption that occurred during the first 6 days of the dosage period and the associated 5% reductions in maternal body weight gains during the entire dosage period are sufficient evidence that a maternally toxic level was tested.

Body weights for female fetuses were minimally but statistically significantly reduced in the 1000- mg/kg/d dosage group compared with concurrent vehicle controls. Although the values were within historical control values for the testing facility, the reduction correlated with a maternally toxic dosage.

The only developmental alteration that occurred at an incidence that exceeded the historical control range of the testing facility was a common variation, an increase in supernumerary thoracic ribs, along with associated increases and decreases in respective thoracic and lumbar vertebrae. This minor variation is often observed at maternally toxic dosages ²⁶ and, because it disappears with continued growth, ^27,28 is generally not considered to be an adverse effect. A minimal reversible delay in ossification ²⁹ of the metatarsal bones in the hindpaws, manifested by a significant reduction (compared with the control group) in the mean number of ossified hindpaw metatarsals, was also observed in fetuses from the 1000-mg/kg/d dosage group. This was not considered of toxicological importance because the value was within the historical range observed at the testing facility, and other ossification sites (fore/hindlimbs, hyoid, sternum, and vertebrae) were not affected.

Because statistically significant maternal and developmental findings were observed in this study at 1000 mg/kg/d, the maternal and developmental NOELs have been identified as 500 mg/kg/d. Maternal and developmental effects observed at 1000 mg/kg/d were considered a threshold and consistent with NOAELs. The current data also indicate that dihydromyrcenol is not a selective developmental hazard in rats and that a margin of safety of 25 000 exists between reversible developmental alterations in rats and the estimated daily human exposure level of 0.02 mg/kg/d.