Abstract
The developmental toxicity of acetyl cedrene (AC), a widely used fragrance ingredient, was evaluated in pregnant Sprague-Dawley rats (25/group). Gavaged dosages of 0 (corn oil), 25, 50, or 100 mg/kg/day were administered on days 7 through 17 of gestation (GDs 7 to 17). First and last day dosing suspensions were analyzed for AC content. All rats were observed daily for viability, clinical signs, abortions, and premature deliveries. Body weights were recorded at frequent intervals. Cesarean-sectioning and necropsy examinations were performed on GD 21. Uteri were 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 also recorded. Fetuses were weighed and examined for gender and gross external changes and soft tissue or skeletal alterations. Totals of 25, 23, 21, and 24 rats became pregnant in the 0 (control), 25, 50 and 100 mg/kg/day groups, respectively, and analysis of dosage preparations verified that administered dosages reflected calculated dosages ±10%. No deaths or premature deliveries occurred in the study. Clinical signs included excessive salivation, which was attributed to the administration of AC. When compared to controls, significant reductions in feed consumption and body weight gains occurred only at 100 mg/kg/day. Both absolute (g/day) and relative (g/kg/day) feed consumption values were significantly decreased on GDs 7 to 12. Relative values were decreased significantly on GDs 15 to 18. Body weight gains were significantly reduced on GDs 7 to 10. Mean maternal body weights remained significantly lower than controls on GDs 9 to 14, but a marked compensatory increase in feed consumption on GDs 15 to 18 prevented further deterioration in body weight gains. No cesarean-sectioning or litter parameters were affected by dosages of AC and necropsy of the dams after cesarean section did not reveal any gross changes attributable to AC. No gross external, soft tissue, or skeletal fetal alterations (malformations or variations) were attributed by dosages AC. The average number of ossifications sites per fetus per litter did not differ among the groups. Based on these data, maternal and developmental no-observable-adverse-effect levels (NOAELs) of 50 and 100 mg/kg/day, respectively, were established for AC.
Acetyl cedrene (AC), also known as methyl cedryl ketone, is a clear, yellowish brown liquid with a warm wood aroma with musky undertones (CAS Registry number 32388-55-9). It is an aliphatic ketone with a molecular weight of 246.39 Da that has been used widely as a fragrance material since the 1950s. AC is prepared by acetylation of the hydrocarbon portion of cedarwood oil in the presence of an acid catalyst. The molecular formula of AC is given in Figure 1. A conservative estimate of the maximum daily dermal exposure to AC in cosmetic products has been calculated to be 0.4 mg/kg/day (IFRA 1998).
The purpose of this study was to determine if exposure to AC during pregnancy could produce any potential adverse effects in pregnant rats or in the developing embryo-fetus and to determine the maternal and developmental no-observable-adverse-effect level (NOAEL) in Sprague-Dawley rats. Requirements of the Food and Drug Administration (US FDA 1994) were used as the basis for study design. All procedures were conducted in compliance with Good Laboratory Practice (GLP) regulations of the Food and Drug Administration (US 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
AC (lot number HRF053136) with an overall purity of 95%, was supplied by the Research Institute for Fragrance Materials, Inc. (Woodcliff Lake, NJ). Mazola corn oil (lot number APR1902A) was the vehicle and control article. Both substances were stored at room temperature. Dosing formulations were prepared daily from bulk materials. Samples from each concentration of the dosing suspensions (first and last days of treatment) were analyzed for AC content by International Flavors & Fragrances.
Animals
Crl:CD (SD) IGS BR VAF/Plus rats (Charles River Laboratories, Raleigh, North Carolina) were used in the study. On the day after arrival at the testing facility, the male rats weighed 272 to 378 g; the females weighed 178 to 219 g. The rats were assigned to individual housing 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 then were assigned to four 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 (gestation day 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 with relative humidity of 30% to 70%; a 12:12-h light-dark lighting cycle was used. Certified Rodent Diet 5002 (PMI Nutrition International, St. Louis, Missouri), as well as reverse-osmosis (R. O). deionized water, were provided ad libitum to the rats.
Procedures
Acetyl cedrene or corn oil was administered orally by gavage to four groups of pregnant rats on days 7 through 17 of gestation (GDs 7 through 17) at dosages of 0 (corn oil), 25, 50, or 100 mg/kg/day. The dosage volume was 1 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 daily for viability and examined for clinical signs, abortions, and premature deliveries. 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 carbon dioxide asphyxiation, cesarean 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 early and late resorptions. The number of corpora lutea in each ovary was also recorded.
Fetuses were removed from the uterus, weighed, and examined for gender and gross external alterations. Live fetuses then were euthanized by intraperitoneal injections of a solution of sodium 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 (Wilson 1965). 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, version SR-2) 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 1967b). Continuous data were analyzed using Bartlett’s test of homogeneity of variances (Sokal and Rohlf 1969a) and the analysis of variance (Snedecor and Cochran 1967a), when appropriate. Dunnett’s test (Dunnett 1955) was used to identify statistical significance of 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 the individual groups. If there were greater than 75% ties, Fisher’s exact test (Siegel 1956) was used to analyze the data.
RESULTS
Results from the analysis of dosage preparations verified that actual dosages reflected the calculated dosages ±10%. Achieved concentrations of AC were 0, 23.62, 44.95, and 102.87 mg/ml.
Totals of 25, 23, 21, and 24 rats became pregnant in the 0 (control), 25, 50, and 100 mg/kg/day groups. No deaths or premature deliveries occurred in the study. Excessive salivation, noted in 5/25 rats at 50 mg/kg/day and in 11/25 rats at 100 mg/kg/day, was attributed to the administration of AC. Necropsy of the dams after cesarian section did not reveal any morphological changes attributable to AC (a slight pelvic dilation of the right kidney in one dam at 100 mg/kg/day was considered to be congenital in origin).
Significantly lower mean body weight, compared to controls, reductions in feed consumption, and resultant body weight gains occurred only in the high dosage (100 mg/kg/day) group of dams. Also, body weight gains were significantly reduced in transient manner on GDs 7 to 10 in this dosage group. All other body weights and body weight gains were generally comparable among the four dosage groups (Table 1). Maternal absolute (g/day) (Table 2) and relative (g/kg/day) (Table 3) feed consumption values were significantly decreased on GDs 7 to 12 and 15 to 18 (relative only) and the entire dosage period (calculated as GDs7 to 18) in the 100 mg/kg/day dosage group.
No uterine, ovarian, or litter parameters were affected by the test articles in doses as high as 100 mg/kg/day (Table 4). The litter averages for corpora lutea, implantations, litter sizes, live fetuses, resorptions, fetal body weights, percent resorbed conceptuses, and percent live male fetuses were comparable among the four dosage groups and did not significantly differ. No dam had a litter consisting of only resorbed conceptuses; there were no dead fetuses.
Fetal evaluations were based on 359, 333, 302, and 360 live, GD-21, cesarean-derived fetuses in 25, 23, 21, and 24 litters in the 0 (vehicle), 25, 50, and 100 mg/kg/day dosage groups, respectively. Each fetus was examined for gross external alterations. Of the respective fetuses, 174, 161, 148, and 174 were examined for soft tissue alterations, and 185, 172, 154, and 186 were examined for skeletal alterations and the status of fetal ossification sites.
There were no fetal gross external alterations (malformations or variations), soft tissue malformations, or skeletal malformations observed in any dosage group. Soft tissue variations included moderate dilation of the lateral ventricles in one fetus in the 100 mg/kg/day dosage group; an absent innominate artery in one control fetus; aberrant umbilical arteries in 4, 2, 3, and 1 fetuses from 4, 2, 2, and 1 litters in the 0, 25, 50, and 100 mg/kg/day dosage groups, respectively; slight or moderate dilation of the renal pelvis from right or left kidneys in one control fetus, one fetus in each of four litters in the 25 mg/kg/day group, and one fetus in the 100 mg/kg/day group; and an undescended testis in one control and one 50 mg/kg/day fetus. Skeletal variations were all reversible delays in fetal ossification, except for the occurrence of cervical ribs, a common variation in this strain of rat (Khera 1981) at maternally toxic dosages.
None of the gross, soft tissue or skeletal alterations was attributed to the test article. There were no significant differences in the incidence of alterations in litters or individual fetuses from the four dosage groups (Table 5).
DISCUSSION
The primary object of this study was to determine if high oral doses of AC during pregnancy could produce potential adverse effects in pregnant rats or their developing embryo-fetuses. Although most current AC usage is topical, oral administration was chosen because exact dosages could be accurately administered. 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 (Poet 2000).
Dosages used in the described full study were determined from results obtained by a previous two-part dosage-range study in which eight pregnant rats per group were treated on GDs 7 through 17. In the first part of the study, four groups of rats were administered gavaged volumes of 0.25, 0.50, 1.0, or 2.0 ml/kg/day neat AC (250, 500, 1000, or 2000 mg/kg/day based on a specific gravity of 1.0). A fifth group (control) was administered 2 ml/kg of water. All rats at the highest dosage died or were sacrificed in a moribund state; one rat died and another delivered prematurely in the 1000 mg/kg/day group; dosage-related reductions in feed consumption and body weight gains occurred in all surviving AC-treated groups. Fetal body weights were reduced in the 500 and 1000 mg/kg groups. Depressed eye bulges were noted in two, one, and one fetuses from single litters in the 250, 500, and 1000 mg/kg/day groups, respectively. The fetus in the 1000 mg/kg/day group also had a cleft snout and palate. These external alterations were not considered to be due to AC because the incidences were within the historical ranges of the testing facility.
Part 2 of the dosage-range study included four groups of pregnant rats that were administered 0 (corn oil vehicle), 50, 100, or 250 mg AC/kg/day in a dosage volume of 1 ml/kg. No deaths occurred in any group; excess salivation, reduced feed consumption, and body weight gains were observed at 100 and 250 mg/kg/day; no cesarean-sectioning or litter parameters were affected by any AC dosage; there were no gross external fetal alterations.
Results from the full developmental toxicity study generally corroborated many of the findings in part 2 of the pilot study: clinical signs were minimal; feed consumption and body weight gains were reduced at 100 mg/kg/day; and cesarean-sectioning, litter, and fetal parameters were unaffected by dosages of test article as high as 100 mg/kg/day. Based on these data, the maternal no-observable-adverse-effect level (NOAEL) of AC is considered to be 50 mg/kg/day (100 mg/kg/day caused significant reductions in body weight gain and feed consumption). The developmental NOAEL for AC is greater than 100 mg/kg/day (no effects were observed at 100 mg/kg/day, the highest dosage tested).