Abstract
The developmental toxicity of 1-(1,2,3,4,5,6,7,8-Octahydro-2,3,8,8-tetramethyl-2-naphthalenyl) ethanone (OTNE), a widely used fragrance ingredient, was evaluated in pregnant Sprague-Dawley rats (25/group) gavaged with dosages of 0 (water), 96, 240, or 480 mg/kg/d on days 7 through 17 of gestation (GDs 7–17). Rats were observed for clinical signs, abortions, premature deliveries, body weights, and feed intake. Caesarean section and necropsy were performed on GD 21. Fetuses were weighed and examined for gender, gross external changes, and soft tissue or skeletal alterations. No deaths or premature deliveries were attributed to OTNE. OTNE-related clinical signs included significantly increased incidences of excessive salivation in all 3 treatment groups, and urine-stained abdominal fur in the high dosage group. Mean body weight gains were significantly reduced by all OTNE dosages on GDs 7–10, while at 480 mg/kg/d, significant reductions continued through the remainder of the dosage period. Feed consumption generally paralleled body weight gains. Fetal body weights were reduced by 480 mg/kg/d, but not to a statistically significant degree. No fetal gross external, soft tissue, or skeletal malformations or variations were attributable to OTNE. Based on these data, maternal and developmental no-observable-adverse-effect-levels (NOAELs) of 240 mg/kg/d were established for OTNE. It was concluded that OTNE is not a developmental toxicant in rats under the conditions of this study, and that a margin of safety greater than 2700 exists between reversible developmental delays in rats and the calculated daily human exposure level of 0.086 mg/kg/d.
OTNE [1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl) ethanone], a mixture of isomeric ketones, is a clear pale yellow liquid having a woody odor. The CAS Registry Number for the major isomer (
The maximum human skin exposure from multiple cosmetic products containing OTNE has been calculated to be 0.52 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. However, in a percutaneous absorption study designed to determine the in vitro human skin (cosmetic reduction skin) penetration rate and distribution of radiolabeled OTNE, only 16.5% of the applied dose was absorbed into the remaining epidermis and the receptor phase after 48 hours. 5,6 As such, correcting for skin absorption, the maximum daily systemic exposure to OTNE resulting from dermal exposure to multiple cosmetic products is 0.086 mg/kg/d.
A review of scientific literature and unpublished studies reported to the Research Institute for Fragrance Materials, Inc. (RIFM) revealed that the existing toxicological data on OTNE did not include developmental or reproductive toxicity studies. Acute toxicity studies in the rat show oral and dermal LD50 values greater than 5 g/kg. 7,8 A subchronic 4-week repeated dose oral (gavage) toxicity study was conducted in male and female rats at 0, 15, 150, and 1000 mg/kg/d OTNE in a corn oil vehicle. The systemic No-Observable-Adverse-Effect-Level (NOAEL) was determined to be 150 mg/kg/d and the No-Observable-Effect-Level (NOEL) was 15 mg/kg/d due to the presence of eosinophilic inclusions in the cortical tubules of kidneys from male rats dosed with 150 and 1000 mg/kg/d. 9 OTNE was not mutagenic in a bacterial reverse mutation (Ames) assay nor in a chromosome aberration assay in human lymphocytes. 10,11
The purposes for the present study were to determine whether exposure to OTNE during pregnancy could produce any potential adverse effects in pregnant rats or in their developing embryo-fetuses and to determine the maternal and developmental No-Observed-Adverse-Effect-Levels (NOAELs) in Sprague-Dawley rats. ICH Guidelines, as published by the U.S. Food and Drug Administration, 12 were used as the basis for study design. The study was conducted in compliance with Good Laboratory Practice (GLP) regulations of the U.S. 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
OTNE, a pale yellow liquid (Lot number 54461) with an overall purity of 97%, was supplied by I.F.F., Inc. (Union Beach, NJ) and stored at room temperature. Deionized water was used as the control article.
Animals
Crl:CD(SD) IGS BR VAF/Plus rats (Charles River Laboratories, Inc., Raleigh, NC) were used in the study. On the day after arrival at the testing facility, the male rats weighed 310–389 g, while the female rats weighed 171–225 g. The rats were assigned to individual housing, 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 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 seminal 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
Methods
Dosages used in the present study were determined from results obtained by a previous pilot study in which dosages of 240, 480, 960, or 1920 mg/kg/d OTNE (at volumes of 0.25, 0.50, 1.0, or 2.0 mL/kg neat OTNE) were given by gavage to groups of 8 presumed pregnant rats on gestational days 7 through 17 (GDs 7–17). A group of 8 control rats were dosed with 2.0 mg/kg/d deionized water by gavage. The highest dosage produced mortality in 1 of 8 dams; the 2 highest dosages produced marked decreases in body weight gains and feed consumption during the entire gestation period after the initiation of dosage administration (GDs 7–17). Caesarean-sectioning or litter parameters were not affected by these dosages. Fetuses were weighed and examined for sex and gross external alterations. No Caesarean-section or litter parameters were affected by dosages of OTNE as high as 1920 mg/kg/d.
In the full study, dosages of 96, 240, and 480 mg/kg/d neat OTNE were administered by gavage to 3 groups of 25 pregnant rats on GDs 7–17 at dosage volumes of 0.1, 0.25, and 0.5 mg/kg/d, respectively, adjusted daily on the basis of the individual body weights recorded directly before treatment and administered at approximately the same time each day. A control group of 25 pregnant rats were administered deionized water by gavage at a volume of 0.5 mL/kg. Animals were observed daily for clinical signs, abortions, and premature delivery. Body weights were recorded before 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 killed by CO2 asphyxiation, 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 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 Beuthanasia®-D Special (Schering-Plough Animal Health) before undergoing further examination. 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 The remaining fetuses in each litter were eviscerated, cleared, stained with alizarin red S, 18 and examined for skeletal alterations.
Data generated during the course of study were recorded either by hand or using the
Results
Totals of 22, 25, 24, and 25 rats were pregnant in the control, 96, 240, and 480 mg/kg/d groups, respectively. No deaths or premature deliveries were produced by OTNE. Two rats from the 240 mg/kg/d dosage group were found dead (1 on GD 17, the other on GD 20), and 1 dam from the same group delivered prematurely (GD 20). These events were considered unrelated to OTNE because they were not dosage-dependent (1 death was due to an intubation accident, the cause of the other death was undetermined). Examination of the uterine contents of the dead dams revealed normal litter sizes; the fetuses, although dead, were well developed for their gestational ages and had no discernable gross abnormalities. The premature delivery produced 15 normal-sized, live fetuses, which indicated that the timing of the mating had been misdiagnosed. All other dams survived to scheduled Caesarean-sectioning. Clinical signs considered to be related to OTNE included the following: a significantly increased incidence of excessive salivation (usually after 6 or 7 dosages in most of the rats at all 3 dosage levels), and a significantly increased incidence of urine-stained abdominal fur (4 rats) in the 480 mg/kg/d dosage group.
Significant reductions in body weight gains were observed in all 3 (96, 240, and 480 mg/kg/d) dosage groups on GDs 7–10, compared to the control group value. After that period, body weight gains continued to be reduced significantly only in the 480 mg/kg/d dosage group throughout the remainder of the dosage period (Table 1). Absolute (g/d) and relative (g/kg/d) feed consumption values also were reduced or significantly reduced during the first half of the dosage period (GDs 7–12) in the 3 OTNE-treated groups, when compared with the control group. After GD 12, absolute and relative feed consumption values were comparable for the control, 96, and 240 mg/kg/d dosage groups, while significant reductions continued at 480 mg/kg/d (data not presented).
All Caesarean-sectioning and litter parameters were unaffected by dosages of the test article as high as 480 mg/kg/d. There were no statistically significant differences among the 4 dosage groups in the litter averages for corpora lutea, implantations, resorptions, litter sizes, fetal body weights, and live or dead fetuses. There was a slight downward trend in fetal body weights, but the results were not statistically significant (Table 2).
All malformations and variations in the fetuses were considered unrelated to OTNE (Table 3). The number of ossification sites per fetus per litter was unaffected by any of the OTNE dosages. Fetal evaluations were based on 321, 372, 306, and 381 live, 21-day Caesarean-derived fetuses from the 22, 25, 21, and 25 litters in the 0, 96, 240, and 480 mg/kg/d dosage groups, respectively. Of these respective fetuses, 154, 181, 148, and 180 were examined for soft tissue alterations, and 167, 191, 158, and 201 were examined for skeletal changes and fetal ossification sites. Gross external malformations were present only in 3 fetuses in the 96 mg/kg/d group. One fetus had a meningocele, depressed eye bulge, low set ears, and agnathia; multiple associated malformations of the skull were also noted during skeletal evaluation. Another fetus had a short trunk, thread-like tail, and soft tissue malformations consisting of fused kidneys with ectopic ovaries. The third fetus had a thread-like tail; soft tissue examination did not reveal additional alterations in this specimen.
Discussion
The primary object of this study was to determine whether exposure to OTNE during gestation could produce any potential adverse effects in pregnant rats or their developing embryo-fetuses. The oral route of administration was chosen, although most current use of OTNE is topical, because maximal exposure was desired and systemic exposure is often limited with topical application as the result of confounding factors such as licking (grooming of application sites) or occlusion, which affect skin absorption, skin metabolism, and skin macromolecules. 26,27
Preliminary results obtained in the pilot developmental range-finding study suggested that OTNE was not a developmental toxicant at maternal doses up to 1920 mg/kg/d, although this dosage produced maternal mortality, and dams were subjected to marked reductions in feed consumption and body weight gains at dosages of greater than or equal to 960 mg/kg/d during gestation. Results from the full developmental toxicity study corroborated and detailed the lack of fetal toxicity at dosages of 96 to 480 mg/kg/d. At these dosages, litter and fetal parameters were unaffected by OTNE, except for a minimal (not statistically significant) downward trend in fetal body weights. For the 480 mg/kg/d dosage group, this was most probably associated with this group’s significantly decreased maternal feed consumption and body weight gains during the entire gestation period. Similar prolonged maternal effects were not observed at 96 or 240 mg/kg/d, although transient decreases in maternal body weight gains and feed consumption occurred early in gestation (GDs 7–10).
Based on the prolonged, significantly decreased body weight gains in dams, and the decrease in fetal body weights at 480 mg/kg/d, a maternal and a developmental No-Observable-Adverse-Effect-Level (NOAEL) of 240 mg/kg/d was established for OTNE. These NOAELs indicate that a margin of safety greater than 2700 exists between the maximum daily human exposure to OTNE from dermally applied fragranced cosmetic products (0.086 mg/kg/d) and the possibility of an adverse fetal exposure. It is concluded that OTNE is not a developmental toxicant in rats under the conditions of this study.
Footnotes
Figure and Tables
Acknowledgements
Portions of this work were presented at the 43rd Annual Meeting of the Society of Toxicology, 2004, Baltimore, MD. The study was conducted at Charles River Laboratories Preclinical Services, Horsham, PA, and funded by the Research Institute for Fragrance Materials, Inc., Woodcliff Lake, NJ.