Abstract
Methyl dihydrojasmonate (MDJ) is a widely used fragrance ingredient. MDJ was evaluated for developmental toxicity in presumed pregnant Sprague-Dawley rats (25/group) at oral dosages of 0, 40, 80 or 120 mg/kg/day in corn oil administered on gestational days 7–20. Dams 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 gender, gross external changes, and soft tissue or skeletal alterations. No maternal or fetal deaths occurred. MDJ-related maternal clinical signs included an increased incidence of sparse hair coat and ungroomed appearance at 120 mg/kg/day. Two dams in this group also had tan areas in the liver and a pale spleen. The 120 mg/kg/day dosage also caused reduced mean maternal body weight gains and body weights during the dosage period and reduced absolute and relative maternal feed consumption for the entire dosage period. No Caesarean-sectioning or litter parameters were affected by dosages of MDJ as high as 120 mg/kg/day, although at the highest dosage a tendency toward slightly reduced, but not statistically significant, fetal mean body weight was observed. No fetal gross external, soft tissue or skeletal changes were attributable to dosages of MDJ as high as 120 mg/kg/day. Based on these data, maternal No-Observable-Adverse-Effect-Level (NOAEL) of 80 and developmental NOAEL of equal to or greater than 120 mg/kg/day were established for MDJ. It is concluded that MDJ is not a developmental toxicant in rats under the conditions of this study.
INTRODUCTION
Methyl dihydrojasmonate (MDJ, CAS# 24851-98-7), also known as methyl 3-oxo-2-pentylcyclopentaneacetate, is a pale yellowish or almost colorless oily liquid with a warm, sweet-floral, jasmine-like fruity odor (Arctander 1969). MDJ’s chemical formula is C13H22O3 and its molecular weight is 226.32. The molecular structure is presented in Figure 1.
MDJ is used primarily as a fragrance in decorative cosmetics and toiletries, and the worldwide volume of use is greater than 1000 metric tons per annum (IFRA 2004). The maximum daily human skin exposure from the use of multiple cosmetic products containing MDJ has been calculated to be 0.7 mg/kg/day for a 60 kg high-end user of these products (IFRA 2007). 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 skin permeation (Ford et al. 2000). Cadby et al. (2002) reported how these data are obtained and how exposure is determined.
The safety aspects of MDJ have been evaluated in a number of in vitro and in vivo toxicity studies. The oral LD50 of MDJ in rats exceeded 5.0 g/kg (RIFM 1976; 1986). By the dermal route, the rabbit LD50 also exceeded 5.0 g/kg (RIFM 1976). By the intraperitoneal route in ICR mice, the LD50 was 1397.2 mg/kg (RIFM 1998), while in rats, an intraperitoneal injection of MDJ in corn oil produced clinical signs at 1000 mg/kg and mortality at 2000 mg/kg (RIFM 2001a).
A subchronic feeding study assessed the potential toxicity of MDJ when administered via a dietary admixture to Sprague-Dawley CD rats (10/sex/group) at dose levels of 0, 10, 50, and 100 mg/kg/day for three months (higher doses could not be used because of palatability problems). The No-Observed-Effect-Level (NOEL) was determined to be 100 mg/kg/day (RIFM 2000a). A battery of mutagenicity studies were also conducted, including bacterial reverse mutation (Ames) tests (RIFM 1987, 2000b, 2000c), an in vitro mouse lymphoma assay (RIFM 2001b), an in vitro chromosome aberration assay in Chinese hamster ovary cells (RIFM 1988), an in vivo rat unscheduled DNA synthesis assay (RIFM 2001a), and an in vivo mouse micronucleus test (RIFM 1998). Based on the negative results from these in vitro and in vivo studies, MDJ was not considered to possess a mutagenic or genotoxic potential.
Lack of developmental or reproductive toxicity studies on MDJ in the scientific literature prompted RIFM to contract the Charles River Laboratories Preclinical Services (Horsham, PA) to conduct a developmental toxicity study. The study was designed to meet requirements of the Food and Drug Administration (FDA 1994) for the evaluation of ICH Harmonized Tripartite Guideline stages C and D of the reproductive process and to determine the maternal and developmental No-Observable-Adverse-Effect-Levels (NOAELs) in Sprague-Dawley rats. All procedures were conducted in compliance with the Good Laboratory Practice (GLP) regulations of the Food and Drug Administration (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
Methyl dihydrojasmonate (MDJ, CAS # 24851-98-7) a clear, colorless liquid (lot number 2F37994), was supplied by Firmenich, Inc. (Plainsboro, NJ). Corn oil (Sigma-Aldrich Inc., 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 and stirred continuously during dosage. Samples from each dosing suspension concentration (first and last days of treatment) were analyzed for MDJ content.
Animals
Crl:CD® (SD) IGS BR VAF/Plus® male and female rats (Charles River Laboratories, Inc., Raleigh, NC) 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 218–246 grams, 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. The presence of spermatozoa and/or a copulatory plug
All cage sizes and housing conditions were in compliance with the
Methods
Dosages of MDJ were selected on the basis of a range-finding study, in which 0, 125, 250, 500, or 1000 mg/kg/day were intubated daily to 8 rats/group on GDs 7–20. No mortality occurred at dosages as high as 1000 mg/kg/day, but adverse clinical signs such as apparent dehydration, ungroomed appearance and excessive salivation were observed at 250 mg/kg/day and higher. Reduced feed consumption and 18 to 59% reductions of body weight were also recorded at dosages of 125 to 1000 mg/kg/day. No fetal gross external alterations were observed, although fetal body weights (total, male and female) were reduced in all MDJ dosage groups (8% to 33% less than the vehicle controls). Based on these data, dosages of 0, 40, 80, or 120 mg/kg/day MDJ were selected for the full developmental toxicity study.
MDJ in a corn oil vehicle was administered orally via gavage to four groups of presumed pregnant rats on GDs 7–20 at dosages of 0, 40, 80, or 120 mg/kg/day. The dosage volume was 10 ml/kg, 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 clinical signs. Body weights were recorded daily during the dosage and post-dosage 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, Caesarean sectioned, and a gross necropsy was performed. Uteri of apparently non-pregnant 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 was also 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; 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 97/2000/XP) and/or The SAS System (version 6.12). The statistical analyses were performed as described in Politano et al. (2008).
RESULTS
Results of all MDJ concentration and homogeneity analyses were within ±15% of calculated concentrations and ≤5% relative standard deviation (RSD), respectively. Room temperature stability of formulations at concentrations of 4 mg/ml corn oil (low dose) was found to be stable for a period of 50 days. Concentrations between 12.5 mg/ml and 100 mg/ml corn oil had previously been found stable for 13 days.
All rats survived until scheduled sacrifice on gestational day 21 (GD 21). One rat in the 80 mg/kg/day dosage group prematurely delivered on the day of scheduled sacrifice. The early delivery was considered unrelated to treatment because it was not dosage-dependent and the incidence was within the historical experience of the Testing Facility. No clinical signs occurred prior to delivery, and there were no gross lesions observed at necropsy. Body weight gains and feed consumption values for this dam were comparable to values for other rats in this dosage group. The litter consisted of 13 delivered pups and one early resorption
Adverse clinical signs possibly associated with the test article were minor and limited to a tendency toward an increase in sparse hair coat and ungroomed appearance in the 120 mg/kg/day dosage group. At necropsy on GD 21, adverse findings were relegated to tan areas in liver lobes and a pale spleen in two rats at 120 mg/kg/day. Although the incidence rate (2/25) was not statistically significant, as compared with the vehicle controls, it was considered a possible effect of MDJ because this finding occurred only in the high dosage group. The tissues were not examined histologically.
Significant reductions in mean maternal absolute feed consumption and body weight gains occurred only in the high-dose group (Table 1). The body weight gain data from three dams with small litters (4, 4, and 9 live fetuses in the 40, 80, and 120 mg/kg/day dosage groups, respectively) were excluded from the data because litter size was unrelated to MDJ but affects maternal body weight gain, particularly late in gestation (GD 18–21) when fetal growth is greatest. Adjusted body weight gains for the entire treatment period (calculated as GDs 7–21) in the 40, 80, and 120 mg/kg/day dosage group were 93%, 92%, and 85% of the vehicle control group value, respectively, and 91%, 92%, and 84% prior to exclusion. Mean maternal body weights were also reduced in the 120 mg/kg/day dosage group on GDs 7–21; these reductions were statistically significant (p ≤ 0.05) on GDs 11–15, when compared to the vehicle control group values (data not shown).
Pregnancy occurred in 23 or 24 rats in each dosage group (Table 2). As a result of one premature delivery in the 80 mg/kg/day dosage group, 24, 23, 22, and 23 pregnant rats with one or more live fetuses were Caesarean-sectioned on GD 21 in the 0, 40, 80, and 120 mg/kg dosage groups, respectively. Of these animals, two rats, one at 40 mg/kg/day and one at 80 mg/kg/day had litters consisting of only four live fetuses and one early resorption, while one rat at 120 mg/kg/day had a litter of nine live fetuses. These reduced litter sizes were considered unrelated to MDJ because the dosage period was not initiated until after implantation occurred and because these litters were smaller than those generally produced by this particular rat strain and source (historically at the Testing Facility, the number of live fetuses is 14.4 with a range of 11.8–15.9 in 92 studies conducted between June 2004 and June 2006). The maternal Caesarean-sectioning and litter values were calculated with and without the values from these dams and litters because such events can skew data distributions (Zambrana and Greenwald 1971).
As indicated in Table 2, the 120 mg/kg/day dosage group had reduced mean fetal body weights, as compared with the vehicle control group (combined fetal body weights averaged 5.12 g, as compared with 5.24 g for the vehicle control group). The reductions in fetal body weights were not considered to be of toxicological significance because the values did not significantly differ from the vehicle control group values and were within the historical ranges (4.96–5.63 g) of the Testing Facility. Apparent increases in fetal body weights in the 40 and 80 mg/kg/day dosage groups were associated with inclusion of the fetal values from small litters, which tend to have larger fetuses. After exclusion of values from these litters, fetal averages for the three respective dosage groups were 101%, 102% and 98% of vehicle control values. No other Caesarean-sectioning or litter parameters were affected by dosages of MDJ as high as 120 mg/kg/day.
Fetal evaluations were based on 342, 308, 293, and 310 live GD 21 Caesarean-delivered fetuses in 24, 23, 22, and 23 litters in the 0, 40, 80, and 120 mg/kg/day dosage groups, respectively. Each of these fetuses was examined for gross external alterations. Of these respective fetuses, 165, 148, 141 and 148 fetuses were examined for soft tissue alterations, and 177, 160, 152 and 162 fetuses were examined for skeletal alterations and fetal ossification site averages.
No gross external, soft tissue or skeletal fetal alterations (malformations or variations) were caused by dosages of MDJ as high as 120 mg/kg/day (Table 3). There were no significant differences in the litter or fetal incidences of any gross external, soft tissues or skeletal alterations. All ossification site averages were comparable among the dosage groups. All values were within the historical ranges of the Testing Facility.
DISCUSSION
The primary purpose of the study was to: 1) determine if daily systemic exposure to MDJ during the formative stages of pregnancy could produce adverse effects in pregnant rats or in the developing embryo-fetus, and 2) determine the oral maternal and developmental No-Observable-Adverse-Effect-Levels (NOAELs). The oral route was used in this study, even though MDJ use in fragrances is topical, because large topical doses in animals are difficult to interpret, due to confounding factors. These factors include licking of application sites, irritation when occluded and variations in skin absorption, metabolism and macromolecules (Poet 2000).
On the basis of data from the present study, the maternal NOAEL of MDJ is 80 mg/kg/day. The 120 mg/kg/day dosage of MDJ caused reduced mean maternal body weight gains and reduced body weights during the dosage period, as well as reduced absolute and relative maternal feed consumption for the entire dosage period.
The developmental NOAEL is equal to or greater than 120 mg/kg/day. The only observation associated with the 120 mg/kg/day dosage of MDJ was a minimal reduction in fetal body weight, which was not considered to be toxicologically important because the value was not statistically significant, as compared with the vehicle control group value, and was within the historical range of the Testing Facility. Based on these data, it is concluded that MDJ is not a developmental toxicant in rats.
Footnotes
Figure and Tables
Portions of this work were presented at the 47th Annual Meeting of the Society of Toxicology, March 2008, Seattle, WA, USA. This study was conducted at Charles River Laboratories Preclinical Services, Horsham, PA, USA and funded by the Research Institute for Fragrance Materials, Inc., Woodcliff Lake, NJ, USA.