Evaluation of the Developmental Toxicity of Alpha-iso-methylionone in Rats

Abstract

Alpha-iso-methylionone, a widely used fragrance ingredient, was evaluated for developmental toxicity in presumed pregnant Sprague-Dawley rats (25/group). Oral dosages of 0, 3, 10, or 30 mg/kg/day alpha-iso-methylionone in corn oil 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. 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. No maternal or fetal deaths occurred. No fragrance ingredient–related clinical signs were observed. Feed consumption, body weight gains, gross tissue changes at necropsy, and caesarean section or litter parameters, as well as fetal developmental morphology, were unaffected by dosages of alpha-iso-methylionone as high as 30 mg/kg/day. Based on these data, maternal and developmental no observed adverse effect levels of equal to or greater than 30 mg/kg/day were established for alpha-iso-methylionone. It is concluded that alpha-iso-methylionone is not a developmental toxicant in rats at maternal doses of up to 30 mg/kg/day.

Alpha-iso-methylionone (AIM; CAS no. 127-51-5; Figure 1), also known as 3-butene-2-one, 3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl), is a clear, pale yellow oily liquid with an extremely versatile odor, ranging from sweet floral to tobacco-like or woody (Arctander 1969). AIM may be found in the fragrances used in decorative cosmetics, fine fragrances, shampoos, toilet soaps, and other toiletries, as well as in noncosmetic products such as household cleaners and detergents. Its fragrance use worldwide is greater than 100 metric tons per annum (IFRA 2004). Because it is a fragrance ingredient found in a variety of consumer products, humans have the potential to be exposed to low but continuous levels of AIM, primarily via the dermal route.

The 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). Using these factors, the total maximum exposure to AIM has been calculated from 10 types of cosmetic products. The 97.5 percentile use level in fragrance formulae for use in cosmetics, data provided by industry, has been reported to be 13% (IFRA 2001). In high-end users of multiple AIM-containing cosmetic products, this would result in a maximal daily systemic exposure of 0.3 mg/kg/day (IFRA 2001). Cadby, Troy, and Vey (2002) reported how these data are obtained and how exposure is determined.

A previous review of scientific literature and unpublished studies reported to the Research Institute for Fragrance Materials, Inc. (RIFM) revealed that the existing toxicological data on AIM did not include developmental or reproductive toxicity studies. Acute toxicity studies show oral LD₅₀ values for AIM in mice of 8.7 g/kg (RIFM 1967) and >10 g/kg (RIFM 1980a), and in rats >5 g/kg (RIFM 1973). The dermal LD₅₀ in rabbits was >5 g/kg (RIFM 1973). In an oral 90-day subchronic toxicity study, feeding rats AIM in the diet at 3.7 mg/kg/day had no adverse effects on body weight gain, feed consumption, clinical pathology parameters, or on the tissues examined histologically (Oser, Carson, and Oser 1965). Daily gastric intubation of 3.4 mg/kg/day AIM for 12 weeks in rats was reported to have no effect; no further details were provided (Bar and Griepentrog 1967). A 90-day subchronic oral (gavage) study has recently been completed by RIFM. The study was begun following the completion of this developmental toxicity study. Rats were dosed daily with 0, 5, 30, or 500 mg/kg AIM in a corn oil vehicle. At 500 mg/kg/day, there was a statistically significant increase in plasma creatine, total protein, and cholesterol in both sexes, and plasma albumin in male rats. Statistically significant increases in liver and kidney weights in both sexes, as well as spleen weights in males, were observed in rats treated with 500 mg/kg/day. Treatment with 500 mg/kg/day AIM resulted in hepatocyte enlargement in both sexes, and a higher incidence of thyroid follicular cell hypertrophy and a higher incidence of lower grades of severity of adipose infiltration of the bone marrow in males. In the kidneys, males treated with 500 and 30 mg/kg/day AIM had a higher incidence of globular accumulations of eosinophilic material in the tubular epithelium, which is consistent with well documented changes that are peculiar to male rats in response to treatment with some hydrocarbons (EPA 1991; Swenberg and Lehman-McKeeman 1999). The no observed effect level (NOEL) was determined to be 30 mg/kg/day for females and 5 mg/kg/day for males, and the no observed adverse effect level (NOAEL) for males was determined to be 30 mg/kg/day (RIFM 2006).

A dermal 90-day subchronic toxicity study was conducted on rats with an unoccluded application of 0, 50, 170, 580, or 2000 mg/kg/day AIM applied to the clipped backs (RIFM 1980b). Several AIM-related dermal irritant effects (erythema, edema, and eschar formation) were observed at all dose levels and were dose dependent. Other treatment-related effects observed were thought to be the result of severe dermal irritation and damage (RIFM 1980b). A second 90-day subchronic dermal study was conducted on rats, in which 0 or 10 mg/kg/day AIM as a 1% solution in phenethyl alcohol was applied to the clipped backs of the animals. To minimize local irritation, each back was divided into a 7-area grid, so that a separate area was treated each day in the week. There was no skin irritation observed. There were no systemic effects, thus the NOEL was determined to be 1% (RIFM 1981).

In an Ames mutagenicity assay, doses of up to 9300 μg/plate AIM had no mutagenic effect in the presence or absence of rat liver S9 fraction (RIFM 1980c). Based on genotoxicity studies conducted on the structurally related methyl ionone, it was concluded that AIM does not possess significant in vivo mutagenic or genotoxic potential (RIFM 2000a, 2000b; Wild et al. 1983).

Due to the high volume of use of AIM (IFRA 2004) and lack of developmental or reproductive toxicity studies available in scientific literature, RIFM contracted with Charles River Laboratories Preclinical Services for a developmental study to evaluate the continued safe use of AIM in fragrances. The study was designed to evaluate ICH Harmonised Tripartite Guideline stages C and D of the reproductive process and to meet the requirements of the Food and Drug Administration (FDA 1994). The purpose of the study was to determine if exposure to AIM during pregnancy could produce any potential adverse effects in pregnant rats or in the developing embryo-fetus, 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 (FDA 1987), the Japanese Ministry of Health and Welfare (MHW 1997), and the Organisation for Economic Cooperation and Development (OECD 1998).

MATERIALS AND METHODS

Materials

Alpha-iso-methylionone (AIM; CAS no. 127-51-5), a clear, pale yellow liquid (lot number RO00619567), was supplied by International Flavors and Fragrances, Inc. (Union Beach, NJ). Corn oil (lot numbers 103K0107 and 074K0025; 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 daily from bulk materials. Samples from each concentration of the dosing suspensions (first and last days of treatment) were analyzed for AIM content by International Flavors and Fragrances, Inc.

Animals

Crl:CD^®(SD) IGS BR VAF^®/Plus male and female rats (Charles River Laboratories, 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 213 to 241 g, 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 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 (Institute of Laboratory Animal Resources 1996). 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-h light-dark lighting cycle was used. Certified Rodent Diet^® R 5002 (PMI Nutrition International, St. Louis, MO) 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 AIM were selected on the basis of the following range-finding study. Based on the results observed in the dermal 90-day subchronic toxicity studies (RIFM 1980b; 1981), dosages of 0, 1.25, 2.5, 5, or 10 mg/kg/day were administered daily to 8 rats/group on GDs 7 to 17. No adverse AIM-related clinical signs were observed. Feed consumption during the dosage period was comparable among the five groups. Although not dosage dependent, body weight gains during the dosage period in the 1.25, 2.5, 5, and 10 mg/kg/day AIM-treated rats were 117.6%, 111.4%, 119.4%, and 111.4%, respectively, of the vehicle control values. During the postdosage period, the body weight gains remained increased. All rats were pregnant and all caesarean sectioning and litter observations were comparable among the five groups. No fetal gross external alterations were observed. Based on these data, a dosage greater than 10 mg/kg/day AIM was recommended for the definitive developmental toxicity study in rats.

AIM in a corn oil vehicle was administered orally via gavage to four groups of presumed pregnant rats on GDs 7 to 17 at dosages of 0, 3, 10, or 30 mg/kg/day. 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 twice daily for viability and examined for abnormal clinical signs, abortions, and premature deliveries before dosage administration and approximately 1 h 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, 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 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, 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 1967a).

Continuous data were analyzed by using Bartlett’s test of homogeneity of variances (Sokal and Rohlf 1969a) and the analysis of variance (Snedecor and Cochran 1967b). 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

The results of all AIM concentration and homogeneity analyses were within ±10% of calculated concentrations and ≤5% relative standard deviation, respectively. The 3 mg/ml concentration level was found to be stable for 10 days when stored at ambient conditions and protected from light.

No mortality occurred during the study. Clinical signs that included occasional incidences of chromorhinorrhea, excess salivation, sparse hair coat, localized alopecia, urine-stained abdominal fur, soft or liquid feces, or decreased activity were observed in a few animals from each group of rats. None of these clinical signs were attributed to AIM because the number of affected rats was not dosage related and did not significantly differ between control and treated groups.

Feed consumption and body weight gains were unaffected by dosages of AIM as high as 30 mg/kg/day (Table 1). For the dosage period, absolute feed consumption at 3, 10, and 30 mg/kg/day was 101.0%, 102.1%, and 102.6% of the control value, respectively, whereas body weight gains were 99.0%, 99.9%, and 101.2% of the control value over the same time period. Body weight gains were also comparable during the post-dosage period (GDs 18 to 21).

Pregnancy occurred in 24 (96%), 25 (100%), 24 (96%), and 21 (84%) of the 25 rats in the 0, 3, 10, and 30 mg/kg/day dosage groups, an event determined before exposure was initiated. No caesarean sectioning or litter parameters were affected by dosages of AIM as high as 30 mg/kg/day (Table 2). The litter averages for corpora lutea, implantations, litter sizes, live fetuses, resorptions, fetal body weights, percentage of dead or resorbed conceptuses, and percentage of live male fetuses were comparable among the four dosage groups, did not significantly differ from the vehicle control group values, and were within the ranges observed historically at the Testing Facility.

Fetal evaluations were based on 353, 363, 336, and 301 live, caesarean-delivered fetuses from the 0, 3, 10, and 30 mg/kg/day dosage groups, respectively. Each of these fetuses was examined for gross external alterations and soft tissue or skeletal alterations (see Table 3 for detailed analyses). Fetal alterations were 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 including reversible delays or accelerations in development).

The only fetal gross external alterations occurred in one control fetus in which a depressed left eye bulge, a fleshy thoracic protrusion, and the presence of an extra hind limb were observed. Skeletal examination of this fetus revealed a small left eye socket; the presence of three femurs, tibia, and fibulas on the right side of the body (partially fused), and extra metatarsals, digits, and three sets of phalanges (four, five, and five) on the right hindlimbs.

No soft tissue malformations occurred in any of the groups. Soft tissue variations were limited to folded retinas in seven fetuses from two different litters at 10 mg/kg/day (variations considered to be artifacts of processing), slight dilation of the renal pelvis in one fetus from the control and one fetus from the 3 mg/kg/day dosage groups, and an aberrant umbilical artery in one and two fetuses at 3 and 10 mg/kg/day, respectively. Skeletal malformations were absent (except in the control fetus already described), and skeletal variations were limited to infrequent incidences of delayed ossification at various bone ossification centers (non–dose dependent) and the presence of cervical ribs at the 7th cervical vertebra in four fetuses in each of the control and 30 mg/kg/day dosage groups.

All fetal gross external, soft tissue, or skeletal alterations (malformations or variations) were considered unrelated to AIM because (1) neither the fetal nor litter incidences were dosage dependent; (2) the incidences did not significantly differ from the control group values; and/or (3) the incidences were within the ranges observed historically at the Testing Facility.

DISCUSSION

The primary purpose of the study was to (1) determine if daily systemic exposure to AIM 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 NOAELs. Although most present AIM use in fragrances is topical, the above objectives are most easily and accurately achieved by gavage administration because AIM produces severe dermal irritation and damage in rats if given in large doses required to test systemic toxicity (RIFM 1980b).

Results from the present definitive developmental toxicity study demonstrated that the maternal and developmental NOAELs for AIM are equal to or greater than 30 mg/kg/day. The data indicate that AIM is not a developmental toxicant in rats under the conditions tested. The dose of 30 mg/kg/day is 100 times higher than the maximum daily human systemic exposure level of 0.3 mg/kg (calculated from exposure to cosmetics on the skin).