Evaluation of the Developmental Toxicity of α -Methyl-3,4-Methylene-Dioxyhydrocinnamic Aldehyde in Rats

Abstract

The developmental toxicity of α-methyl-3,4-methylene-dioxyhydrocinnamic aldehyde (MMDHCA), a widely used fragrance ingredient, was evaluated for developmental toxicity in Sprague-Dawley rats (25/group; cesarean-sectioning identified 21 to 25 pregnant rats/group). Oral dosages of 0 (corn oil), 62, 125, or 250 mg/kg/day were administered by gavage on days 7 through 17 of gestation (GDs 7 through 17). Rats were observed for viability, clinical signs, body weights, and feed consumption. Necropsy and cesarean sectioning occurred on GD 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. Analysis of dosage preparations verified calculated dosages ±10%. No deaths occurred. Excessive salivation occurred in all groups, but the incidence was increased at 250 mg/kg/day. The 250 mg/kg/day dosage also was associated with a significant increase in the incidences of a clear, red or yellow perioral and/or red perivaginal substance and significant reductions in mean feed consumption and body weight gains (11.6% and 7.4%, respectively) during the entire dosage period. No gross changes attributable to MMDHCA were observed at necropsy. Cesarean section or litter parameters, as well as fetal alterations, were not affected by MMDHCA at 250 mg/kg/day or either of the lower dosages tested. Based on these data, maternal and developmental no-observable-effect levels (NOAELs) of 125 and >250 mg/kg/day, respectively, were established for MMDHCA. It is concluded that MMDHCA is not a developmental toxicant in rats under the conditions of this study and dosing regimen.

α-Methyl-3,4-methylene-dioxyhydrocinnamic aldehyde (Figure 1), also known as α-methyl-1,3-benzodioxole-5-propionaldehyde, α-methyl-1,3-benzodioxole-5-propanal or MMDHCA (CAS number 1205-17-0), is a fragrance ingredient used in fine fragrances, decorative cosmetics, shampoos, toilet soaps, and toiletries, as well as in noncosmetic products such as household cleaners and detergents. It is a colorless to slight yellow liquid with a sweet floral and mildly herbaceous odor of “considerable tenacity” (Arctander 1969). Worldwide use of MMDHCA is greater than 100 metric tons per annum (IFRA 2000). Because it is a fragrance ingredient found in a variety of consumer products, humans have the potential to be exposed to low but continual levels of MMDHCA, primarily by the dermal route.

Several authors have documented the general toxicological characteristics of MMDHCA. Genotoxicity studies indicated that MMDHCA was negative in a bacterial mutation assay, positive for induction of structural aberrations in the in vitro Chinese hamster ovary (CHO) chromosome aberration assay, and negative in the in vivo mouse micronucleus assay (Cocchiara, Api, and Jacobson-Kram 2001). In toxicity studies, the acute oral LD₅₀ in rats was calculated to be 3.56 g/kg (RIFM 1985), whereas the dermal LD₅₀ in New Zealand White (NZW) rabbits was ≥2.0 g/kg (RIFM 1993). The undiluted MMDHCA produced some dermal irritation on day 1, but not on day 7, following the 24-h occluded dermal application in rabbits. In human repeated insult patch tests, 0.75%, 20%, or 30% solutions of MMDHCA in various solvents did not produce any irritation (RIFM 2000a, 2000b, 2002).

Results from an in vitro human skin penetration study and a 13-week dermal toxicity study in rats have also been published (Api et al. 2006). In the in vitro study, by 24 and 48 h, 42% and 50%, respectively, of a 1% solution of radio-labeled MMDHCA permeated the epidermal membranes isolated from full thickness human skin samples. In the 13-week toxicity study in rats, dosages of 50, 150, or 300 mg/kg/day (0.043, 0.129, or 0.259 ml/kg/day applied neat to 5-cm² dorsal skin) all produced MMDHCA-related dermal irritation, especially at 300 mg/kg/day, although irritation ranging from “slight” to “marked” improved to “slight” or “resolved completely” after a 4-week recovery period. It was concluded that MMDHCA exhibits moderately high human skin permeation, and that the no-observable-effect level (NOEL) for systemic toxicity following dermal application is greater than 300 mg/kg/day.

The present study was designed to evaluate International Conference on Harmonization (ICH) Harmonised Tripartite Guideline stages C and D of the reproductive process. The purpose of the study was to determine if exposure to MMDHCA during pregnancy produced any potentially adverse effects in pregnant rats or in the developing embryo-fetus, and to determine the maternal and developmental no-observed-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

MMDHCA, a colorless to pale yellow liquid (lot number 413118) with an overall purity of 98.2%, was supplied by International Flavors & Fragrances (Union Beach, New Jersey, USA). Corn oil (lot number 103K0107; Sigma-Aldrich, St. Louis, Missouri) 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. Samples from each concentration of the dosing suspensions (first and last days of treatment) were analyzed for MMDHCA 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 262 to 356 g; the females weighed 188 to 224 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 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, a relative humidity of 30% to 70% and a 12:12-h light-dark lighting cycle. Certified Rodent Diet no. 5002 (PMI Nutrition International, St. Louis, Missouri), as well as reverse-osmosis (R.O.) deionized water, were provided ad libitum to the rats.

Procedures

MMDHCA or corn oil was administered 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), 62, 125, or 250 mg/kg/day. The dosage volume was 10 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 twice daily for viability and examined for clinical signs, abortions, and premature deliveries before dosage administration approximately 1 h later. Body weights were recorded prior to the start of the study and 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 and subjected to cesarean sectioning and gross necropsy. Uteri were excised and examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. Numbers of corpora lutea in each ovary were also recorded.

Fetuses were weighed and examined for gender and gross external alterations. Live fetuses were euthanized by an intraperitoneal injection of pentobarbital before 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). 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 differences among 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 differences among 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%. Results of 10-day stability testing were also within the acceptable range of ±10%.

No mortality occurred during the study. The only clinical signs related to MMDHCA included significantly increased (p ≤ .01) incidences of a clear, red or yellow perioral substance and/or red perivaginal substance in the 250 mg/kg/day dosage group. Excess salivation occurred in all dosage groups, but was most common at 250 mg/kg/day.

Feed consumption and body weight gains were reduced at 250 mg/kg/day (Table 1). Compared to controls, mean absolute (g/day) and relative (g/kg/day) feed consumption were significantly reduced (p ≤ .01) at 250 mg/kg/day for the entire dosage period (DGs 7 to 18), whereas at 125 mg/kg/day a significant reduction was only noted on DGs 10 to 12. For the entire dosage period, absolute feed consumption at 62, 125, and 250 mg/kg/day was 97.9%, 96.8%, and 88.4% of the control value, respectively. In parallel with feed consumption, body weight gains were significantly reduced (p ≤.01) at 250 mg/kg/day on DGs 7 to 10, whereas for the entire dosage period, body weight gain at 62, 125, and 250 mg/kg/day was 100.8%, 102.5%, and 92.4% of the control value, respectively. During the post-dosage period (DGs 18 to 21), body weight gains in all dosage groups were comparable to controls.

Pregnancy occurred in 21 (84.0%), 25 (100.0%), 24 (96.0%), and 23 (92.0%) rats in the four respective dosage groups. One dam in the 125 mg/kg/day dosage group began to deliver just prior to scheduled cesarean sectioning, a non-dose-dependent event unrelated to the test article and within the historical range (5 of 1683 control litters were prematurely delivered in the 2-year period nearest to conduct of this study). As a result, cesarean sectioning observations on DG 21 were based on 21, 25, 23, and 23 pregnant rats with litters including live fetuses. No other cesarean sectioning or litter parameters were affected by dosages of MMDHCA as high as 250 mg/kg/day (Table 2). The litter averages for corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, fetal body weights, percentage of dead or resorbed conceptuses, and percentage of live male fetuses were comparable among the four dosage groups. No significant differences were observed, and all values were within the ranges observed historically. All placentae appeared normal. There were three dead fetuses in one litter from the 250 mg/kg/day dosage group. This observation was considered unrelated to MMDHCA because the incidence was not statistically significant, it occurred in only one high-dosage group litter, and no dead fetuses were observed in the range-finding study. A significant increase (p ≤ .05) in the percentage of live male fetuses was noted in the 125 mg/kg/day dosage group, as compared to the control group value. This finding was considered unrelated to MMDHCA because the finding was not dose dependent and the value was within the range (31.7 to 67.3) observed historically at the Testing Facility.

Fetal evaluations were based on 307, 345, 324, and 318 DG 21 cesarean-delivered live fetuses in 21, 25, 23, and 23 litters in the 0 (vehicle), 62, 125, and 250 mg/kg/day dosage groups, respectively. Each of these fetuses was examined for gross external alterations. Of these respective fetuses, 149, 167, 155, and 152 fetuses were examined for soft tissue alterations, and 158, 178, 169, and 164 fetuses were examined for skeletal alterations and fetal ossification site averages (Table 3). To the extent possible, the three dead fetuses in the 250 mg/kg/day dosage group were examined for gross external, soft tissue, and/or skeletal alterations. None of the dead fetuses had gross external or skeletal alterations; one fetus had a folded retina in the right eye upon soft tissue examination. These results were excluded from tabulation/statistical analysis because they were from dead fetuses.

In the 0, 62, 125, or 250 mg/kg/day dosage groups, litters containing fetuses with alterations numbered 5 (23.8%), 7 (28.0%), 3 (13.0%), and 5 (21.7%), respectively. The numbers of fetuses with any alteration observed were 6 (2.0%), 9 (2.6%), 3 (0.9%), and 5 (1.6%) and the percentages of fetuses with any alteration per litter were 2.2, 2.7, 0.9, and 1.8 in the respective dosage groups. There were no fetal gross external alterations. Fetal soft tissue alterations included: an irregularly shaped brain in one 62 mg/kg/day fetus, and folded retinas, a variation usually associated with tissue processing, in 0, 2, 1, and 2 fetuses in 0, 2, 1, and 2 litters in the four respective dosage groups. There were no additional alterations in these fetuses. There was only one skeletal malformation: a fetus in the 62 mg/kg/day dosage group had fusion of one or more ribs (right, 7th and 8th ribs medially to distally, and 9th and 10th distally), as well as skeletal variations in skull bones, ribs and sternum.

All fetuses appeared normal at external examination. All soft tissue or skeletal alterations (malformations or variations) in the fetuses were considered unrelated to MMDHCA because (1) neither the fetal nor litter incidences were dose-dependent; (2) the incidences did not significantly differ from the vehicle 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 determine if daily high systemic exposure to MMDHCA during the formative stages of pregnancy (first trimester in humans) could produce any potential adverse effects in pregnant rats or in the developing embryo-fetus and to determine the maternal and developmental NOAELs. This objective is most easily and accurately achieved by gavage administration (although most MMDHCA use is topical) because dermal dosages of 50 to 300 mg/kg/day MMDHCA have been shown to produce dermal irritation in rats (Api et al. 2006). The dosing period of GDs 7 through 17 was selected because the rat embryo is most susceptible to developmental changes early in gestation (GDs 8 to 11), whereas late in gestation (GDs 17 to 21) growth is the predominant feature (Wilson and Warkany 1965).

Dosages for this study were selected on the basis of a range-finding study, in which 125, 250, 500, or 1000 mg/kg/day were administered daily on DGs 7 to 17. Adverse clinical signs occurred at 500 and 1000 mg/kg/day, and body weight gains and feed consumption were reduced during the entire dosage period at dosages of 250 mg/kg/day and higher. Following administration of the 1000 mg/kg/day dosage, cesarean sectioning and litter parameters revealed an increase in postimplantation loss, with corresponding reductions in litter size and live fetuses. Reductions in fetal body weights and an increase in fetal external alterations were also observed at this dosage.

Results from the full developmental toxicity study reported here corroborated the fact that 250 mg/kg/day produced mild signs of maternal toxicity by significantly reducing feed consumption and body weight gains during the dosing period and significantly increasing the incidence of a perioral and/or perivaginal substance. However, none of the adverse fetal effects seen at 1000 mg/kg/day were evident at 250 mg/kg/day in either the pilot or the present full developmental toxicity studies. Based on the extensive data from the present study, it was concluded that the maternal NOAEL for MMDHCA is 125 mg/kg/day (250 mg/kg/day caused significant decreases in body weight gain and feed consumption). The developmental NOAEL was considered to be greater than 250 mg/kg/day (no effects were observed at 250 mg/kg/day, the highest dosage tested).

The determinant factors for human systemic exposure to fragrance materials are quantities of cosmetic used, frequency of use, concentration of the fragrance material in these products, and skin permeation (Ford et al. 2000). Using these factors, the total maximum exposure to MMDHCA has been calculated from 10 types of cosmetic products. From exposure data provided by industry, the 97.5 percentile use level in formulae for use in cosmetics in general has been reported to be 4.7% (IFRA 2001). This would result in a maximum daily exposure on the skin of 0.1 mg/kg/day for high-end users. An explanation of how the data are obtained and how exposure was determined has been reported by Cadby, Troy, and Vey (2002). Based on the skin permeation data (50%) reported by Api et al. (2006), the daily systemic exposure for high end users can be estimated to be 0.05 mg/kg/day.

In conclusion, the calculated NOAELs indicate that MMDHCA is not a developmental hazard because adverse effects to the fetus would occur only at dosages that greatly exceed the maternal NOAEL and thus be toxic to the dam. The data also indicate that a large safety factor exists between normal daily human exposure and possible adverse fetal exposure. On a mg/kg basis, human daily exposure would have to be ≥15 g/day, based on a 60-kg human, to reach the fetal NOAEL, which is not probable because daily dermal systemic fragrance exposure is usually less than 1 mg/kg/day.