Methyleugenol is a naturally occurring flavoring and perfuming agent, and is also used as an attractant for the oriental fruit fly. This study was performed due to the potential for human exposure to MEUG and the lack of pertinent developmental toxicity data.
Dose selection was based on a screening study in which (CD) rats were treated by gavage with 0, 31.25, 62.5, 125, 250 or 500 mg MEUG/kg body weight/day on gestational days 6 through 19 (NTP, 1998). In the screening study, evidence of developmental toxicity was limited to a decreasing dose-response trend for average fetal body weight per litter. Maternal toxicity was noted at ≥125 mg/kg/day (NTP, 1998).
In this study, female Sprague-Dawley-derived (CD) rats were dosed by gavage with MEUG (80, 200, or 500 mg/kg/day) or its vehicle (0.5% aqueous methylcellulose) on gd 6 through 19. The dose volume was 5 ml/kg. Twenty-five timed-mated rats were assigned to each group. Dams were monitored at regular intervals throughout gestation for clinical signs, feed and water consumption, and body weight. At necropsy on gd 20, the following were recorded: maternal clinical condition; body, liver and gravid uterine weights; pregnancy status; and number of corpora lutea. In the gravid uterus, the numbers of resorbed, dead, or live fetuses were recorded. All live fetuses were weighed, sexed, and examined for external morphological anomalies. Approximately one-half of the fetuses were examined for visceral anomalies, including internal head structures, and the remaining fetuses were examined for skeletal anomalies.
Confirmed pregnancy rates were 72-92% per group. No treatment-related maternal deaths occurred in this study. Clinical signs associated with MEUG exposure included evidence of sedation (lethargic or prone) at 500 mg/kg/day. Aversion to the dose formulation was indicated by a dose-related incidence of rooting behavior among MEUG-treated dams. For example, rooting was observed in 0, 10, 14 and 19 dams from the control through high-dose groups, respectively, on gd 11.
Maternal body weight and weight gain were decreased in a dose-related manner. The high dose was associated with decreased maternal body weight (gd 9, 12, 15, 18, 19 and 20), and decreased maternal weight gain (gd 6-9, 15-18, and 6-20). Maternal body weight gain during gestation (absolute and corrected for gravid uterine weight) was reduced at both the mid- and high-dose groups. Gravid uterine weight was not affected.
Increased maternal liver weight (absolute and relative) was treatment related and significant at all doses. Ten randomly selected maternal livers per group were submitted for histopathology. Hepatocellular cytoplasmic vacuolization was noted at ≥200 mg/kg/day and hepatocellular degeneration at 500 mg/kg/day.
Maternal relative feed consumption (g/kg/day) was significantly decreased in the high-dose group: 61% of control consumption during early treatment (gd 6-9), 83% during late treatment (gd 18-19), and 91% for the treatment period as a whole (gd 6-20). In the low and mid-dose groups, feed consumption was occasionally elevated (104-107% of controls). Maternal relative water consumption was only affected during early treatment (gd 6-9), when the high dose group consumed 72% as much as controls.
Average fetal body weight per litter was significantly reduced at the high dose (86% of the average control weight). MEUG did not affect any indices of prenatal viability. Live litter size in MEUG-treated groups was 104-109% of the control mean. Incidences of fetal morphological anomalies were statistically equivalent among groups, except for an increased incidence of unossified sternebra(e), a skeletal variation, at the high dose.
In summary, maternal effects including increased liver weight and aversion to dosing were noted at ≥80 mg MEUG/kg/day on gd 6 to 19. Thus, the maternal toxicity lowest observed adverse effect level was 80 mg/kg/day and the maternal toxicity no observed adverse effect level was not determined in this study. Reduced fetal body weight and an increased incidence of unossified sternebra(e) were found at 500 mg MEUG/kg/day. Thus, the developmental toxicity LOAEL was 500 mg/kg/day based on intrauterine growth retardation and mildly delayed skeletal ossification. The developmental toxicity NOAEL was 200 mg/kg/day.