* Chemistry and Life Sciences, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709-2194; and
Developmental and Reproductive Toxicology Group, National Toxicology Program, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709
Received September 5, 2000; accepted December 6, 2000
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ABSTRACT |
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Key Words: isoeugenol; developmental toxicity; teratogenicity; rats; morphological development.
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INTRODUCTION |
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Concentrations in soaps, deodorants, detergents, lotions, or perfumes have ranged from 0.0127 to 0.2%, the previously recommended maximum concentration for perfumes (Johansen et al., 1996; Rastogi et al., 1996
, 1998
). Contact sensitization to isoeugenol has been reported and it is frequently used in patch testing for fragrance sensitivity (Bertrand et al., 1997
; Buckley et al., 2000
; Frosch et al., 1995
; Hilton et al., 1996
; Johansen and Menné, 1995
). Thus, the recommended maximum concentration of isoeugenol has recently been lowered to 0.02% (0.2 mg/g) in consumer products due to its allergenic potential (White et al., 1999
). Additional evaluation of the concentration of isoeugenol in children's products has been suggested (Rastogi et al., 1999
).
Used as a flavoring agent in nonalcoholic beverages, baked goods, and chewing gum, concentrations range from 0.3 ppm to 1000 ppm (Furia and Bellanca, 1975). Isoeugenol also inhibits the growth of food-contaminating fungi (Pauli and Knobloch, 1987
). Daily per capita intake (eaters only) has been estimated as 0.8 µg/kg (Stofberg and Grundschober, 1987
). Isoeugenol has been detected in mainstream smoke from nonfiltered cigarettes at 315 µg/cigarette (IARC, 1986
). In 1992, total United States import of eugenol/isoeugenol was 330 metric tons (Chemical Economics Handbook, 1996
). Low-level human exposure potentially occurs in certain industrial settings; from tobacco, wood, and marijuana smoke; from ingested foods, chewing tobacco, and snuff; from soaps, lotions, detergents, and perfumes; and from contamination of water by industrial runoff. Data from the National Occupational Exposure Survey (NOES), conducted by the National Institute for Occupational Safety and Health (NIOSH) between 1981 and 1983, estimated that 35,167 workers, including 24,978 female employees, were potentially exposed to isoeugenol in the workplace (NTP, 2000
). The permissible exposure limit (PEL), threshold limit value (TLV), or recommended exposure limit (REL) for isoeugenol have not been established.
Isoeugenol is selected for or is currently on test by the National Toxicology Program (NTP) in the areas of in vivo carcinogenicity, in vitro studies in human, murine, and rat cells, and reproductive toxicity assessed by the continuous breeding protocol in rodents (NTP, 2000). No evaluation of the developmental toxicity of isoeugenol in humans was found in the literature. The developmental toxicity of isoeugenol had been evaluated in a preliminary study in rats (NTP, 1993
). Doses of 0, 50, 200, 400, 600, and 800 mg isoeugenol/kg/day in corn oil were administered by gavage to timed-mated female CD® Sprague-Dawley rats (1011/group) on gestational days (gd) 6 through 15. However, only four to seven pregnant animals per group were available for evaluation of developmental effects due to the low fertility rate in that study (NTP, 1993
). Signs of toxicity were noted in all animals at doses of 400 mg/kg/day and greater. Clinical signs increased in severity in a dose-related manner and ranged from transient slight sedation immediately postdosing at 400 mg/kg/day to more prolonged sedation and lethargy at the high dose. No maternal deaths were observed, and maternal weight change was limited to a transient decrease in the high-dose group. Isoeugenol at doses up to 800 mg/kg/day had no adverse effect on any measure of postimplantation viability, growth, or external morphological development (NTP, 1993
).
The data produced in the 1993 NTP-sponsored preliminary study was considered inconclusive, due to the small group size. Therefore, isoeugenol was selected for additional evaluation of developmental toxicity in rats (NTP, 2000). The results of this investigation are expected to provide information relevant to the safety assessment of isoeugenol exposure during pregnancy, with particular focus on in utero growth, viability, and morphological development. In addition, this study was designed to include exposure of pregnant rodents during the entire embryo/fetal period and examination at the end of pregnancy, as specified by current guidelines (U.S. EPA, 1997
, 1998
) or proposed guideline revisions (Collins et al., 1999
; U.S. FDA, 1993
) for prenatal toxicity testing. The present NTP-sponsored study was designed to establish NOAELs and LOAELs for maternal and developmental toxicity following daily oral exposure throughout the embryo/fetal period, as conducted using a standardized protocol.
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MATERIALS AND METHODS |
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The experimental animals were female Sprague-Dawley outbred albino rats [Crl:CD® (SD)BR VAF/Plus®] (Charles River Laboratories, Inc., Raleigh, NC), 810 weeks old upon arrival. The CD® rat was chosen by the NTP for this developmental toxicity evaluation and its screening study to conform to the requirement of the use of a rodent species for prenatal developmental toxicity testing (Collins et al., 1999; U.S. FDA, 1993
). It was also the test animal used in the NTP-sponsored range-finding study (NTP, 1993
). Animals were individually identified by ear tag with dam identification numbers (National Band and Tag Co., Newport, KY) during a 10-day quarantine. These identification numbers were used throughout the study. After quarantine, individual females were placed overnight in the home cage of a singly-housed male of the same stock for mating, and then examined the next morning for the presence of sperm in the vaginal lavage (Hafez, 1970
). During the study, females were housed singly in solid-bottom, polycarbonate cages with stainless steel lids (Laboratory Products, Rochelle Park, NJ) and Certified Sani-Chip® hardwood cage litter (P. J. Murphy, Montville, NJ). Food (Purina Certified Rodent Chow (#5002), PMI, St. Louis, MO) and tap water (Durham, NC city water) were provided ad libitum throughout the study. The light cycle (12 h light, 12 h dark) and temperature and relative humidity in the animal rooms were controlled and monitored (Siebe/Barber-Coleman Network 8000® System with SIGNAL® Software [Version 4.1], Siebe Environmental Controls (SEC)/Barber-Colman Company, Loves Park, IL) throughout the study. Temperature and relative humidity fell within their respective target ranges (6975°F and 3565%).
Twenty-five timed-mated female rats were assigned to each dose group by stratified randomization for body weight on gd 0 (i.e., day of vaginal sperm detection). On gd 0, timed-mated females weighed from 232 to 275 g, and body weight did not differ among treatment groups. Females assigned to isoeugenol groups showed lower weight gain during the pretreatment period (gd 0 to 6), but there was no difference among groups for maternal body weight on gd 6 (i.e., body weight taken prior to initiation of treatment; Table 1).
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Isoeugenol [obtained and characterized by Battelle, Columbus, OH through the sponsor from Penta Manufacturing Company, Livingston, NJ, Lot No. 46928) (CAS No. 97-54-1)] of > 99% purity was dissolved in corn oil. Identity and purity of the test material were confirmed by Battelle, Columbus, OH (NIEHS Contract N01-ES-55395). Identity was confirmed by infrared (IR) spectroscopy as well as proton and C13 nuclear magnetic resonance spectroscopy, and the test material was characterized as a 9:1 mixture of the trans- and cis-isomers, respectively. Purity (> 99%) was determined by gas chromatography. Within 14 days of the screening study start, relative purity was 97.9% when compared to a frozen reference sample by high performance liquid chromatography (HPLC) with ultraviolet detection (NTP, 1999).
Stability of the dosing solutions was verified for the period of use, and two sets of dosing solutions were verified to be within 99.1107.1% of their theoretical concentrations HPLC prior to the period of administration (NTP, 1999). Each dosing solution was coded so that treatment and examination of animals were performed without knowledge of the dose levels. The dose volume was 5 ml/kg based on body weight taken just prior to each daily dose.
Observations.
Animals were weighed on gd 0, daily during treatment from gd 6 through 19, and on gd 20 (in life and at termination). Food and water weights were taken at 1- to 6-day intervals throughout the study beginning on gd 0. Clinical signs were recorded once daily prior to initiation of treatment, and three times daily during the treatment period (at dosing and at approximately 20 min and 2 h postdosing). Animals were randomized with respect to dose group prior to termination. Following termination by CO2 asphyxiation on gd 20, maternal liver and gravid uterine weights were measured and corpora lutea were counted. Uterine contents were evaluated for the number of implantation sites, resorptions, late fetal deaths (i.e., fetuses with discernible digits and weighing greater than 0.9 g, but displaying no vital signs at the time of uterine dissection), and live fetuses. The uterus was stained to reveal possible early resorptions (Salewski, 1964) when visible evidence of pregnancy was not apparent. Live fetuses were dissected from the uterus and anesthetized by inducing hypothermia (Blair, 1971
; Danneman and Mandrell, 1997
; Lumb and Jones, 1973
; Wixson and Smiler, 1997
). Each live fetus was weighed and examined for external morphological abnormalities, including cleft palate. Approximately one-half (50%) of the fetuses were terminated by decapitation and the remaining fetuses by evisceration.
Approximately one-half (50%) of the fetal carcasses were sexed and examined for visceral morphological abnormalities using a fresh-tissue dissection method (Staples, 1974; Stuckhardt and Poppe, 1984
). The same fetal carcasses were decapitated prior to dissection. Fetal heads were fixed and decalcified in Bouin's solution and subsequently examined using a freehand sectioning technique (Wilson, 1965
). All fetal carcasses were eviscerated (and sex determined for those not scheduled for a full visceral morphological examination), and the skeletons macerated and stained with alcian blue/alizarin red S stain (Marr et al., 1988
). Intact fetal skeletons (i.e., those fetuses that were not decapitated) were examined for skeletal morphological abnormalities.
Statistical analyses.
The unit for statistical measurement was the pregnant female or the litter. Quantitative continuous data were compared among treatment groups by parametric tests if Bartlett's test for homogeneity of variance was not significant. If Bartlett's test indicated a lack of homogeneity (p < 0.001), then nonparametric tests were applied (Winer, 1962). Statistical analyses were based on SAS® software (SAS Institute, Inc., 1989a
, b
; 1990a
, b
, c
; 1992
; 1996
; 1997
).
General Linear Models (GLM) were applied to the Analyses of Variance (ANOVA) and the Tests for Linear Trend. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data (Snedecor and Cochran, 1967). For litter-derived percentage data, the ANOVA was weighted according to litter size. If a significant (p < 0.05) main effect for dose occurred, then a one-tailed Dunnett's Multiple Comparison Test (Dunnett, 1955
; 1964
) was used to compare each treatment group to the control group for that measure, except that a two-tailed test was used for significant maternal body and organ weight parameters, maternal feed and water consumption, fetal body weight, and percent males per litter.
Nonparametric tests for continuous variables with nonhomogeneous data included the Kruskal-Wallis one-way analysis of variance by ranks for among-group differences and, if significant (p < 0.05), the Mann-Whitney U test for pairwise comparisons to the vehicle control group (Siegel, 1956). A two-tailed Mann-Whitney U test was used for significant maternal weight change parameters (gd 12 to 15), maternal feed consumption (gd 12 to 15, 18 to 19), and a one-tailed test was applied to the percent fetuses with unossified sternebra per litter. Jonckheere's test for k independent samples (Jonckheere, 1954
) was used as the nonparametric test for detection of dose-response trends.
All nominal scale measures were analyzed by Chi-Square Test for Independence for differences among treatment groups (Snedecor and Cochran, 1967) and by the Cochran-Armitage Test for Linear Trend on Proportions (Agresti, 1990
; Armitage, 1955
; Cochran, 1954
). None of these tests were statistically significant.
The alpha level for each statistical comparison was 0.05, and the significance levels for trend tests and pairwise comparisons were reported as p < 0.05 or p < 0.01.
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RESULTS |
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Isoeugenol-induced sedation was observed in all treated groups. Evidence was minimal at 250 mg/kg/day, since only one female per day was lethargic (gd 11, 12, 13, 17, and 18), and none were found prone. At 500 mg/kg/day, five females were lethargic on gd 6, and 12 each day were lethargic on gd 919; furthermore, two females were found prone on gd 17. Sedation was most evident at the high dose, with 9 females lethargic and 16 females prone on gd 6. Throughout the remainder of the dosing period (gd 719), 07 females were lethargic and 619 females were prone per day (data not shown).
Maternal body weight exhibited significant dose-related decreasing trends beginning on gd 9 and continuing throughout treatment. Maternal body weight was significantly depressed at the mid- and high-dose groups on gd 9, and at all three dose groups beginning on gd 12 (data not shown). On gd 20, maternal body weight was 95, 92, and 90% of the control value in the low-, mid-, and high-dose group, respectively (Table 1). Maternal body weight gain was also affected by isoeugenol exposure. Maternal weight gain for the treatment (gd 6 to 20) and gestation (gd 0 to 20) periods was significantly reduced at all doses (Table 1
). Corrected weight gain (i.e., gestational weight gain minus gravid uterine weight) showed a significant decreasing trend (72, 52, and 34% of controls), and reductions were significant at all dose levels (Table 1
).
Absolute (grams per day) and relative (grams per kilogram per day) maternal feed and water consumption were comparable across treatment groups prior to initiation of treatment (Table 1). Absolute maternal feed consumption was significantly decreased at the 500 and 1000 mg/kg/day dose levels on gd 620. However, maternal relative feed consumption was significantly reduced only at 1000 mg/kg/ day from gd 6 to 20 (Table 1
). Absolute and relative maternal water consumption both exhibited an increasing trend for gd 6 to 20, but significant increases above the control group were noted only for relative maternal water consumption at 500 and 1000 mg/kg/day (Table 1
).
Gravid uterine weight and maternal absolute liver weight both exhibited a decreasing dose-response trend. At the mid and high doses, reductions in gravid uterine weight were 9091% of the concurrent control weight (both statistically significant). Maternal relative liver weight exhibited an increasing dose-response trend, with statistical significance at all doses (Table 1).
At termination on gd 20, there were no differences among groups for the number of implantation sites per dam, or for the incidences of resorptions or late fetal deaths (Table 2). Likewise, average live litter size and percentage of male fetuses per litter did not differ among treatment groups (Table 2
).
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There were no statistically significant differences among groups for the incidences of fetal malformations or variations when they were analyzed by total incidence or by the main subgroupings (external, visceral, or skeletal; Table 2). The incidence of malformations was low, with only one fetus (1/373) in the 500 mg/kg/day dose group exhibiting an external malformation (gastroschisis), one fetus (1/179) in the 1000 mg/kg/day dose group exhibiting a visceral malformation (unilateral hydronephrosis), and one fetus in each of the 250 (1/180) and 500 (1/184) mg/kg/day dose groups exhibiting a skeletal malformation (bipartite centrum, Table 3
). No external variations were observed in any dose group (Table 3
). Visceral variations included unilateral enlargement of the lateral ventricle (brain), enlarged nasal sinus, agenesis of the innominate artery, and distended ureter, but these did not occur in a dose-related pattern (Table 3
). Skeletal variations, including short rib, wavy rib and delayed development of the thoracic or lumbar centra, were observed, but none of these occurred with a dose-related incidence (Table 3
). However, the incidence of one skeletal variation, unossified sternebrae, was significantly increased at the high dose, with 0.5% (1/184), 2.2% (4/180), 1.6% (3/184), and 7.8% (14/179) fetuses in the 0, 250, 500, and 1000 mg/kg/day dose groups, respectively, when analyzed separately (Tables 2 and 3
).
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DISCUSSION |
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In the preliminary study, pregnant Sprague-Dawley rats were administered isoeugenol (0, 50, 200, 400, 600, or 800 mg isoeugenol/kg/day in corn oil) by gavage during major organogenesis (gd 615) (NTP, 1993). Signs of toxicity (primarily sedation) were noted in all animals at doses of 400 mg/kg/day and greater, and increased in severity in a dose-related manner. There were no maternal deaths, and maternal body weight was not consistently affected. There was no effect of treatment on any index of developmental toxicity.
In the screening study, pregnant Sprague-Dawley (CD®) rats were dosed by gavage with isoeugenol (200, 400, 600, 800, or 1000 mg/kg/day) or vehicle (corn oil) on gd 6 through 19 (NTP, 1998). There were no maternal deaths. Evidence of sedation was dose-related and the incidence of piloerection was increased at
400 mg/kg/day. Maternal body weight and weight gain were reduced at 1000 mg/kg/day. Corrected body weight gain was reduced at
800 mg/kg/day. Relative liver weight was significantly increased at 600 and 800 mg/kg/day, with a similar increase at 1000 mg/kg/day, which did not reach statistical significance. Isoeugenol did not affect prenatal viability, fetal weight, or external fetal morphology (NTP, 1998
).
Maternal body weight effects in the screening study (NTP, 1998) exceeded those previously reported in the preliminary study (NTP, 1993
). In the preliminary study, maternal body weight was transiently reduced (800 mg/kg/day, gd 8), but long-term indices of maternal body weight gain (i.e., treatment period, gestation, or corrected weight gains) were not affected. This difference may be due to differing lengths of exposure, and/or postdosing recovery as previously reported for formamide (Price et al., 1999
). Specifically, the preliminary study (NTP, 1993
) included a 5-day recovery period between the end of dosing and termination, whereas only 24 h elapsed between the final dose and termination of rats in the screening study (NTP, 1998
). In addition, statistical power was lower in the preliminary study (NTP, 1993
) due to the small number of pregnancies per group.
In the present developmental toxicity study (NTP, 1999), timed-mated Sprague-Dawley (CD®) rats were dosed by gavage with isoeugenol (0, 250, 500, or 1000 mg/kg/day) from gd 6 through 19. Aversion to treatment was noted at all isoeugenol doses, as evidenced by an increased incidence of rooting in the cage bedding after dosing, and the incidence of piloerection was increased at
500 mg/kg/day. In addition, dose-related evidence of sedation occurred in all isoeugenol-exposed groups. Clinical signs observed in this study are consistent with previous reports of rotorod performance deficits (myorelaxation) or sleep induction (anesthesia) in mice, as well as hypothermia in rats following sufficiently high doses of isoeugenol administered by the ip route (Dallmeier and Carlini, 1981
; Sell and Carlini, 1976
).
Maternal body weight and body weight gain were decreased at all doses in a dose-related manner. Maternal body weight gain also decreased in a manner that was related to dose and length of exposure, with the most persistent effects being observed in the high-dose group. Exposure to isoeugenol also decreased maternal relative food intake in the high-dose group, and increased maternal relative water intake in the mid- and high-dose groups. Relative maternal liver weight was increased at all doses.
Isoeugenol did not affect prenatal viability or the incidence of fetal malformations (external, visceral, or skeletal) at any dose. At 1000 mg/kg/day, average fetal body weight per litter was significantly reduced, and the incidence of unossified sternebra(e), a skeletal variation, was increased. Thus, evidence of developmental toxicity was limited to intrauterine growth retardation, including a mild delay in skeletal ossification, at 1000 mg/kg/day.
In the present study, all doses were clearly within the pharmacologically active range for isoeugenol administered orally to rats (i.e., based on clinical evidence of sedation) and were several orders of magnitude higher than the presumed per capita exposure to isoeugenol due to its use as a flavoring ingredient (0.8 µg/kg). Furthermore, mild developmental delay in the rat conceptus was found at doses at least four times higher than those associated with pharmacological or toxicological effects in the adult pregnant female rat. Pharmacological activity (sedation), maternal toxicity (reduced body weight and corrected weight gain), and aversion to dosing were noted at 250 mg isoeugenol/kg/day on gd 6 through 19. Thus, the maternal toxicity LOAEL was 250 mg/kg/day and the maternal toxicity NOAEL was not determined in this study. Reduced fetal body weight and an increased incidence of unossified sternebra(e) were found at 1000 mg isoeugenol/kg/day. Thus, the developmental toxicity LOAEL was 1000 mg/kg/day based on intrauterine growth retardation and mildly delayed skeletal ossification. The developmental toxicity NOAEL was 500 mg/kg/day on gd 6 through 19.
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ACKNOWLEDGMENTS |
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NOTES |
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This study was presented in part at the 40th Annual Meeting of the Teratology Society, Palm Beach, Florida [published in Teratology 61(6), 483 (Abstract P33). The published abstract indicated that fetal body weight was reduced by > 10% at 1000 mg/kg/day in the developmental toxicity study. This was true in the screening study (NTP, 1998), where reductions in fetal body weight were 11% (both sexes), 11% (males), and 10% (females) below concurrent controls. However, in the developmental toxicity study (NTP, 1999
), reductions in fetal body weight were 8% (both sexes), 7% (males), and 9% (females) below concurrent controls.
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