Evaluation of the Developmental Toxicity of Formamide in New Zealand White Rabbits

Julia D. George*,1, Catherine J. Price*, Melissa C. Marr*, Christina B. Myers* and Gloria D. Jahnke{dagger}

* Chemistry and Life Sciences, Center for Life Sciences and Toxicology, Herman Laboratory Building, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709–2194; and {dagger} 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 December 18, 2001; accepted April 29, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Naturally mated female New Zealand White (NZW) rabbits (24/group) received formamide (35, 70, or 140 mg/kg/day) or vehicle (1 ml/kg deionized/distilled water) by gavage on gestational days (GD) 6 through 29. The study was conducted using a 2-replicate design. Maternal food consumption (absolute and relative), body weight, and clinical signs were monitored at regular intervals throughout gestation. One and four maternal deaths occurred at the low and high doses, respectively. Abortions or early deliveries were noted in 0, 2, 2, and 8 females in the 0, 35, 70, and 140-mg/kg/day dose groups, respectively. Other clinical signs associated with formamide exposure were minimal: primarily reduced or absent fecal output at the high dose (2–13 animals/day). Also at the high dose, maternal body weight was significantly depressed on GD 21, 24, and 27 (87–90% of the control value); maternal body weight gain was significantly reduced for GD 12 to 15, 18 to 21, and 21 to 24 (treated animals gained less than 1 g, or lost up to 100 g). In addition, maternal body weight gain was reduced at the middle dose for GD 18 to 21. Maternal body weight gain, corrected for gravid uterine weight, was unaffected. Relative maternal food consumption in the high-dose group was 34–59% of control intake from GD 12 through GD 24, but was comparable to controls thereafter. At termination (GD 30), confirmed-pregnant females (9–20 per group) were evaluated for clinical status, liver weights, and gestational outcome; live fetuses were examined for external, visceral, and skeletal malformations and variations. Maternal liver weight (absolute or relative to body weight) was unaffected by treatment, but gravid uterine weight at the high dose was 71% of the control value. A significantly increasing trend was noted for the percent non-live implants per litter. In addition, although not statistically significant from the control group, the values for the percent late fetal deaths per litter and percent non-live implants per litter in the 140-mg/kg/day group were higher than maximum historical values, suggesting an increase in late gestational deaths in the surviving high-dose animals. Formamide decreased the mean number of live fetuses per litter at the high dose to 66% of the control value. Mean fetal body weight per litter for males and the sexes combined was significantly decreased at the high dose; mean female fetal body weight was also decreased, although the difference did not reach statistical significance. There was no effect of treatment on the incidence of external, visceral, or skeletal malformations or variations in animals surviving to scheduled necropsy. In summary, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 70 mg/kg/day and the lowest-observed-adverse-effect level (LOAEL) was 140 mg/kg/day under the conditions of this study. Similarly, the NOAEL for developmental toxicity was 70 mg/kg/day and the LOAEL was 140 mg/kg/day.

Key Words: formamide; developmental toxicity; teratogenicity; rabbits; morphological development.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Formamide (FORM) is used as an industrial solvent in organic synthesis reactions, and as an intermediate in the manufacture of dyes and pigments (Budavari et al., 1996Go; ITII, 1988Go; NTP, 2000Go; Sax and Lewis, 1987Go). It is also used as a softener for paper, gums, and animal glues, as an ionizing and pharmaceutical solvent, in ink solutions, and as a versatile synthetic reagent. ACGIH (1999) has set the threshold limit value (TLV) for FORM at 10 ppm, and the NIOSH time-weighted average (TWA) is also 10 ppm (15 mg/m3 for skin; NIOSH, 2000Go). Documented incidences of exposure or quantitative occupational monitoring for FORM exposures were not found in the recently published literature, except by indirect exposure to alkylformamides (see below).

FORM is generated in vivo as a metabolite of structurally related alkylformamides, which have medicinal and/or industrial uses. In mice, FORM was one of several metabolites found in the plasma and urine after exposure to N-methylformamide (Gescher et al., 1982Go; NMF; Ross et al., 1981Go), a potential antitumor medication studied in recent clinic trials (Cody et al., 1992Go; Del Bufalo et al., 1994Go). NMF is also a potential photoproduct of the aqueous herbicide, fluridone, but environmental concentrations are likely to be quite low (ppb range; West and Turner, 1988Go). More importantly, FORM is found as one of the major metabolites following exposure to N,N-dimethylformamide (DMF; Major et al., 1998Go; Mraz et al., 1989Go; Ogata et al., 1997Go; Saillenfait et al., 1997Go), another widely used industrial solvent (Cheng et al., 1999Go; Lareo and Perbellini, 1995Go; Sakai et al., 1995Go; ). In human volunteers exposed to DMF by inhalation, a common route of occupational exposure, FORM accounted for ~8–24% of the total dose excreted in the urine (Mraz et al., 1989Go). In laboratory animals (mouse, rat, hamster), FORM accounted for ~8–38% of the total dose (Mraz et al., 1989Go).

The literature pertaining to the toxic effects of alkylformamides and their metabolites is extensive, and a thorough review exceeds the scope of the present manuscript. Developmental toxicity of NMF and DMF has been reported in laboratory animals; see the review by Kennedy (2001) and more recent studies, including those of Hellwig et al.(1991); Kelish et al.(1995); Rickard et al.(1995); Liu et al.(1989); and Saillenfait et al.(1997). For instance, NMF has been shown to cause increased embryolethality after nose-only inhalation exposure of maternal CD® rats to 150 ppm, 6 h/day on GD 7 to 16 (vaginal sperm = GD 1) (Rickard et al., 1995Go). In addition, reduced fetal weight was observed at 50 and 150 ppm, while increases in fetal malformations, including microphthalmia, anophthalmia, fused ribs and/or vertebrae, and distended brain ventricles were noted at 150 ppm. Oral gavage administration of NMF to pregnant rats on GD 6 to 15 (sperm detection = GD 0) produced reduced fetal viability and body weight, and an increased incidence of cephalocele and sternoschisis at 75 mg/kg/day in the presence of decreased maternal food consumption and weight gain (Liu et al., 1989Go). In rabbits, oral gavage administration of 50 mg/kg/day NMF on GD 6 to 18 produced similar results (Liu et al., 1989Go). Rabbit fetuses exhibited increased incidences of gastroschisis, cephalocele, domed head, and anomalies of the skull and sternum. Hellwig et al.(1991) reported that oral gavage administration of 0, 166, 503, and 1510 mg/kg/day DMF to rats, on GD 6 to 15, produced increased mid-gestation embryolethality at the high dose, in the presence of reduced maternal body-weight gain. Surviving fetuses had one or more anomalies including anasarca, tail aplasia, micrognathia, and anomalies of the ribs, sternum, and vertebral column. NMRI mice, exposed orally to 182 or 548 mg/kg/day DMF by gavage on GD 6 to 15, exhibited decreased fetal body weight at both doses and an increase in the total incidence of anomalies (malformations and variations) at the high dose (Hellwig et al., 1991Go).

The reproductive and developmental toxicity of FORM has previously been investigated in mammalian species, including rats, mice, and rabbits (BASF, 1974aGo,bGo; DuPont, 1967, 1992; Fail et al., 1998Go; Gliech 1974Go; Kennedy, 2001Go; Merkle and Zeller, 1980Go; Oettel and Frohberg, 1964Go; Oettel and Wilhelm, 1957Go; Thiersch, 1962Go, 1971Go). However, treatment periods in previous developmental toxicity testing and research focused either on critical periods of morphological development or covered the period of organogenesis. Merkle and Zeller (1980) evaluated pregnant Chbb:HM rabbits exposed to FORM by gavage on GD 6 through 18, at doses of 0, 23, 79, or 227 mg/kg/day. No significant maternal or developmental toxicity was observed at 23 mg/kg/day. Decreased maternal body weight, decreased fetal-body weight, and an increase in fetal morphological anomalies were observed at 79 mg/kg/day, whereas maternal mortality (80%) and no live litters were observed at 227 mg/kg/day.

None of the developmental toxicity studies conducted prior to 1998 exposed animals throughout the embryo/fetal period, as recommended by current guidelines for prenatal developmental toxicity testing (U.S. EPA, 1997Go, 1998Go; U.S. FDA, 1994bGo). Thus, current testing strategies call for a longer gestational exposure period and thereby eliminate the recovery period for potentially reversible endpoints such as maternal or embryo/fetal body weight. In rats, for example, oral exposure to FORM by gavage during major organogenesis (GD 6–15) was associated with decreased fetal body weight and increased fetal malformations in rats, including malformations of the vertebral column and ribs at 318 mg/kg/day (Covance Research Products, Inc., Denver, PA).), but not at 177 mg/kg/day (BASF, 1974bGo, 1983Go). However, in a more recent study, FORM was evaluated for developmental toxicity in Sprague Dawley rats after gavage administration of 0, 50, 100, or 200 mg/kg/day on GD 6 through 19 (George et al., 2000Go; NTP, 1998bGo). Using this extended treatment regimen, FORM did not affect prenatal viability or incidences of fetal malformation or variations, although average fetal body weight/litter was decreased at 100 and 200 mg/kg/day. Fetal body weight was affected at lower daily doses than in previous studies, possibly due to the longer total exposure period and lack of a recovery period between cessation of treatment and termination. The maternal toxicity NOAEL was 100 mg/kg/day and the LOAEL was 200 mg/kg/day. The developmental toxicity NOAEL was 50 mg/kg/day and the LOAEL was 100 mg/kg/day. Thus, modification of the study design may influence dose range selection as well as the overall pattern of results, including the critical endpoints that define the NOAEL/LOAEL values.

The widespread uses of FORM, as well as prior evidence of developmental toxicity for FORM and structurally related alkylformamides, warranted determination of developmental toxicity LOAEL and NOAEL values based on a study in nonrodents, designed in accordance with current regulatory guidelines. The results of the proposed investigation provide additional information relevant to the safety assessment of FORM exposure during pregnancy, with particular focus on in utero growth, viability, and morphological development. The present study was designed to establish NOAELs and LOAELs for maternal and developmental toxicity in a nonrodent species, following daily oral exposure by gavage throughout the embryo/fetal period. Because FORM had been previously studied using the shorter exposure period (i.e., major organogenesis) required by predecessor testing guidelines, the influence of the longer exposure period on dose-range selection and outcome for various toxicity endpoints in rabbits was also of interest.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and animal husbandry.
This study was conducted in accordance with the Food and Drug Administration Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (U.S. FDA, 1994aGo, with subsequent revisions). Copies of the final study report (NTP, 2001Go) are available for a fee from the National Technical Information Service, Springfield, VA 22161.

The experimental animals were female Hra:(NZW)SPF; New Zealand White (NZW) rabbits (Covance Research Products Inc., Denver, PA). Prior to arrival at RTI, animals were individually identified by the supplier with a unique animal identification number, through the use of a subcutaneously implanted microchip (Avid, Norco, CA). A separate shipment of 48 animals was used for each replicate of the study. Prior to shipment to RTI, females were naturally mated at the vendor's facilities. For each replicate, animals arrived at RTI on GD 1 (n = 24) or GD 2 (n = 24). Animals were maintained in quarantine for 3 days after arrival at RTI. Female rabbits were assigned to treatment groups by stratified randomization for body weight on GD 0, as reported by the vendor, so that mean body weight on GD 0 did not differ among treatment groups (Fig. 1Go). Maternal body weight for confirmed pregnant females used in this study ranged from 2894 to 3919 g on GD 0 in Replicate I, and 3255 to 4185 g on GD 0 in Replicate II. The study design included two consecutive breeding dates for each replicate. Females were housed singly in stainless steel cages with mesh flooring (Hoeltge, Inc., Cincinnati, OH). Food (Purina Certified Rabbit Chow [#5322], PMI, St. Louis, MO) was rationed at ~65 g for the first 24 h after arrival (GD 1 or 2). For the following 24 h, females received ~125 g of food, which was available ad libitum for all females from GD 3 to scheduled sacrifice. Tap water (City of Durham, NC) was provided ad libitum to all females throughout the study. The light cycle (12 h light:12 h dark), temperature, and relative humidity in the animal rooms were monitored, recorded, and controlled (Siebe/Barber-Coleman Network 8000® System with SIGNAL® Software [Revision 4.1], Siebe Environmental Controls (SEC)/Barber-Colman Company, Loves Park, IL) throughout the study. The ranges for temperature and relative humidity were 63.9–71.3°F and 30.7–80.4% relative humidity, respectively, for Replicate I, and 63.9–69.4°F and 27.0–69.7% RH, respectively, for Replicate II.



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FIG. 1. Maternal gestational body weight. Formamide caused a significant decreasing trend for maternal body weight on GD 15, 18, 21, 24, 27, 29, and 30 (test for linear trend). On GD 21, 24, and 27, maternal body weight was significantly decreased at the high dose (ANOVA and Dunnett's test). Data are presented as mean ± S.E.M. (n = 9–22 pregnant dams per group; also see Table 1Go, footnote b).

 
Test chemical and treatment.
The doses chosen for this study were 0, 35, 70, and 140 mg FORM/kg/day. FORM the bulk chemical [formamide, Lot No. 15231AN, Aldrich Chemical Company Inc., Milwaukee, WI] was obtained by Midwest Research Institute under a separate contract to the Sponsor) of 99% purity (identity and purity of the test material were confirmed by Midwest Research Institute under a separate contract to the Sponsor [NIEHS Contract N01-ES-55385]. Identity was confirmed by infrared (IR) spectroscopy and 1H nuclear magnetic resonance spectroscopy. Purity was determined by gas chromatography (GC) and GC/mass spectrometry (NTP, 2001Go). Bulk chemical reanalyses (BCR) were performed approximately 5 months prior to study initiation and again between the in-life phases of Replicates I and II. Identity was reconfirmed by IR spectroscopy and relative purity was 98.7% when compared by HPLC to a frozen reference sample for both BCRs (NTP, 2001Go). was dissolved in deionized/distilled water. Stability of the dosing solutions was verified for the period of use, and dosing solutions were verified to be within 98.0–103.1% of their theoretical concentrations by high performance liquid chromatography (HPLC) prior to and after the period of administration (NTP, 2001Go). Each dosing solution was coded so that treatment and examination of animals was performed without knowledge of the dose levels. The volume administered (1 ml/kg) was based on body weight taken daily prior to dosing, during the dosing period.

Dose selection was based on a screening study in which NZW rabbits (8 animals per group) were exposed to FORM (0, 10, 20, 40, 80, or 120 mg/kg/day) by gavage from GD 6 through 29 (NTP, 1998cGo). At 120 mg/kg/day, 3 females aborted (GD 25) or delivered early (GD 27 or 29). One maternal death occurred at each of the 40- and 80-mg/kg/day groups, but there were no deaths in any other group. At 120 mg/kg/day, maternal relative food intake was decreased during all measurement periods between GD 12 and 24, but the data were insufficient to evaluate this high-dose effect between GD 24 and 30. Maternal body gain was significantly decreased at the high dose for GD 12 to 15. Incidences of prenatal mortality and morphological anomalies were not affected. Gravid uterine weight and live litter size were unaffected. Average fetal body weight per litter was 106, 107, 104, 94, and 83% of the control weight in the 10-through 120-mg/kg/day groups, respectively. Although this apparent dose response did not reach statistical significance, the reduction of fetal body weight at the high dose was considered to be a biologically relevant response to FORM exposure. There were no external morphological anomalies noted in that study. Based on these results, doses of 0, 35, 70, and 140 mg/kg/day were selected for the present developmental toxicity study. The high dose of 140 mg/kg/day was chosen since no persistent clear-cut maternal toxicity was noted at 120 mg/kg/day in the screening study.

Observations.
Naturally mated does were weighed on GD 0 (vendor), and food and body weights were recorded on GD 3, 6, 9, 12, 15, 18, 21, 24, 27, 29, and 30. In addition, body weight was recorded immediately following termination on GD 30. Clinical signs were recorded once daily prior to initiation of treatment, and twice daily during the treatment period. Following termination by intravenous injection (marginal ear vein) of sodium pentobarbital on GD 30, maternal liver and gravid uterine weights were measured. Uterine contents were evaluated for the number of implantation sites, resorptions, late fetal deaths (i.e., fetuses with discernible digits and weighing greater than 10.0 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, 1964Go) when visible evidence of pregnancy was not apparent.

Live fetuses were dissected from the uterus and immediately terminated by intraperitoneal injection of sodium pentobarbital. Each live fetus was counted, weighed, and examined for external morphological abnormalities, including cleft palate. Fetal carcasses were sexed and examined for visceral morphological abnormalities using a fresh tissue dissection method (Staples, 1974Go; Stuckhardt and Poppe, 1984Go). Approximately one-half (50%) of the fetal carcasses were decapitated prior to dissection. Fetal heads were fixed and decalcified in Bouin's solution and subsequently examined using a free-hand sectioning technique (Wilson, 1965Go). All fetal carcasses were eviscerated and the skeletons macerated and stained with alcian blue/alizarin red S stain (Marr et al., 1988Go). All fetal skeletons were examined for skeletal morphological abnormalities. Due to the fact that ~50% of the fetal carcasses had been decapitated, the skeletal structures of the head were only examined for intact carcasses.

Statistical analyses.
The unit for statistical measurement was the pregnant female or the litter. Quantitative continuous data (e.g., maternal body weights, fetal body weights, feed consumption, etc.) were compared among treatment groups by parametric statistical tests whenever Bartlett's test for homogeneity of variance was not significant. Statistical analyses were based on SAS® software (SAS Institute, Inc., 1989aGo,bGo, 1990aGo, bGo,cGo, 1992Go, 1996Go, 1997Go) available at RTI.

General linear models (GLM) procedures 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: e.g., percent resorptions per litter, percent malformations per litter, and percent variations per litter (Snedecor and Cochran, 1967Go). For litter-derived percentage data, the ANOVA was weighted according to litter size. When a significant (p < 0.05) main effect for dose or replicate occurred, Dunnett's multiple comparison test (Dunnett, 1955Go, 1964Go) was used to compare each treatment group to the control group for that measure. A one-tailed test (i.e., Dunnett's) was used for all pair-wise comparisons to the vehicle control group, except that a 2-tailed test was used for maternal body and organ weight parameters, maternal feed consumption, fetal body weight, and percent males per litter.

Data for any measure that showed a significant (p < 0.05) dose x replicate interaction in a 2-way (dose x replicate) ANOVA were presented as mean ± SEM for each cell in the ANOVA design. Dose effects within each replicate were further evaluated using a one-way ANOVA, test for linear trend, and Dunnett's test.

All nominal scale measures were analyzed by a Chi-square test for independence for differences among treatment groups (Snedecor and Cochran, 1967Go) and by the Cochran-Armitage test for linear trend on proportions (Agresti, 1990Go; Armitage, 1955Go; Cochran, 1954Go).

The alpha level for each statistical comparison was 0.05, and the significance levels for trend tests and pair-wise comparisons were reported as p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Twenty-four naturally mated female rabbits (12 per group per replicate) were assigned to each treatment group in this study. One animal was found dead in the low-dose group (GD 24), and 4 females were found dead or moribund in the high-dose group (GD 24, 25, 27, or 28) (Table 1Go). Necropsy of the animal in the low-dose group revealed no live fetuses and only one resorption, hemorrhagic lungs with signs of infection, and empty stomach and intestines. The dead or moribund animals in the high-dose group had fetuses that were small or normal size for gestational age. Two females in the low- and mid-dose groups, and 8 females in the high-dose group aborted or delivered early, and additional females were removed from the study, due to technical errors or because pregnancy was not confirmed. Thus, at termination (GD 30), pregnancy was confirmed in 19/22 (86%), 19/19 (100%), 20/22 (91%), and 9/10 (90%) of the surviving females in the control, low-, mid-, and high-dose groups, respectively (Table 1Go).


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TABLE 1 Maternal Toxicity in New Zealand White Rabbits Exposed to Formamide on Gestational Days 6 through 29
 
Other clinical signs associated with FORM exposure were minimal (data not shown). Observation of reduced fecal output was more often recorded at the high dose (2–13 animals per day), which was most likely related to reduced feed consumption (Fig. 2Go). Maternal body weight exhibited a decreasing trend beginning on GD 15, and was significantly reduced, compared to the control group, at 140 mg/kg/day on GD 21 (90% of controls), GD 24 (87% of controls) and 27 (87% of controls; Fig. 1Go). On GD 29 and 30, maternal body weight for surviving animals in the high-dose group was still reduced (90–91% of controls), although the difference was not statistically significant (Fig. 1Go). Weight gain in the high-dose group was significantly less than the control group for GD 12–15, 18–21, and 21–24 (Table 1Go). At the mid-dose, weight gain was transiently decreased compared to the control group, from GD 18 to 21 (Table 1Go). Maternal weight gain across the entire treatment period (GD 6 to 29), or the gestation period (GD 0 to 30) was unaffected by treatment (Table 1Go). For surviving animals in the high-dose group, the apparent decrease in weight gain during treatment or gestation was not accompanied by significant trend tests or pair-wise comparisons to controls. Gravid uterine weight was significantly reduced in the high-dose group (71% of the control value; Table 1Go). Following correction for the weight of the gravid uterus, maternal gestational weight gain was not significantly reduced at any dose (Table 1Go). Examination of the data revealed that corrected maternal weight gain was similar in the mid- and high-dose groups, compared to the control group (–212.6 or –228.3 g vs. –230.4 for the control group; Table 1Go). In light of the significantly reduced gravid uterine weight, these data suggest that reduced gravid uterine weight was a primary contributor to reduced maternal body weight and gestational weight gain during the latter portion of this study.



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FIG. 2. Maternal gestational feed intake. Formamide was associated with a decrease in relative maternal feed consumption for GD 9 to 12 (trend only), and GD 12 to 15, 15 to 18, 18 to 21, and 21 to 24 (trend and ANOVA). Relative maternal feed consumption was decreased at the high dose on GD 12 to 15, 15 to 18, 18 to 21, and 21 to 24. Data are presented as mean ± SEM for 8–21 pregnant dams per group (also see Table 1Go, footnote b).

 
Maternal relative feed consumption was significantly decreased in the high-dose group (34–59% of control intake) for GD 12 to 15, 15 to 18, 21 to 24 (Fig. 2Go), and 18 to 21 (Fig. 2Go). However, feed consumption was equivalent across treatment groups for other intervals, including GD 6 to 29 (treatment) and GD 3 to 30 (gestation), which included only high-dose animals surviving until scheduled necropsy (Table 1Go). Maternal liver weight (relative to body weight) did not differ among groups on GD 30 (Table 1Go).

The number of implantation sites per litter exhibited a decreasing trend that appeared to be due to effect in the surviving animals in the high-dose group (Table 2Go). No significant differences were observed among groups for the percent resorptions per litter, percent late fetal deaths per litter, and percent non-live (late fetal deaths and resorptions) implants per litter, although a significant increasing trend was noted for the percent non live implants per litter (Table 2Go). Values for these parameters were higher and exhibited more variability than the control group. Examination of historical control data indicated that the values for the percent late fetal deaths per litter and percent non-live implants per litter were higher than maximum historical values (1.99 ± 1.12 and 16.15 ± 10.34, respectively), suggesting an increase in late gestational deaths in the surviving high-dose animals. The average numbers of live fetuses per litter in the low- and mid-dose FORM-treated groups were 98 and 89% of the control mean, respectively, and neither group was significantly different from the controls (Table 2Go). However, the average number of live fetuses per litter was significantly reduced to 66% of the control value in the high-dose group. Male and female fetal body weight per litter exhibited decreasing trends and were both reduced to 85% of their control values at the high dose (Table 2Go). This reduction was statistically significant for males and for both sexes combined, but not for females. The lack of statistical significance for the reduction in female fetal body weight at the high dose, most likely due to a smaller number of litters with female fetuses (7 vs. 8 for males or 9 for both sexes combined), does not invalidate the biological significance of this effect.


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TABLE 2 Developmental Toxicity in NZW Rabbit Fetuses following Maternal Exposure to Formamide on Gestational Days 6 through 29
 
When considered collectively (all types) or grouped by type (external, visceral, skeletal), there was no difference among groups in the incidence of fetal malformations or variations for litters examined on GD 30 (Tables 2 and 3GoGo). Rare fetal malformations (macrophthalmia, fused and misplaced kidneys, or misshapen brain) were noted in this study, but their individual incidences were low and not dose-related (Table 3Go). Skeletal malformations included anomalies of the vertebral column, ribs, or sternebrae, but there were no apparent dose-related patterns for these findings (Table 3Go).


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TABLE 3 Morphological Abnormalities in NZW Rabbit Fetuses following Maternal Exposure to Formamide on Gestational Days 6 through 29
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prior to the initiation of this developmental toxicity study (NTP, 2001Go) and its related screening study (NTP, 1998cGo), the developmental toxicity potential of FORM had undergone investigation in rats exposed during post-implantation gestation (GD 6–19; NTP, 1998aGo, 1998bGo; George et al., 2000Go). However, no developmental toxicity studies were found in which nonrodents had been exposed to FORM by the oral route during the entire post-implantation period of embryo/fetal development. Thus, the present developmental toxicity study and associated screening study were the first to use an extended period of exposure in rabbits combined with termination for developmental assessment on GD 30.

In the present developmental toxicity study, naturally mated NZW rabbits were dosed by gavage with FORM (0, 35, 70, or 140 mg/kg/day) on GD 6 through 29. Maternal clinical signs of toxicity were limited to the reduction in or absence of fecal output, primarily at the high dose. However, maternal mortality during late gestation was increased at the high dose. In addition, abortion or early delivery was observed to be 4-fold higher in the high-dose group compared to the low- or mid-dose groups. Maternal body weight and weight gain, relative maternal feed consumption, and gravid uterine weight were also decreased at the high dose. In the absence of any other indicators of maternal toxicity (feed consumption, body weight, etc.), the transient decrease in maternal body weight on GD 18 to 21 was not considered to be a significant indication of maternal toxicity at this dose level. Liver weight (absolute and relative) was unaffected by treatment with FORM. FORM decreased fetal survival, live litter size (66% of control) and male fetal-body weight (85% of control) at the high dose, but did not affect the incidence of fetal malformations (external, visceral or skeletal) among survivors in the high-dose group, or at any other dose. Under the conditions of the present study, and based on the mg/kg/day treatment level, the rabbit conceptus was not more sensitive than the adult to the adverse effects of FORM administered orally throughout the embryo/fetal period of gestation. Thus, in the present developmental toxicity study, the NOAELs for both maternal and developmental toxicity were 70 mg FORM/kg/day administered on GD 6 through 29.

Based on similar treatment levels and similar parameters, the results of the present study can be compared to those of Merkle and Zeller (1980; Table 4Go). In the Merkle and Zeller study (1980), pregnant Chbb:HM rabbits were exposed to FORM by gavage on GD 6 through 18, at doses of 0, 23, 79, or 227 mg/kg/day. The results of the two studies agree well at the low doses and the high doses. No significant maternal or developmental toxicity was observed at either 23 mg/kg/day (Merkle and Zeller, 1980Go) or at 35 mg/kg/day in the present study. At 140 mg/kg/day, the high dose in the present study, maternal mortality, a decrease in maternal body weight, a decrease in gravid uterine weight, and decreased fetal viability and fetal body weight were observed, whereas Merkle and Zeller (1980) observed 80% maternal mortality and no live litters at their higher dose of 227 mg/kg/day. Although the mid doses for these studies are close (70 mg/kg/day for the present study vs. 79 mg/kg/day for Merkle and Zeller, 1980Go), the data do not agree as well as would be expected. In the present study, no persistent adverse maternal or developmental effects were observed at 70 mg/kg/day, whereas Merkle and Zeller (1980) observed decreased maternal body weight, decreased fetal body weight, and an increase in fetal morphological anomalies. The reason for the lack of concordance in these two studies at the mid dose is not clear, but could likely be due to the use of rabbits of different strains. The addition of comparative toxicokinetic data for these two strains of rabbit would aid in delineating actual differences in exposure to the test compound.


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TABLE 4 Maternal and Developmental Toxicity in Evaluations of Formamide Administered to CD® Rats
 
Comparison of the present study along with the study by Merkle and Zeller (1980)Go to the study previously conducted in this laboratory in rats (NTP, 1998bGo; George, et al., 2000Go), suggests that, based on the mg/kg/day treatment level, maternal rabbits are more sensitive than maternal rats with regard to the general toxicity of FORM, since the maternal NOAEL and LOAEL in rats were 100 and 200 mg/kg/day, respectively. However, toxicokinetic data would be needed to confirm this observation. Whereas developmental toxicity is observed in rats at doses of FORM lower than those causing maternal toxicity, rabbit fetuses are not uniquely sensitive to the developmental toxicity of FORM, even after extended exposure during gestation (Table 4Go). Maternal mortality was observed in rabbits at >=140 mg/kg/day but not in rats, even at the high dose of 200 mg/kg/day. In addition, in both the Merkle and Zeller studies (1980) and the present study, the LOAEL for developmental toxicity in rabbits was the same as the maternal LOAEL. Fetal rabbits exhibited a reduction in fetal body weight at >=79 mg/kg/day in the Merkle and Zeller (1980) study, and a reduction in fetal viability at >=140 mg/kg/day in the present study, whereas the fetal rat exhibited a decrease in body weight at >=100 mg/kg/day (NTP, 1998bGo). Similar observations of species sensitivity have been made with regard to the toxicity of NMF and DMF (Liu et al., 1989Go; Hellwig et al., 1991Go).


    ACKNOWLEDGMENTS
 
The present study was conducted at Research Triangle Institute (RTI), Research Triangle Park, North Carolina, under contract to the National Toxicology Program and the National Institute of Environmental Health Sciences (NIEHS/NTP Contract N01-ES-65405). We express our appreciation for chemistry support to Cynthia S. Smith of NIEHS, as well as Evelyn Murrill, Robert E. Smith, Roger K. Harris, Robert Moore, Linda Siemann, Michael Kozak, and J. Michael Cannon of Midwest Research Institute. In addition, we thank the following RTI professional and technical personnel who contributed to the completion of this study: Patricia A. Fail, Donald B. Feldman, Frank N. Ali, Frieda S. Gerling, Vickie I. Wilson, Betty T. McTaggart, Lawson B. Pelletier, Marian C. Rieth, Dee A. Wenzel, Angela D'Antonio, M. Michael Veselica, Randy A. Price, T. Douglas Burnette, and Donald L. Hubbard.


    NOTES
 
This research was presented at the 41st Annual Meeting of the Teratology Society, Montreál, Québec, Canada, June 23–28, 2001.

1 To whom correspondence should be addressed. Fax: 919–541–6499. E-mail: jdg01{at}rti.org. Back


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 DISCUSSION
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