Evaluation of the Developmental Toxicity of Formamide in Sprague-Dawley (CD) Rats

Julia D. George1, Catherine J. Price, Melissa C. Marr, Christina B. Myers and Gloria D. Jahnke*

Chemistry and Life Sciences, Research Triangle Institute, Post Office Box 12194, Research Triangle Park, North Carolina 27709–2194 * Developmental and Reproductive Toxicology Group, National Toxicology Program, National Institute of Environmental Health Sciences, Post Office Box 12233, Research Triangle Park, North Carolina 27709

Received April 10, 2000; accepted June 28, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Timed-pregnant CD® outbred albino Sprague-Dawley rats received formamide (50, 100, or 200 mg/kg/day) or vehicle (5 ml/kg deionized/distilled water, po) on gestational days (gd) 6 through 19. Maternal food and water consumption (absolute and relative), body weight, and clinical signs were monitored at regular intervals throughout gestation. At termination (gd 20), confirmed-pregnant females (21–23 per group) were evaluated for clinical status and gestational outcome; live fetuses were examined for external, visceral, and skeletal malformations and variations. There were no maternal deaths and no dose-related clinical signs. At 200 mg/kg/day, maternal body weight on gd 20, weight gain, and gravid uterine weight were significantly decreased. Maternal weight gain, corrected for gravid uterine weight, liver weight (absolute or relative), and food and water consumption (absolute or relative), were not affected. Formamide did not affect prenatal viability or incidences of fetal malformations or variations. 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 previously published studies, possibly due to the longer total exposure period and/or lack of a recovery period between cessation of exposure and termination. In summary, the maternal toxicity no-observed-adverse-effect level (NOAEL) was 100 mg/kg/day and the low observed adverse effect level (LOAEL) was 200 mg/kg/day under the conditions of this study. Similarly, the developmental toxicity NOAEL was 50 mg/kg/day and the LOAEL was 100 mg/kg/day.

Key Words: formamide; developmental toxicity; teratogenicity; rats; 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 (ITII, 1988Go; Sax and Lewis, 1987Go; Windholz et al., 1983Go). 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 TLV for formamide at 10 ppm, and the NIOSH TWA is also 10 ppm (15 mg/m3 for skin) (NIOSH, 2000Go). Documented incidences of exposure or quantitative occupational monitoring for formamide exposures were not found in the recently published literature, except by indirect exposure to alkylformamides (see below).

Formamide is generated in vivo as a metabolite of structurally related alkylformamides, which have medicinal and/or industrial uses. In mice, formamide was one of several metabolites found in the plasma and urine after exposure to N-methylformamide (NMF; Ross et al., 1981; Gescher et al., 1982), a potential antitumor medication studied in recent clinic trials (Cody et al., 1992Go; Del Bufalo et al., 1994Go). N-methylformamide is also a potential photoproduct of the aqueous herbicide, fluridone, but environmental concentrations are likely to be quite low (ppb range; Liu et al., 1989; West and Turner, 1988). More importantly, formamide is found as one of the major metabolites following exposure to N,N-dimethylformamide (DMF; Mraz et al., 1989; Saillenfait et al., 1997), another widely used industrial solvent (Cheng et al., 1999Go; Lareo and Perbellini, 1995Go; Major et al., 1998Go; Ogata et al., 1997Go; Sakai et al., 1995Go). Human volunteers were exposed to DMF by a common route of occupational exposure (inhalation), and formamide accounted for ~8–24% of the total dose excreted in the urine, and in laboratory animals (mouse, rat, hamster), formamide accounted for ~8–38% of the total dose (Mraz et al., 1989Go). The literature pertaining to the biological and toxicological 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 a review by Kennedy 1986, and more recent studies including Hellwig et al., 1991; Kelich et al, 1995; Liu et al., 1989; Rickard et al., 1995; Saillenfait et al., 1997).

The reproductive and developmental toxicity of FORM has also been investigated in mammalian species, including rats, mice, and rabbits (BASF, 1974aGo, 1983Go; Fail et al., 1998Go; Gliech 1974Go; Kennedy, 1986Go; Klauss et al., 1967, Merkle and Zeller, 1980Go; Oettel and Frohberg, 1964Go; Oettel and Wilhelm, 1957Go; Stula et al., 1992Go; Thiersch, 1962Go, 1971Go). However, treatment periods in previous developmental toxicity studies did not extend throughout the embryo/fetal period, as suggested by current guidelines for prenatal developmental toxicity testing (U.S. EPA, 1997GoU.S. EPA, 1998Go; U.S. FDA, 1994bGo). For example, oral exposure to formamide (318 mg/kg/day) 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 (BASF, 1974bGo, 1983Go).

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 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 following daily oral exposure throughout the embryo/fetal period. Because formamide had been previously studied using the shorter exposure period required by predecessor testing guidelines (i.e., major organogenesis), the influence of the longer exposure period on dose-range selection and outcome for various toxicity endpoints 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`s "Good Laboratory Practice Regulations for Nonclinical Laboratory Studies" (U.S. FDA, 1988GoU.S. FDA, 1994aGo). Copies of the final study report (NTP, 1998bGo) are available for a fee from the National Technical Information Service, Springfield, VA 22161.

The experimental animals were Sprague-Dawley-derived outbred albino rats [Crl:CD® (SD)BR VAF/Plus®] (Charles River Laboratories, Inc., Raleigh, NC). Animals were individually identified by ear tag (National Band and Tag Co., Newport, KY) during a 10-day quarantine. 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 by vaginal lavage the next morning for the presence of sperm (Hafez, 1970Go). Female rats weighed from 222 to 273 g on gd 0 (i.e., day of vaginal sperm detection). 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 were provided ad libitum throughout the study. The light cycle (12-h light:dark), temperature, and relative humidity (RH) in the animal rooms were monitored, recorded, and controlled (Siebe/Barber-Colman Network 8000® System with SIGNAL® Software [Version 4.1]) throughout the study. The means (ranges) for temperature and humidity were 72°F (70.7–73.4°F) and 53.5% (48.2–58.5% RH), respectively.

Time-mated females were assigned to dose groups by stratified randomization for body weight on gd 0, so that mean body weights across dose groups were not significantly different on gd 0.

Test chemical and treatment.
The doses chosen for this study were 0, 50, 100, and 200 mg FORM/kg/day. FORM (Obtained by Midwest Research Institute through the sponsor, from Aldrich Chemical Company, Inc., Lot No. 15231AN) (CAS No. 75–12–7) of >99% purity was dissolved in deionized/distilled water. Identity and purity of the test material were confirmed by Midwest Research Institute (NIEHS Contract NO1-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, 1998bGo). Bulk chemical re-analysis was performed within one week of study initiation. Identity was reconfirmed by IR spectroscopy and relative purity was 103.6% when compared by HPLC to a frozen reference sample (NTP, 1998bGo). Stability of the dosing solutions was verified for the period of use, and they were verified to be within 97.9–100.3% of their theoretical concentrations by high performance liquid chromatography, prior to and after the period of administration (NTP, 1998bGo).

Each dosing solution was coded so that treatment and examination of animals were performed without knowledge of the dose levels. The volume administered (5 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 CD® rats were exposed to FORM (0, 62, 125, 250, 500, or 1000 mg/kg/day) by gavage from gd 6 through 19 (NTP, 1998aGo). At 1000 mg/kg/day, excessive maternal toxicity resulted in termination of all dams by gd 14. At >=250 mg/kg/day, maternal food and water intake were transiently reduced during early treatment, with full recovery after gd 15. Maternal body weight and weight gains, including corrected maternal weight gain, were reduced at >=250 mg/kg/day. Incidences of prenatal mortality and morphological anomalies were not affected. However, two fetuses in the 500-mg/kg/day group exhibited unusual malformations (either cleft palate or agenesis of the tail). Gravid uterine weight was reduced at 500 mg/kg/day. Average fetal body weight per litter was 96, 90, 80, and 58% of the control weight in the 62 through 500-mg/kg/day groups, respectively. Thus, fetal body weight was significantly lower than controls at >=125 mg/kg/day (NTP, 1998aGo). Based on these results, the target doses for the definitive study were 0, 50, 100, and 200 mg/kg/day.

Observations.
Animals were weighed on gd 0, then daily during treatment from gd 6 through 19, and on gd 20. Food and water weights were taken at 1–6-day intervals throughout the study, beginning on gd 0. Clinical signs were recorded once daily prior to initiation of treatment, and twice daily during the treatment period. Following termination by CO2 asphyxiation on gd 20, maternal liver weight and gravid uterine weight were measured and ovarian 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, 1964Go) when visible evidence of pregnancy was not apparent. Live fetuses were dissected from the uterus and anesthetized by inducing hypothermia (Blair, 1971Go; Danneman and Mandrell, 1997Go; Lumb and Jones, 1973Go; Wixson and Smiler, 1997Go). Each live fetus was counted, weighed, and examined for external morphological abnormalities, including cleft palate. Approximately one-half of the fetuses were terminated by decapitation and the remaining by evisceration under terminal cold anesthesia.

Approximately one-half of the fetal carcasses were sexed and examined for visceral morphological abnormalities using a fresh tissue dissection method (Staples, 1974Go; Stuckhardt and Poppe, 1984Go). The same 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 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., 1988Go). 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 (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, 1989aGo,bGo; 1990aGo,bGo,cGo; 1992Go; 1996Go; 1997Go) available at RTI.

General Linear Models (GLM) procedures (SAS Institute, 1989aGo,bGo; 1990aGo,bGo,cGo; 1992Go; 1996Go; 1997Go) 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., percentages resorptions per litter, malformations per litter, and 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 occurred, Dunnett's Multiple Comparison Test (Dunnett, 1955Go; 1964) was used to compare each treatment group to the control group for that measure. A one-tailed test (i.e., Dunnett's Test) was used for all pairwise comparisons to the vehicle control group, except that a two-tailed test was used for maternal body and organ weight parameters, maternal feed and water consumption, fetal body weight, and percent males per litter.

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). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Twenty-five timed-mated female rats were assigned to each treatment group in this study, and pregnancy was confirmed in 84–92% per group (Table 1Go). The incidence of clinical signs (primarily alopecia and clinical weight loss) did not appear to be associated with formamide exposure (data not shown). No maternal deaths or morbidity occurred in this study.


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TABLE 1 Maternal Toxicity in CD® Rats Exposed to Formamide on Gestational Days 6 through 19
 
Maternal body weight was reduced at 200 mg/kg/day on gd 18, 19 (data not shown), and 20 (Table 1Go). In addition, maternal body weight gain was significantly decreased in the high-dose group during the first 3 days of treatment (gd 6 to 9), from gd 15 to 18 (data not shown), for the treatment period as a whole (gd 6 to 20) and for the gestational period (gd 0 to 20) as a whole (Table 1Go). Gravid uterine weight was decreased in a dose-related manner (100%, 94%, and 87% of control weight, at 50, 100, and 200 mg/kg/day, respectively), and the reduction was significant at the high dose (Table 1Go). Following correction for the weight of the gravid uterus, maternal gestational weight gain was not significantly reduced at any dose (Table 1Go), suggesting that reduced gravid uterine weight was the primary contributor to reduced maternal body weight and late gestational weight gain in this study. Maternal consumption of food or water was unaffected (data not shown). Maternal liver weight (absolute or relative to body weight) did not differ among groups on gd 20 (Table 1Go).

FORM did not significantly affect any endpoints related to prenatal viability (Table 2Go). The percent resorptions per litter and the percent litters with one or more resorptions per litter did not differ significantly among groups. There were no reported late fetal deaths in this study. Average live litter size in FORM-treated groups was between 97–101% of the control mean. Average fetal body weight per litter in FORM-treated groups was decreased in a dose-related manner, and this reduction was statistically significant at the mid and high doses for both sexes or either sex alone (Table 2Go). Thus, the mean fetal body weight per litter was 98, 93, and 85% of the average control weight for the low-, mid-, and high-dose groups, respectively, for the sexes combined.


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TABLE 2 Developmental Toxicity in CD® Rat Fetuses following Maternal Exposure to Formamide on Gestational Days 6 through 19
 
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 (Tables 2 and 3GoGo). The only individual finding with a distinctive dose-response incidence pattern was unossified sternebra (I, II, III, and/or IV) which was noted as a skeletal variation in 1, 3, 9, and 12 fetuses in the control through high-dose groups, respectively (Table 3Go). A slight increase in the incidence of enlarged lateral ventricles was noted at the mid and high doses (Table 3Go). Consistent with the observed reductions in fetal body weight, these specific morphological findings indicate a mild developmental delay associated with formamide exposure.


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TABLE 3 Morphological Abnormalities in CD® Rat Fetuses Following Maternal Exposure to Formamide on Gestational Days 6 through 19
 
Rare fetal malformations—cleft palate, cleft lip, exencephaly, open eye lid, agenesis or malformation of the tail, dextrocardia, double-outlet right ventricle, and interventricular septal defect—were noted in this study, but their individual incidences were low and not dose-related (Table 3Go). Multiple malformations were observed in several low-weight fetuses in different dose groups, and were considered to be spontaneous events rather than treatment related (Table 3Go). Some of these (for example, cleft palate) were consistent with treatment-related malformations reported at higher oral doses in an earlier investigation (BASF, 1974bGo, 1983Go). However, there was no clear dose relationship for such findings in the present study.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prior to the initiation of this study, the reproductive and developmental toxicity of FORM had been investigated in the rat, mouse, and rabbit, but the exposure period had never encompassed the entire embryo/fetal period of development, except for the previously referenced screening study performed in this laboratory (NTP, 1998aGo). Thus, the present definitive developmental toxicity study and associated screening study were the first to use an extended period of exposure combined with termination for developmental assessment on gd 20. In this definitive study, CD® rats were dosed by gavage with FORM (0, 50, 100, or 200 mg/kg body weight/day) from gd 6 through 19. Minimal evidence of maternal toxicity was noted at 200 mg/kg/day (i.e., reduced body weight and weight gain), and the absence of an effect on maternal corrected-weight gain suggests that developmental toxicity was a contributing factor. This study demonstrated that the maternal toxicity LOAEL was 200 mg/kg/day, and the NOAEL was 100 mg/kg/day. Clear evidence of developmental toxicity in the definitive study was limited to reduction of fetal body weight at >=100 mg/kg/day. The dose response for decreased fetal body weight was highly consistent across the screening study and the definitive study. Fetal body weights (both studies) were 98, 96, 93, 90, 85, 80, and 58% of control weights for doses of 50, 62, 100, 125, 200, 250, or 500 mg/kg/day. Although the percent resorptions per litter appeared increased at 200 mg/kg/day in this study (4.16 vs. 1.40 for the control group), there was no statistically significant difference from control, and the value was well within the range of historical control values for this parameter (1.18–6.82 percent resorptions per litter). Thus, under the conditions of this study, the developmental toxicity for the formamide LOAEL was 100 mg/kg/day and the NOAEL was 50 mg/kg/day.

Except for the length of exposure, the study design of previously reported studies (BASF, 1974bGo, 1983Go) was quite similar to the present definitive study and the associated screening study (NTP, 1998aGo). Results of these studies are summarized in Table 4Go. In the BASF study, pregnant Sprague-Dawley rats were administered technical grade FORM (0, 156, 280, 463, 700, or 1400 µl/kg/day) by gavage during major organogenesis (gd 6–15 day; BASF, 1974bGo, 1983Go). Doses were equivalent to 0, 177, 318, 525, 794, or 1588 mg/kg/day. Maternal weight gain was depressed at all doses (i.e., >=177 mg/kg/day), and other indicators of maternal toxicity were reported at >=794 mg/kg/day. Corrected maternal weight gain was not reported; the contribution of decreased fetal weight to decreased maternal gravid weight is not known. Nearly all (99%) of the fetuses died at 1588 mg/kg/day, and some prenatal mortality was reported at 794 mg/kg/ day. No prenatal mortality was reported at 525 mg/kg/day, a dose more than twice that used in the present NTP-sponsored definitive study. Reduced fetal body weight, as well as an increased incidence of fetal malformations, were reported at >=318 mg/kg/day. Skeletal malformations, including vertebral cleaving, aplasia and hypoplasia, and fused ribs were noted, although data were not provided. Thus, in the BASF study, maternal toxicity was noted at all doses >=177 mg/kg/day (maternal LOAEL), the developmental toxicity NOAEL was equivalent to 177 mg/kg/day, and the developmental toxicity LOAEL was equivalent to 318 mg/kg/day.


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TABLE 4 Maternal and Developmental Toxicity in Evaluations of Formamide Administered to CD® Rats
 
In contrast to the BASF study, the results of the study described in this manuscript indicate that the conceptus is more sensitive than the adult to the adverse effects of FORM when lower doses are used and the exposure period is extended over the full period of embryo/fetal development. Fetal body weight was the most sensitive indicator of developmental toxicity, exhibiting significant reductions at >= 100 mg/kg/day formamide. A significant increase in malformations was not seen at this dose level. Thus it appears that these two developmental endpoints (fetal weight/growth and malformations) respond differently to the presence of formamide. Whereas the dose-response curve for fetal weight/growth exhibited a downshift at lower doses, the dose-response curve for malformations did not. The observation of fetal body weight as the most sensitive endpoint for the developmental effects of formamide is not surprising, since fetal body weight is often the most sensitive developmental endpoint in rodents (Schwetz and Harris, 1993Go). The extended treatment period in this study (gd 6–19) was also associated with significant developmental toxicity at lower NOAEL and LOAEL values relative to those reported in the BASF studies (BASF 1974bGo, 1983Go) after exposure during major organogenesis (gd 6–15). Thus, in the BASF studies, the developmental toxicity NOAEL was 177 mg/kg/day (gd 6–15) vs. 50 mg/kg/day (gd 6–19) in the present study. Likewise, BASF identified a developmental toxicity LOAEL at 318 mg/kg/day (gd 6–15), whereas the present study sets the developmental LOAEL at 100 mg/kg/day (gd 6–19). Although the possible influence of other experimental factors cannot be ruled out entirely, it seems likely that the observation of the developmental toxicity NOAEL and LOAEL at lower-dose levels in this study reflect the combined impact of a longer exposure period on embryo/fetal growth, as well as no recovery period (versus 5 days for the BASF study) between the end of exposure and termination of the experiment. The comparison of results between studies illustrates the potential influence of experimental design on dose selection, relative sensitivity of the maternal and developing organism, and characterization of the toxic response.

In summary, the maternal toxicity LOAEL was 200 mg/kg/day, and the NOAEL was 100 mg/ kg/day in this study. The developmental toxicity LOAEL and NOAEL were 100 and 50 mg/kg/ day, respectively. These data provide a more complete evaluation of the developmental toxicity of formamide in rats.


    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 NO1-ES-65405). The authors express their appreciation for chemistry support to Cynthia S. Smith of NIEHS, as well as Evelyn Murrill, Robert E. Smith, Robert Moor, James G. Greaves, Linda Siemann, Michael Kozak, and J. Michael Cannon of Midwest Research Institute. In addition, our appreciation goes to 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, Mal-Selika H. Perry, Marian V. Cheesborough, Dee A. Wenzel, M. Michael Veselica, Randy A. Price, T. Douglas Burnette, and Donald L. Hubbard.


    NOTES
 
This study was presented at the 39th Annual Meeting of the Teratology Society, Keystone, Colorado [Teratology 59(6), 412 (Abstract p. 49) 1999].

1 To whom correspondence should be addressed at Hermann Laboratory Building, P.O. Box 12194, Research Triangle Institute, Research Triangle Park, NC 27709–2194. Fax: (919) 541-6499. E-mail: jdg01{at}rti.org. Back


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 DISCUSSION
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