Embryo-Fetal Developmental and Reproductive Toxicology of Vinyl Chloride in Rats

Suzanne R. Thornton*, Raymond E. Schroeder{dagger}, Rodney L. Robison*, Dean E. Rodwell*, David A. Penney{ddagger}, Kenneth D. Nitschke§ and Wendy K. Sherman,1

* Huntingdon Life Sciences, Inc., East Millstone, New Jersey 08875; {dagger} MPI, Mattawan, Michigan 49071; {ddagger} CONDEA Vista Company, Houston, Texas 77079; § The Dow Chemical Company, Midland, Michigan 48674; and American Chemistry Council, 1300 Wilson Boulevard, Arlington, Virginia 22209

Received January 23, 2001; accepted March 11, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vinyl chloride (VC) exposure is primarily via inhalation in the workplace. The primary target organ of VC toxicity is the liver and occupational exposure to VC leads to hepatic angiosarcoma. However, based on epidemiological studies, researchers have been unable to ascertain the effect of occupational VC exposure on embryo-fetal development or reproductive function. A limited number of animal studies available in the literature have examined the effect of VC on embryo-fetal development, however, there are no published studies on the effect of VC exposure on reproductive capability. The current study was designed to assess the potential maternal and/or embryo-fetal developmental and 2-generation reproductive toxicity of inhaled VC in CD® Sprague-Dawley rats at exposure levels of 0, 10, 100, and 1100 ppm. In the embryo-fetal/developmental toxicity study, the female rats were exposed to VC daily from gestation day (GD) 6 through 19. In the reproductive toxicity study, the F0 generation male and female rats were exposed to VC for a 10-week premating and 3-week mating periods. The F0 generation male rats were exposed to VC until terminal euthanasia. The F0 generation female rats were exposed from GD 0 through GD 20 and lactation day (LD) 4 through LD 25. Our results indicate that up to 1100 ppm VC exposure did not adversely affect embryo-fetal developmental or reproductive capability over 2 generations in rats. The primary target organ of VC, the liver, was affected as evidenced by an increase in liver weight and/or histologically identified cellular alterations, such as centrilobular hypertrophy at 100 and 1000 ppm. Based on the results of these studies, the no observed adverse effect level (NOAEL) for embryo-fetal/development is 1100 ppm, and the NOAEL for reproduction is 1100 ppm. The results from the current studies, which are a more comprehensive embryo-fetal/developmental and reproduction study, may be incorporated into future risk assessments of occupational exposure to VC where concerns regarding the effects of VC exposure remain.

Key Words: vinyl chloride; embryo-fetal; developmental; reproduction; two-generation; rat; Sprague-Dawley.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vinyl chloride (VC) is a manufactured substance primarily used to make polyvinyl chloride (PVC), which is used in a variety of plastic products, including pipes, wires, cable coatings, and furniture upholstery. Inhalation in the workplace during the PVC fabrication process is the primary route of exposure. The current American Conference of Government Industrial Hygienists (ACGIH) threshold limit value (TLV) for vinyl chloride is 1 ppm (ACGIH, 2000Go). Organ systems affected following acute or subchronic occupational VC exposure include the respiratory and central nervous (CNS) systems. CNS effects include the anesthesia/narcosis signs of ataxia, drowsiness, visual disturbances, and other CNS effects such as numbness and/or tingling in the extremities (Easter and Von Burg, 1994Go). Hepatotoxicity is the primary result of chronic occupational VC exposure. Cytochrome P450 2E1 converts VC into the active metabolite 2-chloroethylene oxide. This metabolite covalently binds to nucleic acids and proteins, resulting in genotoxicity that ultimately leads to hepatic angiosarcoma (Easter and Von Burg, 1994Go).

While chronic occupational VC exposure has been shown to produce hepatotoxicity, the effects of this exposure on parental reproductive function and on the developing human fetus have not been clearly delineated. Several authors have attributed birth defects in children to the chronic occupational VC exposure of the mothers' husbands. Other birth defects have been attributed to women working in close proximity to polymerization facilities. However, these authors have been unable to demonstrate a positive correlation between VC exposure and birth defects. One epidemiological study on men linked occupational exposure to narcosis-producing concentrations of VC with a higher incidence of impotence (Suciu et al., 1975Go). Many epidemiological studies on fetal development and reproductive function have been criticized by others conducting similar studies. They state that inappropriate methods and improper statistical analysis have made it difficult to positively correlate parental exposure with changes in reproductive performance and fetal development. Thus, the potential effects of VC exposure on the human reproductive system remain uncertain.

Animals exposed to VC exhibit toxicity in numerous organs including the liver, kidneys, endocrine, dermal, ocular, immune, and reproductive systems. The liver is the most prominent organ system affected in the animal studies, and the results correspond to human case reports (ATSDR, 1997Go). Hepatotoxicity in rats from VC exposure included hepatocellular degeneration (Sokal et al., 1980Go; Torkelson et al., 1961Go), hypertrophy (Wisniewska-Knypl et al., 1980Go), changes in metabolic activity (Du et al., 1979Go; Wisniewska-Knypl et al., 1980Go), and an increase in the liver:body weight ratio (Torkelson et al., 1961Go; Sokal et al., 1980Go).

Research on the epidemiology of VC exposure on human fetal development and reproductive performance has been criticized for the lack of correlation between parental exposure and toxicity. Yet, animal studies have revealed potential targets within the reproductive system that could account for the reports of toxicity in humans. Few embryo-fetal/developmental toxicity studies are available in the literature. The most comprehensive study was reported by John et al. (1977, 1981). The strength of these studies is that they utilized multiple species (mice, rats, and rabbits), and investigated the embryo-fetal/developmental effects at comparable VC exposure levels. Results from these studies indicate that mice appeared to be more sensitive to VC toxicity than rats or rabbits. Mice exposed to 500 ppm VC showed maternal toxicity evidenced by decreases in body weight, and an increase in mortality. Because of the maternal toxicity associated with 500 ppm VC, there was a decrease in the litter size. There were also fetal toxicities including decreases in fetal weight and delayed ossification of skull bones. In rats exposed to 500 ppm VC, the only observed sign of maternal toxicity was a decrease in body weight gains compared to the control. In rabbits exposed to 500 ppm VC, the only observed sign of maternal toxicity was a decrease in feed consumption.

While there are a couple of embryo-fetal/developmental toxicity studies available in the literature, there are no published studies available within the literature in which the effect of VC exposure on the reproductive performance of parental animals has been investigated. There are dominant lethal studies in mice and chronic toxicity studies in rats that have examined the effect of VC on the male reproductive system. The results of the dominant lethal study in mice were negative when the mice were exposed to 5000 ppm for 4 h/day for 10 weeks (Himeno et al., 1983Go). Chronic toxicity studies with VC in rats have shown that chronic VC exposure produces damage to the seminiferous tubules, depletion of spermatocytes, damage to the spermatogenic epithelium, and disorders of spermatogenesis that lead to a decrease in testicular weight (Bi et al., 1985Go; Sokal et al., 1980Go). While the dominant lethal study in mice does not support effects of VC on the male reproductive system, the chronic rat study indicates that the male reproductive system may be a target of VC. Caveats to the effect of VC on the male reproductive system are that the researchers in the chronic toxicity rat study did not investigate whether these effects led to impotency and/or the inability of the male rats to sire offspring, nor did they investigate whether the observed effects of VC were reversible after VC exposure was terminated.

In an attempt to clarify the effect of VC exposure on the developing fetus and on reproductive performance, the current study was designed to provide a more comprehensive embryo-fetal/developmental and reproductive assessment of VC using current regulatory guidelines. The study was designed to assess maternal and/or embryo-fetal developmental and two-generation reproductive toxicity in rats that had been exposed to the same VC levels so that a more direct comparison of the study results would be possible.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vinyl chloride exposure.
For both the embryo-fetal/developmental and reproductive studies, Sprague-Dawley rats were exposed to VC via whole-body inhalation for 6 h/day at targeted exposure levels of 10, 100, and 1100 ppm. These exposure levels correspond to oral equivalent doses of approximately 0, 9.2, 92, and 1012 mg/kg/day assuming ventilation rates of 1 l/min/kg, 100% absorption, and a 6 h/day exposure. The high concentration of VC was selected based on the oral equivalent of the regulatory "limit dose" of 1000 mg/kg body weight/day (OECD, 1981Go). The control rats received air only (0 ppm). During exposures, all animals were individually housed in stainless steel, wire mesh cages within a 6 m3 (6000 liter) stainless steel and glass whole-body exposure chamber. The placement of the animals within the whole-body exposure cages was rotated at each exposure to ensure uniform exposure of the animals. No feed was provided during exposures, but water was available ad libitum via an automated watering system. Exposure chamber temperature and relative humidity were recorded every half-hour during exposure. The temperature and relative humidity during exposure were maintained within 16–28°C and 29–79%, respectively. Exposure chambers were operated dynamically under slight negative pressure at a minimum airflow rate of 1200 liters per minute (lpm). The specified flow rate provided 1 complete air change every 5.0 min and a 99% equilibrium time (T99) of 23 min. For the control (0 ppm) exposure group, ambient air was fed into the 6 m3 exposure chamber at a rate of 1329 lpm. For the VC exposed groups, ambient air was fed into the 6 m3 exposure chamber at a rate of 1239–1267 lpm. Vinyl chloride was delivered from a compressed gas cylinder to a Scott Specialty Gases regulator equipped with inlet and outlet back pressure gauges. The regulator was attached to flowmeters that delivered flow rates of 12.36 ml/min, 128.3 ml/min, and 1312 ml/min for the 10, 100, and 1100 ppm exposure groups, respectively. All animals remained in the exposure chamber for a minimum of 30 min following the exposure to allow the VC to be removed at the same airflow rate as used during the exposure.

Measurements of airborne concentrations of VC were made using a MIRAN® Ambient Air analyzer equipped with a strip chart recorder. Test atmosphere samples were taken hourly during the exposure periods. The exposure levels were determined by comparison of the measured absorbance to a calibrated response curve.

Embryo-fetal/developmental study.
This embryo-fetal/developmental study was conducted in accordance with the U.S. Environmental Protection Agency TSCA Test Guidelines (U.S. EPA, 1985Go), Organization for Economic Co-operation and Development (OECD) Guidelines for testing of chemicals, Section 4, Health Effects (OECD, 1981Go), and European Economic Community (EEC) Methods for the determination of toxicity (EEC, 1988Go). See Figure 1AGo for schematic of the experimental design. Briefly, 100 nulliparous female CD® Sprague-Dawley Crl: CD® BR rats (25/group), 57 days of age and weighing 172–252 g, were received from Charles River Laboratories (Portage, MI). Female rats were acclimated for approximately 2 weeks and then cohabitated (1:1) nightly with male CD® Sprague Dawley Crl: CD® BR rats from an in-house breeding colony (Huntingdon Life Sciences, East Millstone, NJ). Female rats were considered pregnant if the vaginal smear performed each morning following cohabitation contained sperm or if a vaginal plug was present. GD 0 was defined as the day on which evidence of mating was observed. On GD 0, each mated female rat was assigned to an exposure group using a computer randomization program that randomly assigned animals to most nearly equalize GD 0 body weights between groups. Female rats were exposed to VC daily from GD 6 through 19 (see Vinyl Chloride Exposure section for further details).



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FIG. 1. Experimental design. (A) Embryo-fetal/developmental study; (B) reproductive study.

 
During nonexposure periods, all animals were housed individually in suspended stainless steel cages with wire mesh floors, except during mating. The animal rooms were maintained on a 12-h light/dark cycle with temperature and humidity kept within the specified ranges (20–24°C and 40–70%, respectively). All animals were provided feed (Certified Rodent Diet No. 5002, meal) supplied by PMI Feeds, Inc. (St. Louis, MO) and water via an automated water system ad libitum.

All animals were observed twice daily for mortality and toxicological effects. Detailed physical examinations were performed on GD 0 and GD 6 through 20. Body weight and feed consumption values were recorded on GD 0, 6, 9, 12, 15, and 20. Postexposure observations were performed approximately a half hour after exposure. On GD 20, all female rats were euthanized via carbon dioxide inhalation, and a cesarean section and a macroscopic postmortem examination were performed on each. During the macroscopic postmortem examination, gravid uterus (with ovaries attached), kidneys, and liver weights were recorded. Uterine implantation data including the position and number of live and dead fetuses, early and late resorptions, implantation sites, and the number of corpora lutea were recorded. Fetuses were removed from the uterus, weighed, their gender was determined, and they were examined for external malformations. Approximately half of the fetuses from each litter were processed for soft tissue (visceral) evaluations using a micro-dissection procedure (Staples, 1974Go), and half of the fetuses from each litter were processed for skeletal and ossification evaluations after evisceration and staining with Alizarin Red S. Uteri without grossly visible implantation sites were stained according to the procedure of Salewski (1964) to identify the presence of early resorption sites. If no stained implantation sites were present, the rat was considered not pregnant.

Reproduction study.
This reproduction study was conducted in accordance with the U.S. Environmental Protection Agency TSCA Test Guidelines (U.S. EPA, 1985Go), Organization for Economic Cooperation and Development (OECD) Guidelines for testing of chemicals, Section 4, Health Effects (OECD, 1981Go), and European Economic Community (EEC) Methods for the determination of toxicity (EEC, 1988Go). See Figure 1BGo for schematic of the experimental design.

F0 generation.
One hundred twenty male and 120 nulliparous female CD® Sprague-Dawley Crl: CD® BR rats (30/sex/group) were received from Charles River Laboratories (Portage, MI). The male and female rats were approximately 4 weeks of age at receipt. Both the male and female rats were acclimated for approximately 2 weeks prior to exposure to VC. Body weights for the male and female rats were 207–273 and 137–177 g, respectively. The F0 male and female rats were assigned to the exposure groups using a computerized randomization program, which randomly assigned animals to most nearly equalize body weights between groups. The F0 generation male and female rats were exposed to VC for a 10-week premating period and a 3-week mating period. The F0 generation male rats continued exposure through the postmating period. Female rats were exposed during gestation through GD 20. After GD 20, exposure to VC for the female rats was discontinued to allow delivery of litters, resumed on LD 4, and continued throughout the remainder of lactation. Unmated female rats continued exposure to VC until euthanized (see Vinyl Chloride exposure section for further details.)

During nonexposure periods, all animals were housed individually in suspended stainless steel cages with wire mesh floors, except during mating. The animal rooms were maintained on a 12-h light/dark cycle with temperature and humidity kept within the specified ranges (20–24°C and 40–70%, respectively).The female rats, beginning on GD 20 and continuing throughout lactation, were individually housed in plastic shoebox cages containing certified hardwood shavings (Lab Aspen Shavings, North Eastern Products Corporation, Warrenburg, NY). All animals were provided Certified Rodent Diet No. 5002 (meal) supplied by PMI Feeds, Inc. (St. Louis, MO) and water via an automated water system ad libitum.

Estrous cycle determination via a vaginal smear procedure were performed on the first 15 F0 generation female rats per group for the 3-week period prior to mating and continued until mating was confirmed. During the mating period, 1 male rat was cohoused with 1 female rat from the same exposure group nightly until evidence of mating was observed or for 14 consecutive days. Female rats were considered mated if the vaginal smear performed each morning following cohabitation contained sperm or if a vaginal plug was present. GD 0 was defined as the day on which evidence of mating was observed. Due to poor mating performance (primarily in the control group), unmated female rats in all groups were randomly assigned to a male rat known to inseminate another female rat from the same exposure group for an additional 6 days. Once mated, the female rats were removed from the mating unit and housed individually for the remainder of gestation.

All F0 generation animals were observed twice daily for mortality and toxicity. Detailed physical examinations were performed and body weight and feed consumption recorded weekly during the premating period for the male and female rats and during the postmating period for the male and unmated female rats. For mated female rats, body weights and feed consumption were recorded on GD 0, 7, 14, and 20 and LD 0, 4, 7, 10, 14, and 25. F1 litters were examined for the presence of live and dead pups as soon as possible after delivery on LD 0 until weaning on LD 25. On LD 4, litters were culled to 8 pups/litter with equal gender number/litter, where possible.

F0 generation rats were euthanized via carbon dioxide inhalation. The male rats were euthanized after parturition completion and the female rats were euthanized on day 2 of diestrus after weaning, assessed by daily vaginal smears, whenever possible. Female rats with no estrous cyclicity or irregular estrous cycling, as determined by vaginal smears, were euthanized as a group after the confirmed diestrous female rats had been euthanized. All parental animals were given a macroscopic postmortem examination. Reproductive tissues from the male rats (testes, left epididymis, prostate, seminal vesicles with coagulating glands and their fluids) and female rats (uterus and ovaries) were weighed and preserved in 10% neutral buffered formalin, except for the testes and epididymis, which were preserved in Bouin's fixative. In addition, the adrenal glands, brain, kidneys, liver, lungs, spleen, and thymus were weighed for all F0 male and female rats. Other tissues preserved in 10% neutral buffered formalin from the F0 generation parental animals were mammary glands, nasal tissues (turbinates), pituitary, and trachea. All preserved tissues were embedded in paraffin, sectioned on a microtome (4–7 µm thickness), mounted on glass slides, stained with hematoxylin and eosin, and examined by light microscopy for histopathological abnormalities. While staging of the seminiferous tubules was not conducted, the testes cross-sections were examined by a pathologist for potential qualitative histopathological changes.

F1 pups.
F1 pups had physical examinations performed, body weights recorded, and gender determinations performed on LD 0, 4, 7, 14, and 25. The F1 pups were weaned on postnatal day (PND) 25, at which time 2 pups/sex per litter were chosen at random to become the pool of animals from which the F1 generation was selected. Each litter contributed at least 1 pup/sex, when possible, for a total of 30 pups/sex/group. The F1 pups selected to become the F1 generation were housed in pairs, 2 littermates of the same sex/cage, in stainless steel cages with wire mesh floors until the last F1 litters weaned, after which, they were individually housed. All F1 weanlings selected for the F1 generation were assessed for postweaning developmental landmarks, vaginal patency and preputial separation, beginning on PND 28 and 40, respectively. At weaning, 15 male and female pups from each exposure level were selected for macroscopic and microscopic examinations. Pups were given a macroscopic postmortem examination following euthanasia via carbon dioxide inhalation. The reproductive organs (testes or ovaries), adrenal glands, brain, kidneys, liver, spleen, and thymus were weighed. All other pups, including those found dead, culled, or not selected for the F1 generation were given an external examination following euthanasia via carbon dioxide inhalation and if within normal limits, discarded.

F1 generation.
One hundred twenty male and 120 female rats (30/sex per group) selected from the F1 pups began daily exposure to VC on PND 26. The F1 generation male and female rats were exposed daily to VC for a 10-week premating period and a 3-week mating period. The F1 generation male rats continued exposure through the postmating period. Female rats were exposed during gestation through GD 20. After GD 20, exposure to VC for the female rats was discontinued to allow delivery of litters, resumed on LD 4 and continued throughout the remainder of lactation. Unmated female rats continued exposure to VC until euthanized. During nonexposure periods, all animals were housed, provided feed, water, and the animal room maintained the same as the F0 generation.

Estrous cycle determination via a vaginal smear procedure was performed on the first 15 F1 generation female rats per group for the 3-week period prior to mating and continued until mating was confirmed. During the mating period, 1 male rat was cohoused with 1 female rat from the same exposure group nightly until evidence of mating was observed or for 14 consecutive days. In the mating of the F1 generation, brother-sister pairings were avoided. Female rats were considered mated if the vaginal smear, performed each morning following cohabitation, contained sperm or if a vaginal plug was present. GD 0 was defined as the day on which evidence of mating was observed.

All F1 generation animals were observed twice daily for mortality and toxicological effects. Detailed physical examinations were performed and body weight and feed consumption recorded weekly during the premating period for the male and female rats and during the postmating period for the male and unmated female rats. For mated female rats, body weights and feed consumption were recorded on GD 0, 7, 14, and 20 and LD 0, 4, 7, 10, 14, and 21. F2 litters were examined for the presence of live and dead pups as soon as possible after delivery on LD 0 until weaning on LD 21. On LD 4, litters were culled to 8 pups/litter with equal gender number/litter, where possible.

F1 generation rats were euthanized via carbon dioxide inhalation. The male rats were euthanized after parturition completion and the female rats were euthanized on day 2 of diestrus, assessed by daily vaginal smears, whenever possible. Female rats with no estrous cyclicity or irregular estrous cycling, as determined by vaginal smears, were euthanized as a group after the confirmed diestrous female rats had been euthanized. All parental animals were given a macroscopic postmortem examination. Reproductive tissues from the male rats (testes, left epididymis, prostate, seminal vesicles with coagulating glands and their fluids) and female rats (uterus and ovaries) were weighed and preserved in 10% neutral buffered formalin, except for the testes and epididymis, which were preserved in Bouin's fixative. In addition, the adrenal glands, brain, kidneys, liver, lungs, spleen, and thymus were weighed for all F1 male and female rats. Other tissues preserved in 10% neutral buffered formalin from the F1 generation parental animals were mammary glands, nasal tissues (turbinates), pituitary, and trachea. All preserved tissues were embedded in paraffin, sectioned on a microtome (4–7 µm thickness), mounted on glass slides, stained with hematoxylin and eosin, and examined by light microscopy for histopathological abnormalities. While staging of the seminiferous tubules was not conducted, cross-sections of the testes were examined by a pathologist for potential qualitative histopathological changes.

F2 pups.
F2 pups had physical examinations performed, body weights recorded, and gender determinations performed on LD 0, 4, 7, 14, and 21. At weaning (PND 21), 15 male and female pups from each exposure level, from the F1 litters were selected for macroscopic and microscopic examinations. Pups were given a macroscopic postmortem examination following euthanasia via carbon dioxide inhalation. The reproductive organs (testes or ovaries), adrenal glands, brain, kidneys, liver, spleen, and thymus were weighed. All other pups, including those found dead, culled, or not selected for the F1 generation were given an external examination following euthanasia via carbon dioxide inhalation and if within normal limits, discarded.

Sperm analysis.
Sperm motility, caudal epididymal sperm count, and sperm morphology were assessed in 15 male rats per group from the F0 and F1 generation, including male rats that did not impregnate a female rat. Sperm evaluations were performed by Pathology Associates International (PAI) (Frederick, MD) using an automated Hamilton Thorne IVOS sperm analyzer. The male rats were euthanized via carbon dioxide inhalation followed by exsanguination. Following euthanasia, an incision in the abdominal cavity was made and the reproductive organs exposed. Sperm motility evaluations were conducted on semen collected from the right vas deferens. For the evaluations, the right vas deferens was dissected from the testes and immediately placed in a warmed solution of phosphate buffered saline containing 1% bovine serum albumin. After a 3-min "swimout" period was allowed, the sample was placed in the Hamilton Thorne IVOS automated sperm analyzer and 5 fields evaluated for percent motility for each animal. Total sperm count determinations were conducted on the right epididymis. For the sperm count evaluation, the right epididymis was dissected from the surrounding tissues and immediately frozen and shipped to PAI (Frederick, MD). The right epididymis was thawed and the caudal section trimmed, weighed, and mechanically homogenized. A sample of the homogenized epididymis was stained with disbenzimide (Hoescht, H33342) which uniquely stains the heads of the sperm and 20 fields evaluated by the automated sperm analyzer for each animal. The total number of sperm in the caudal epididymis was calculated and normalized based on the caudal epididymal weight. Sperm morphology was assessed from 2 samples prepared from the prehomogenized caudal epididymis following eosin staining. The 2 samples (minimum of 200 sperm cells/animal) were evaluated for head and tail morphological irregularities (Filler, 1993Go).

Mean and SD values for sperm motility, total count, and sperm morphology data were calculated and compared across groups using the Kruskal-Wallis nonparametric ANOVA test. If a significant effect occurred, the Wilcoxon (Mann-Whitney U) test was used for pairwise comparisons of each exposed group to the control group; p values less than 0.05 were considered statistically significant.

Statistical analyses.
As a result of different computerized data capture systems utilized during the embryo-fetal/developmental and reproductive toxicity studies, different standard statistical packages were used to assess the statistical significance of the experimental parameters. Maternal body weight and body weight gains during gestation and lactation, fetal and pup body weights, mean gestation length, number of corpora lutea, number of fetuses or pups, fetal body weight, early and late resorptions, and organ weights (embryo-fetal/developmental study) were statistically evaluated for equality of means using a one-way ANOVA, followed by a post hoc test, if needed (Dunlap et al., 1981Go). If ANOVA was significant, Dunnett's test was performed to determine which data differed from the control (Dunnett, 1955Go, 1964Go); p values less than 0.05 were considered statistically significant.

Premating, mating, and postmating body weight and body weight gains, feed consumption, organ weights (reproduction study), and mean age to vaginal patency and preputial separation were statistically evaluated for equal variance using Bartlett's test (Snedecor and Cochran, 1967Go). If variances were equal, the parametric procedures, a one-way ANOVA using the F distribution to assess significance was performed. If significant differences were indicated a Dunnett's test was used to determine which means were significantly different from the control (Dunnett, 1955Go, 1964Go). If variances were not equal, the nonparametric procedures, Kruskal-Wallis test followed by Dunn's summed rank test to determine which exposures differed from control was performed (Hollander and Wolfe, 1973Go); p values less than 0.05 were considered statistically significant.

Mating indices, pregnancy rates, male rat fertility indices, pup survival indices, and mortality rates were statistically evaluated using a Fisher Exact Test with Bonferroni correction to identify differences in incidence data between groups (Dunlap and Duffy, 1975Go; Hollander and Wolfe, 1973Go; Siegel, 1956Go); p values less than 0.05 or 0.01 were considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure Analysis
For both the embryo-fetal/developmental and reproductive studies, infrared (IR) and gas chromatographic (GC) analyses during the exposure periods revealed that target concentrations and calculated nominal levels were within 91–100% of the targeted exposure level (data not shown). Particle size measurements indicated that VC was completely vaporized in the chamber air during exposure. Chamber temperatures ranged from 16–28°C and the relative humidity ranged 29–79% for both studies.

Embryo-Fetal/Developmental Study
All animals, including the control, survived to scheduled euthanasia on GD 20 (data not shown). During GD 15–20 and GD 6–20, VC exposure caused a slight, but statistically significant suppression in maternal body weight gains, at all exposure levels (Table 1Go). However, pregnancy rates, clinical observations, feed consumption, macroscopic postmortem findings (data not shown), uterine implantation data, fetal gender distribution and body weight, fetal malformations and variations remained comparable between the VC and control groups (Tables 2–5GoGoGoGo). Maternal absolute and relative kidney and liver weights in the 10 ppm VC exposed groups were comparable to control (Table 6Go). In the 100 and 1100 ppm groups, the absolute kidney and liver weights were comparable to the control. However, in the 100 ppm exposed group, the kidney relative to body weight ratio was statistically significantly increased, while in the 1100 ppm exposed group, the organ relative to body weight ratios for both the kidney and liver were statistically significantly increased.


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TABLE 1 Summary of Maternal Body Weight Gains and Gravid Uterine Weights for Rats Exposed to Vinyl Chloride during GD 6–19
 

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TABLE 2 Summary of Cesarean Section Data for Rats Exposed to Vinyl Chloride during GD 6–19
 

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TABLE 3 Summary of Fetal Data for Fetuses Exposed to Vinyl Chloride in Utero during GD 6–19
 

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TABLE 4 Summary of Fetal Malformations following Exposure to Vinyl Chloride during GD 6–19
 

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TABLE 5 Summary of Fetal Variations following Exposure to Vinyl Chloride during GD 6–19
 

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TABLE 6 Summary of Maternal Organ Weights from Rats Exposed to Vinyl Chloride during GD 6–19
 
Reproduction Study
F0 generation.
Overall mortality was unaffected by VC in the F0 generation. However, 2 animals were found dead, 1 male rat in the 10 ppm group and 1 female rat in the control. The male rat in the 10 ppm group was found dead during study week 15 and had exhibited clinical signs of swollen paws, impaired movement of the limbs, and yellow anogenital staining. Macroscopic postmortem examination of the male rat revealed urinary bladder distention, bilateral kidney enlargement, and bilateral dilated renal pelvis. The female control rat was found dead during study week 20, and the death was considered accidental. Clinical observations, body weight, and body weight gains for the F0 generation male and female rats during premating, mating, postmating periods were also unaffected by VC exposure, as these parameters remained comparable to the control group (data not shown).

During the premating period for the F0 generation female rats, the control and VC exposure groups experienced 4 to 6 estrous intervals over the 22-day evaluation period (data not shown). The initial mating period for the F0 generation was 14 days during which the mating indices for the control and VC groups was 80 and 87–97%, respectively. Because of the low mating index of the control group, the mating period for all groups was extended for 6 additional days for a total of 20 days of mating. The overall 20-day F0 generation female mating indices at the end of the mating period for the control, 10, 100, and 1100 ppm VC groups were 93, 97, 93, and 100%, respectively. Even though there was an initial decrease in the mating index for the control group in the F0 generation, overall female and male mating indices for the F0 generation were not significantly affected by VC exposure (Table 7Go). Pregnancy rates for the F0 generation were comparable between control and VC exposed groups (Table 7Go).


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TABLE 7 Summary of F0 and F1 Generation Cohabitation Data for Rats Exposed to Vinyl Chloride
 
Body weight, body weight gains, and feed consumption during gestation and lactation for the F0 generation were comparable between the control and VC exposure groups (data not shown). The gestation index and duration of gestation were comparable between the control and VC exposure groups (Table 8Go). The live birth index was significantly decreased, while the number of stillborn pups was significantly increased in the F0 generation group exposed to 1100 ppm VC (Table 8Go). These differences are not considered exposure-related since no dose response was observed, and while the values differ from F0 control values, they are comparable to historical control values from the conducting laboratory (93.6–99.8%) and comparable to the F1 control and exposed group values. Pup viability index to LD 4 for the F1 pups exposed to 10 and 100 ppm VC was statistically significantly decreased (Table 8Go). However, there were no effects on these parameters at 1100 ppm. Pup gender distribution and pup body weights were unaffected by VC exposure in the F1 and F2 litters (data not shown).


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TABLE 8 Summary of F1 Litter Data for Rats Exposed to Vinyl Chloride
 
There were no VC exposure-related macroscopic postmortem findings for the F0 generation male and female rats (data not shown). Sperm motility, total caudal epididymal sperm count, and sperm morphology for the F0 generation male rats were unaffected by VC exposure (Table 9Go). The mean % and SD for morphologically abnormal sperm were elevated for F0 generation male rats exposed to 100 ppm. This elevation was not considered to be exposure-related and was the result of 1 male rat having only 42 normal sperm and 158 sperm with amorphous heads. In the F0 generation male rats, absolute and relative liver weights were significantly increased in all VC exposure groups (Table 10Go). Also in the male rats exposed to 100 ppm VC there was a statistically significant increase in the absolute epididymis and kidney weights (data not shown). In the F0 generation female rats exposed to 10 ppm VC, there was a significant increase in the absolute and relative spleen weight (data not shown). There were no other VC exposure-related effects on organ weights or histopathology, including the reproductive organs (testes, ovaries), in the F0 generation male and female rats. Histopathological evaluations of the male and female rat livers revealed centrilobular hypertrophy in male and female rats exposed to 100 and 1100 ppm (Figs. 2A and 2BGo). Of interest is that there was no observed effect of VC on the liver weight of F0 female rats, but there were histological alterations in the liver at 10, 100, and 1100 ppm. The hepatocytes were enlarged with increased acidophilic cytoplasm within the centrilobular areas of the liver (Table 11Go).


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TABLE 9 Summary of F0 and F1 Generation Sperm Analysis for Male Rats Exposed to Vinyl Chloride
 

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TABLE 10 Summary of F0 Generation Organ Weights for Rats Exposed to Vinyl Chloride
 


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FIG. 2. Liver photomicrographs of F1 generation female rats. (A) Control liver showing normal hepatocytes in centrilobular and periportal regions; (B) centrilobular hypertrophy in liver from 1100 ppm exposed female rat; (C) basophilic focus from 1100 ppm exposed female rat; (D) acidophilic focus from 1100 ppm exposed female rat. Abbreviations: cv, central vein; pv, portal vein. Bar = 100 µ, hematoxylin and eosin stained paraffin sections.

 

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TABLE 11 Summary of F0 and F1 Generation Liver Histopathology in Rats Exposed to Vinyl Chloride
 
F1 generation.
Overall mortality was unaffected by VC in the F1 generation. In the F1 generation, 1 male rat in the control group was found dead during the second week of the premating period. The rat did not exhibit any clinical signs prior to death, and since there were no macroscopic postmortem findings, the cause of death is unknown. Clinical observations, body weight, or body weight gains for the F1 generation male and female rats during premating, mating, and postmating periods were also unaffected by VC exposure, as these parameters remained comparable to the control group (data not shown). No VC exposure-related effects on body weight or postweaning developmental landmark data, preputial separation, and vaginal patency during the growth and development period of the F1 generation were observed (Table 12Go).


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TABLE 12 Summary of F1 Pup Postweaning Developmental Landmarks
 
During the premating period for the F1 generation female rats, the control and VC exposure groups experienced 4 to 6 estrous intervals over the 22-day evaluation period (data not shown). Female and male mating indices for the F1 generation were not significantly affected by VC exposure (Table 7Go). Pregnancy rates for the F1 generation were comparable between control and VC exposed groups (Table 7Go).

Body weight, body weight gains, and feed consumption during gestation and lactation for the F1 generation were comparable between the control and VC exposure groups (data not shown). The gestation index and duration of gestation were comparable between the control and VC exposure groups (Table 13Go). In the F2 litters, there was a statistically significant decrease in the mean number of pups delivered in the 1100 ppm exposed group (Table 13Go). These differences in the mean number of pups delivered is not considered exposure-related since no dose response was observed, and while the values were lower than respective F1 control group values, they are comparable to the F0 control group values. Viability indices for the F2 pups exposed to 10, 100, and 1100 ppm VC were increased when compared to the control group. These increases were considered incidental and unrelated to VC exposure. Pup gender distribution and pup body weights were also unaffected by VC exposure in the F2 litters (data not shown).


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TABLE 13 Summary of F2 Litter Data for Rats Exposed to Vinyl Chloride
 
Sperm motility, total caudal epididymal sperm count, and sperm morphology for the F1 generation male rats were unaffected by VC exposure (Table 9Go). In the F1 generation, there was a statistically significant increase in the absolute and relative liver weight for male rats exposed to 100 and 1100 ppm VC (Table 14Go). Also in the male rats exposed to 1100 ppm, there was a significant increase in the absolute and relative spleen weight (data not shown). Female rats of the F1 generation exposed to 100 and 1100 ppm exhibited an increase, though not statistically significant, in absolute liver weight (Table 14Go). There were no other VC exposure-related effects on organ weights or histopathology, including the reproductive organs (testes, ovaries), in the F1 generation male and female rats. As with the F0 generation, there was histopathological evidence of centrilobular hepatocyte hypertrophy and altered foci in the male and female rats (Table 11Go; Figs. 2A and 2BGo). The severity of centrilobular hypertrophy and altered foci was very slight at all dose levels and appeared to be more prevalent in females than males. In addition, hepatocellular altered foci, including acidophilic, basophilic, and clear cell foci, were observed in the livers from the male rats exposed to 100 and 1100 ppm and in the female rats exposed to 1100 ppm VC (Table 11Go, Figs. 2C and 2DGo).


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TABLE 14 Summary of F1 Generation Organ Weights for Rats Exposed to Vinyl Chloride
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our results indicate that VC exposure as high as 1100 ppm during organogenesis (GD 6–19) in rats was not maternally toxic or developmentally toxic (teratogenic). Although maternal body weight gain was significantly suppressed during GD 15–20 and GD 6–20 in rats exposed to 100 and 1100 ppm, the effect does not appear to be VC exposure-related. In comparing the number of live fetuses and the mean combined fetal body weight, it becomes apparent that the control group gave birth to approximately 1 additional fetus than the 100 and 1100 ppm VC exposed groups (Table 2Go). Because fetuses gain most of their weight during GD 15–20 (the observation period where the body weight gain suppression was observed in the VC exposed groups), and because the control group had approximately 1 additional fetus, it is probable that the statistically significant suppression of body weight gain observed in the 100 and 1100 ppm VC exposed groups was not a result of the VC exposed groups gaining less weight, but rather that the control group gained more weight. Although there were differences in body weight gains between the control and VC exposed groups, there was no significant difference in gravid uterine weight or corrected body weight gain between the control and VC exposure groups (Table 1Go). This is substantiated by the finding that maternal absolute body weights throughout gestation did not differ significantly between the control and VC exposed groups.

There was no embyro-fetal or developmental toxicity at any exposure levels as evidenced by the lack of malformations, variations, and significant effects on fetal body weight. John et al. (1977, 1981) also reported a lack of embryo-fetal/developmental toxicity in rats exposed to VC. They found comparable fetal body weight, external, visceral, and skeletal variations in control rats and those exposed to VC levels of 500 and 2500 ppm during GD 6–15. They also reported that mice appeared to be more sensitive to VC exposure than rats or rabbits. In our study, we exposed rats to levels of VC that were approximately 10–1100 times higher than permissible occupational exposure levels of 1 ppm. The embryo-fetal/developmental results indicate that exposure of VC up to 1100 times the permissible occupational exposure level via inhalation during pregnancy did not result in adverse developmental effects. These results may provide an explanation for the epidemiological researcher's inability to demonstrate a positive correlation between exposure and the development of birth defects in children born to women whose husbands were occupationally exposed to VC and/or to women within close proximity to polymerization facilities.

In the embryo-fetal developmental study, the observed effects of VC on maternal kidney and liver weight relative to body weight can be directly correlated with the amount of VC exposure. These tissues were not examined histopathologically, thus it is unknown what changes occurred that led to the significant increases in kidney weights. One study in mice noted that the kidney may be a target of VC exposure (Feron and Kroes, 1979Go). However, it should be noted that no human epidemiological studies have identified the kidney as a potential target organ. Hepatotoxicity is one result of prolonged exposure to VC.

The two-generation reproductive toxicity results indicate that VC exposures of up to 1100 ppm over multiple generations in rats did not affect body weight, feed consumption, ability to reproduce, gestation index or length, preweaning or postweaning developmental landmarks. Furthermore, the sperm analysis for either the F0 or F1 generation animals was unaffected by VC exposure. To our knowledge this is the first study that directly measured sperm counts, as well as sperm motility and head and tail morphology following VC exposure. This conflicts with an earlier study in which chronic VC exposure damaged the seminiferous tubules, depleted spermatocytes, and damaged spermatogenic epithelium, leading to a decrease in testicular weight (Bi et al., 1985Go; Sokal et al., 1980Go). However, unlike our study, the rats were chronically exposed to narcosis producing concentrations significantly greater than 1000 ppm. Humans are very unlikely to be exposed to such high concentrations for even short periods of time.

In this study, the liver was the primary site of toxicity from VC exposure. In both the F0 and F1 generation, male and female rats exhibited changes in liver weights and/or histopathological alterations. Histopathological alterations consisted of centrilobular hypertrophy and acidophilic staining of hepatocytes in the F0 generation animals. F1 generation animals exhibited centrilobular hypertrophy and acidophilic, basophilic, or clear cell foci. Hepatotoxicity occurs by the actions of cytochrome P450 2E1 to metabolize VC into 2-chloroethylene oxide, which covalently binds to nucleic acids and proteins to produce genotoxicity (Easter and Von Burg, 1994Go). Ultimately, this leads to hepatic angiosarcoma. Centrilobular hypertrophy is a common compensatory reaction following toxic insult to the liver and was observed in all VC treated groups. However, it was more prevalent in the F0 and F1 generation male and female rats exposed to 100 and 1100 ppm. Others have similarly shown that subchronic/chronic VC exposure in rats can lead to hepatotoxicity, including hepatocellular degeneration (Sokal et al., 1980Go; Torkelson et al., 1961Go), hypertrophy (Wisniewska-Knypl et al., 1980Go), and increases in the liver to body weight ratio (Sokal et al., 1980Go; Torkelson et al., 1961Go).

In summary, our results indicate that VC up to 1100 ppm exposure did not adversely affect embryo-fetal/developmental and/or reproductive capability over two generations in rats. However, the liver was affected as evidenced by increases in liver weight and/or histologically identified cellular alterations, such as centrilobular hypertrophy. Based on our results, the NOAEL for embryo-fetal/development is 1100 ppm, and the NOAEL for reproduction is 1100 ppm. We believe that this study provides a more systematic and comprehensive assessment of embryo-fetal/developmental and reproductive effects of chronic VC exposure than previous publications. It is hoped that these results will be incorporated into future risk assessments examining occupational exposure to VC.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (703) 741-6091. E-mail: wendy_sherman{at}americanchemistry.com. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACGIH (2000). Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices. American Conference of Government Industrial Hygienists, Cincinnati, OH.

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Dunnett, C. W. (1964). New tables for multiple comparisons with a control. Biometrics 20, 482–491.[ISI]

Easter, M. D., and Von Burg, R. (1994). Vinyl chloride. J. Appl. Toxicol. 14, 301–307.[ISI][Medline]

EEC (1988). Methods for the determination of toxicity. European Economic Community. Off. J. European Communities 31(L133).

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Filler, R. (1993). Methods for evaluation of rat epididymal sperm morphology. In Methods in Toxicology (R. E. Chapin and J. J. Heindel, Eds.), pp. 334–343. Academic Press, San Diego, CA.

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John, J. A., Smith, F. A., Leong, B. K., and Schwetz, B. A. (1977). The effects of maternally inhaled vinyl chloride on embryonal and fetal development in mice, rats, and rabbits. Toxicol. Appl. Pharmacol. 39, 497–513.[ISI][Medline]

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Sokal, J. A., Baranski, B., Majka, J., Rolecki, R., Stetkiewicz, J., Ivanova-Chemishanska, L., Vergieva, T., Antonov, G., Mirkova, E., Kolakowski, J., Szendzikowski, S., and Wroblewska, K. (1980). Experimental studies on the chronic toxic effects of vinyl chloride in rats. J. Hyg. Epidemiol. Microbiol. Immunol. 24, 285–294.[ISI][Medline]

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