Document n9K6Qbm3KBv7ogD5JvQ63aejm

R&S 022825 ,&^JL 0 TO*ICOt>GY RESEARCH LABORATORY um|TTEC> CHAROC Manuf. Chem. Assoc 998-0060100 K i(*- /f>6 nffifiF nnPY DOW CHEMICAL U S.A. jo*rc December 19, 1975 Ww* II . II H V W V HET 3.4-16-10 K M UW tR * K-001711 -ft O') -- THE EFFECTS OF MATERNALLY INHALED VINYL CHLORIDE ON EMBRYONAL AND. FETAL DEVELOPMENT UfaMIgE, RATS AND RABBITS J.A.1 John, B.A,'ScHueez, B.K.J.' Leottg,. FTki^sSlth, K.P. Nitsehkii;'B.D-^riab'erstroh, F.J*.. Murray, p- ]; ' reported BY; M.F.'fealmar and F.J. Gahring checked bv: P. JGehring IHFOWMATIVC tUUMART WITH CONCLUSION, Hill) OH TUI (AMTLi WCCCIVCO. AOOItlOH.I. IMTOWMATIOH INC(.UOIHS T( IICT| OF RCPtATKO CXPQ1UKC MAY RCQUIflCO A* *P*Cl*tC AMO FORMUL*TM>N| ARC O(V(.OR|0 OR IW AOCKfl CMANQII CCCUA. These studies evaluated the effects of inhaled vinyl chloride on mouse, rat and rabbit embryonal and fetal development. Groups of pregnant SwissWebster mice, Sprague-Dawley rats and New Zealand white rabbits were exposed to 500 ppm vinyl chloride 7 hrs daily during the period of major organo-' ` genesis. Subsequently, other groups of mice were similarly exposed to 50 ppm vinyl chloride and rats and rabbits were exposed to 2500 ppm vinyl chloride. While maternal toxicity was observed, vinyl chloride alone did not cause significant embryonal or fetal toxicity and was not teratogenic in any of the species at the concentrations tested. Maternal toxicity was more prominent among mice than among rats and rabbits. Simultaneous exposure of some of the pregnant animals to vinyl chloride by inhalation plus 15% ethanol in the drinking water resulted in toxic effects greater than those associated with exposure to vinyl chloride alone in the three species. The maternal toxicity was enhanced to an extent greater than the embryotoxicity. J 5i~orcri oW<= ^ /39/ "T*V .ilWifit|||,H-|H;W<Wi ** THE EFFECTS OF MATERNALLY INHALED VINYL CHLORIDE ON EMBRYONAL AND FETAL DEVELOPMENT IN MICE, RATS AND RABBITS BY: j.A. John, B.A. Schwetz, B.K.J. Leong, F.A. Smith K.D. Nitschke, H.D. Haberstroh, F.J. Murray M.F. Balmer and P.J. Gehring REVIEWED BY: P. J. Gehring December 19, 1975 Toxicology Research Laboratory Health and Environmental Research Dow Chemical U-S.A. Midland, Michigan 48640 R&S 022826 > r i-- j. L-: i t i, ;a I THE EFFECTS OF MATERNALLY INHALED VINYL CHLORIDE ON EMBRYONAL AND FETAL DEVELOPMENT IN MICE, RATS AND RABBITS INTRODUCTION Vinyl chloride is widely used in the preparation of poly vinyl chloride resin and as a copolymer in plastics, and to a lesser extent as a solvent and as a chemical intermediate. A report of the effect of single exposures of mice, rats and guinea pigs to vinyl chloride by Mastromatteo et al., (1960) indicates that this compound has very low acute toxicity. Anesthesia is the primary significant effect of acute exposure to high concentrations (75,000-100,000 ppm). The effect of repeated exposure of laboratory animals to vinyl chloride has been reported by Torkelscn, et al., (1961). Repeated exposure for six months to 200 ppm resulted in histoiogic changes in the centrilobular area of the livers of rabbits but not in rats, guinea pigs or dogs. In a study reported by Viola et a_l. , (1971), rats were exposed to 30,000 ppm vinyl chloride vapor for 12 months. Findings on these rats were reported to include severe chronic hepatitis, inter stitial pneumonia, as well as tumors of the skin, lungs and bones. Maltoni and Lefemine (1974) reported the oncogenic effects of repeated exposure to inhaled vinyl chloride in rats and mice. Angiosarcomas, zymbal gland carcinomas and nephroblastomas developed in rats exposed to concentrations of vinyl chloride ranging from 50-10,000 ppm, 4 hr/dav, 5 2- - days/week for 12 months, and subsequently maintained and observed until death. Pulmonary adenomas, mammary carcinomas and liver angiosarcomas were observed among mice exposed to the same range of concentrations for 7 months. Similarly, Keplinger, et: al. , (1975), reported neoplasms in mice and the tentative diagnosis of tumors in hamsters and rats exposed to 50, 200 or 2,500 ppm ''ir.yl chloride. The carcinogenic potential of inhaled vinyl chloride has also been studied by exposing animals in utero (Maltoni, 1975), but observations to determine effects which are more in line with a classical teratology study were not made. Reports on the embryotoxic potential of vinyl chloride in laboratory animals have not been found in the literature. Thus, the purpose of the studies described in this report was to assess .the potential of inhaled vinyl chloride to have a deleterious effect on embryonal and fetal development in mice, rats and rabbits. Since previous studies in this laboratory suggested that the primary metabolic pathway for vinyl ciAoride is blocked by ethanol (Hefner, et al_. , 1975) , iu was considered possible that administration of ethanol in the drinking water of animals exposed to vinyl chloride might alter its metabolism in a manner which would enhance its toxic or teratogenic potential. To assess this possibility, some of the vinyl -3- chloride-exposed animals were given 151 ethanol in their drinking water during the days of exposure to vinyl chloride. The teratogenic potential of 15% ethanol in the drinking water in mice, rats and rabbits is being reported separately by Schwetz, e_t al^. , (1975), and is summarized in Table 11 of this report. The majority of this study was conducted under the auspices of the Manufacturing Chemists Association. METHODS Animals and Test Material. Female CF-1 nice (Carworth, Portage, Michigan), weighing 25 to 30 g, Sprague-Dawley rats (Spartan-substrain, Haslett, Michigan) weighing,approximately 250 g and Mew Zealand white rabbits (Langshaws Rabbi try, Augusta, Michigan) weighing 3.5 to 4.5 kg were used in this study. The day on which a vaginal plug was observed or the day on which sperm were seen in a vaginal smear was considered day zero of pregnancy for mice and rats, respectively. The day of hand mating was considered day zero for rabbits. Between daily exposures, animals were housed in wire-bottom cages in a room controlled for temperature, humidity and light cycle. Commercial laboratory animal food (Ralston Purina Company, St. Louis, Missouri) and water were available. Food consumption was measured at three day intervals for mice and rats and at two day intervals for rabbits. R&S 022829 4- Exposuro of bred animals was conducted in stainless steel chambers of 3.7 cubic meter volume under dynamic conditions. The atmosphere of vii'.yl chloride was generated by diluting gaseous vinyl chloride with filtered room air at a rate calculated to give the desired concentration. Samples of inhibited vinyl chloride monomer (chloraothylene) obtained from Mathcsor. Gas Products, Joliet, Illinois, were used for the exposures. The actual concentration was measured with an infrared spectrophotometer (Perkin Elmer 12A) with a multipath gas cell. In addition, the concentration in the chamber was continuously monitored using a recording combustion analyzer to assure the absence cf significant deviations from the desired levels. Experimental Design. In the initial experiment, groups of 30-40 bred mica, 20-35 bred rats and 15-20 bred rabbits were exposed to 500 ppm vinyl chloride for 7 hrs daily on days 615 (mice and rats) or 6-18 (rabbits) of gestation. Some of the mice were also given 15* ethanol in the drinking water. Subsequently, additional groups of mice were exposed to 50 ppm vinyl chloride. Some of these mice also received 15% ethanol in the drinking water. Additional groups of rats and rabbits were exposed to 2,500 ppm vinyl chloride. Some of these rats and rabbits also received 15% ethanol in the drinking water. The experimental design for each of the three species is summarized in Table 1. i3 11 R&S 022830 * r s. . Maternal and Fetal Observations. All animals were observed daily throughout pregnancy c.nd maternal body weights were recorded on gestation days 6, 12, 15 and 18 for mice and on days 6, 10, 16 and 21 for rats. Maternal body weights for rabbits were recorded on days 6, 12, 18, 22 and 29 of gestation Pregnant mice and rats were sacrificed by carbon dioxide inhalation on day 18 and 21 of gestation, respectively. Pregnant rabbits were sacrificed on day 29 of gestation. The uterine horns were exteriorized through a mid-line incision in the abdominal wall and the number and position of live, dead and resorbed fetuses were noted. After being weighed, measured (crown-rump length) and sexed (mice Imdrats), the fetuses were examined for external anomalies. One-third of each litter was immediately examined for evidence of soft tissue anomalies by dissection under a low power microscope. Rabbit fetuses were sexed on the basis of examination of internal genitalia. All fetuses were then eviscerated, preserved in alcohol and subsequently cleared and stained with Alizarin red-S (Dawson, 1926) for examina- ) tion for skeletal anomalies. Statistical Evaluation. The Fisher exact probability test (Siegel, 1956) was used to evaluate the incidence of resorp tions among litters. Maternal and fetal body weights and body measurements and maternal liver weights were analyzed statistically, by an analysis of variance and Dunnett's test (Steel and Torrie, I960). The incidence of fetal anomalies was analyzed by the Wilcoxon test as modified by Haseman and Hoel (1975). Controls. In these studies the effect of inhaled vinyl chloride alone was compared to the effect of vinyl chloride in combination with ethanol in the drinking water. Thus, the group of animals which was exposed only to vinyl chloride served as the controls for those animals which were exposed to vinyl chloride in combination with 15% ethanol in the drinking water. The controls for animals exposed to vinyl chloride alone were exposed concurrently to filtered room air in inhalation chambers. RESULTS Maternal Toxicity. Among mice exposed to 500 ppm vinyl chloride by inhalation, there was a decrease in maternal weight gain during gestation and in the absolute liver weight at the time of cesarean section compared to control values (Table 2). This effect was not observed among mice exposed to 50 ppm vinyl chloride. Mice exposed to a combina tion of 500 ppm vinyl chloride by inhalation and 15% ethanol in their drinking water also showed a decrease in weight gain and liver weight (absolute and relative) on day 18 of gestation. Ethanol in combination with 50 ppm vinyl chloride resulted in a decrease in maternal weight gain throughout gestation and a decrease in absolute liver weight on day 18 of gestation. Food consumption throughout gestation was decreased for mice exposed to both concentrations of vinyl chloride in combination with ethanol. Food consumption was decreased for mice exposed to 500 ppm vinyl chloride alone but not 50 ppm during days 6-15 of gestation. Except for an apparent decrease in maternal weight gain in rats (Table 3) and a decrease in food consumption for rabbits (Table 4), no signs of toxicity were observed in the adult rats or rabbits during exposure to 500 ppm vinyl chloride. The apparent decrease in maternal weight gain among rats exposed to 500 ppm vinyl chloride is most likely due to the lower body weight of the control animals on day 6 and subse quently a higher weight gain of these animals during the later days of gestation. Among rats exposed to 2,500 ppm vinyl chloride, both the absolute and relative liver weight were significantly increased on day 21 of gestation, but maternal weight gain was no different from that of rats exposed to filtered room air. Maternal food consumption was, however, lower than among control rats in this group. The relative liver weight on day 21 of gestation was signi ficantly increased among rats exposed to 2,500 ppm vinyl chloride and 15% ethanol. Maternal weight gain for this group of rats was significantly decreased during days 6-10 8- - of gestation and food consumption was decreased during the exposure period. No effect on maternal weight gain or liver weight was observed among rabbits exposed to 2,500 ppm vinyl chloride by inhala tion. A decrease in maternal weight gain during days 612 and 12-18 of gestation was observed among rabbits exposed to 2,500 ppm vinyl chloride in combination with 15% ethanol (Table 4), but total weight gain in this group was not different from that among rabbits exposed to 2,500 ppm vinyl chloride alone. Food consumption was, however, significantly decreased among these rabbits exposed to 2,500 ppm plus ethanol. Observations made at the tine of Cesarean Section. Observations made at the time of cesarean section of mice, rats and rabbits are presented in Tables 5-7. Maternal deaths were observed among mice exposed to 500 ppm vinyl chloride alone and in combination with ethanol (Table 5). The percent pregnancy was reduced among mice exposed to 500 ppm vinyl chloride and ethanol. There was an increase in the incidence of resorptions and the fetal body weights were lower than in controls for the 500 ppm vinyl chloride group. Litter size was also reduced. These effects were augmented among mice exposed to 500 ppm vinyl chloride in combination with 15% ethanol. Two litters in this group were totally resorbed. In addition, the number of implantation sites per dam and -9- fetal crown-rump length were significantly lower in the ethanol group as compared to mice receiving 500 ppm vinyl chloride alone. Among mice exposed to 50 ppm vinyl chloride, the fetal crown-rump length was significantly greater than among controls; a decrease in fetal body weight and crownrump length was observed in the 50 ppm vinyl chloride plus ethanol group. The incidence of resorptions was not signi ficantly greater among mice treated with 50 ppm vinyl chloride in combination with ethanol although 2 litters were totally resorbed. One maternal death was observed among rats exposed to 2,500 ppm vinyl chloride by inhalation (Table 6). There was no significant effect on litter size, the number of implantation sites per dam or the incidence of resorptions among any of the exposed groups of rats. The pregnancy wastage was significantly lower among rats exposed to 500 ppm vinyl chloride than among the control rats. Percent pregnancy was unaffected among rats exposed to vinyl chloride alone or in combination with 15% ethanol in the drinking water. Fetal body weight and crown-rump length were significantly reduced among rats exposed to 2,500 ppm vinyl chloride in combination with ethanol. A significant reduction in fetal body weight was also observed among rats exposed to 500 ppm vinyl chloride alone, but not among those exposed to 2,500 ppm. Signifi cant decreases in the number of corpora lutea per dam were observed among rats exposed to 500 ppm vinyl chloride alone ' }K f lit and among those exposed to 2,500 ppm in combination with 15% ethanol. A significant increase, however, in the number of corpora lutea per dam was observed among rats exposed only to 2,500 ppm vinyl chloride. Since the number of corpora lutea is established prior to day 6 of gestation, these differences in the number of corpora letoa per dam among rats exposed to vinyl chloride is not considered a treatment-related effect, but rather a measure of the reproductive status of the animals prior to the beginning of the experiment. Among rabbits, a significant increase in the incidence of resorptions was observed in the high concentration (2,500 ppm) plus ethanol group where 7 litters were totally resorbed (Table 7). Exposure of rabbits to either 500 or 2,500 ppm vinyl chloride alone did not alter the incidence of resorptions A decrease in the number of live fetuses per litter was observed among rabbits exposed to 500 ppm vinyl chloride alone, but not among those exposed to 2,SCO ppm vinyl chloride alone or in combination with 15% ethanol in the drinking water. This decrease in litter size was associated with a decrease in the number of corpora lutea and implantation sites in these does and therefore is probably not due to exposure to vinyl chloride. No differences in fetal body weight or crown-rump length were observed in any of the exposed groups of rabbits. -11- Incidence of Anomalies. The incidence of anomalies among litters of mice, rats and rabbits exposed to vinyl chloride by inhalation are indicated in Tables 8, 9 and 10, respectively. The incidence of gross anomalies observed by external examination of fetuses from mice was not significantly greater than among control litters (Table 8). One fetus among the litters of mice exposed to 500 ppm vinyl chloride in combination with 151 ethanol exhibited anopthalmia. The incidence of cleft pala.e in this group was slightly higher than that observed among mice receiving vinyl chloride alone. No soft tissue anomalies occurred at an incidence signifi cantly greater than control in mice. Two fetuses from cr.e litter among mice exposed to 50 ppm vinyl chloride- plus ethanol exhibited a small thymus. Significant increases in the incidence of delayed ossification of sternebrae (=5) and bones of the skull were observed among litters of mice exposed to 500 ppm vinyl chloride. The incidence of unfused centers of ossification of sternebrae was also significantly higher in this group. No skeletal anomalies were observed at an incidence significantly greater than in controls amonc mice exposed to 50 ppm vinyl chloride. Among mice exposed to vinyl chloride in combination with 15% ethanol, however, several skeletal anomalies occurred at an incidence signi ficantly greater than among mice exposed to vinyl chloride R&S 022837 -12- alone. In mice exposed to 500 ppm plus ethanol, increases in the incidence of delayed ossification of bones of ^the skull, sternebrae (if' s 2-6) and arches of the cervical vertebrae were observed. Significant increases in the incidence of lumbar spurs, missing centra of cervical vertebra and missing 5th sternebra were observed in this group as well. The incidence of extra ribs was slightly increased compared to the controls. Among litters of mice exposed to 50 ppm vinyl chloride plus ethanol, increases in the inci dence of delayed ossification of bones of the skull and sternebrae (#'s 4-6) were again observed upon skeletal examination. The incidences of unfused occiptal, unfused sternebrae (#'s 5 and 6) and forked atlas were also signifi cantly increased in the 50 ppm plus ethanol group. In rats, no gross anomalies occurred at an incidence signi ficantly greater than among control animals (Table 9). Among litters of rats exposed to 2,500 ppm vinyl chloride, the incidences of unilateral and bilateral dilated ureter were significantly higher than among control litters. The incidence of dilated ureter was not affected by concomitant exposure to 2,500 ppm vinyl chloride and 15% ethanol. Petechial hemorrhage in the kidney was observed at a slightly higher incidence than control among litters of rats exposed to 500 ppm but not 2,500 ppm vinyl chloride. Among litters i w t n >' j it i *i ; t, !/ JJ fi CO oro io 00 w 00 iM___________________________________________________ VM |l| ifT rt -13- of rats exposed to vinyl chloride, only minor skeletal variations wore observed at an incidence higher than that of controls. The incidence of lumbar spurs was increased among litters exposed to 500 ppm vinyl chloride. No increases in the occurrence of skeletal anomalies wore observed among litters of rats exposed to 2500 ppm vinyl chloride. The incidences of delayed ossification of bones of the skull and unfused centers of ossification of skull and sternebrae were significantly decreased among litters of this group. The incidences of lumbar spurs and missing centers of the cervical vertebrae were, however, increased among litters of rats exposed to 2500 ppm vinyl chloride in combination with 15% ethanol in the drinking water. In rabbits, no gross anomalies were observed at an incidence greater than among control litters, though a cleft palate was observed in one fetus among litters of rabbits exposed to 2,500 ppm vinyl chloride plus ethanol (Table 10). Dilated renal pelvis was also observed in two fetuses from one litter among rabbits exposed to 2,500 ppm plus ethanol. Two fetuses from this group also exhibited an enlarged right atrium of the heart. One fetus with 3 kidneys of normal size was observed among litters of rabbits exposed to 500 ppm vinyl chloride. Delayed ossification of the 5th and 6th sternebrae wore the only skeletal anomalies observed in i r ; i j ii f i i' t 30 0) ortoo oo oo <0 | ^' -14- rabbits at a significantly different incidence from controls. Among litters of rabbits exposed to 500 ppm vinyl chloride, the incidence of delayed ossification of the 5th sternebra was increased whereas that of the 6th sternebra was decreased Delayed ossification of sternebrae did not occur at a higher incidence than control among litters of rabbits exposed to 2,500 ppm vinyl chloride alone or in combination with 15* ethanol in the drinking water. DISCUSSION The results of these studies indicate that exposure of pregnant mice, rats or rabbits to vinyl chloride by inhalation at concentrations sufficiently high to cause maternal toxicity was not teratogenic in any of the three species. The responses of mice, rats and rabbits are summarized in Table 11. Less maternal toxicity was observed among rats and rabbits than mice during exposure or at the time of cesarean section. Among rats one maternal death and an increase in liver weight was observed at 2,500 ppm while only a slight decrease in maternal weight gain was observed at 500 ppm. Among rabbits, one maternal death was observed at 2,500 ppm and there was a decrease in food consumption at 500 ppm. In comparison, 500 ppm was quite maternally toxic to mice as evidenced by the significantly decreased weight gain and food consumption and by the occurrence of a number of maternal deaths. -15- Exposure to vinyl chloride was not consistently embryotoxic in the three species studied. A significant increase in the percent resorptions was observed among mice exposed to 500 ppm vinyl chloride, but not among rats or rabbits exposed to 500 ppm or 2500 ppm vinyl chloride. Some decreases in fetal body weight and crown-rump length were observed in rats and mice but not rabbits. A teratogenic response to maternally inhaled vinyl chloride was not observed in nice, rats or rabbits. With the exception of unilateral and bilateral dilated ureter among litters of rats exposed to 2500 ppm vinyl chloride, no external or soft tissue anomalies were observed at an incidence significantly higher than control in any of the three species. Examination of the skeletons revealed only minor skeletal variants; no skeletal malformations were found at an incidence significantly greater than in the control groups. 30 W oto N) 00 4* i Ingestion of 15% ethanol in the drinking water enhanced some of the effects of inhaled vinyl chloride. In each of the three species tested, maternal weight gain and food consumption 1 was lower than among animals exposed to vinyl chloride alone. The percent resorptions was slightly increased among ; \ mice and significantly increased among rabbits exposed to vinyl chloride in combination with 15% ethanol in the drinking water. Fetal body measurements were lower among litters of ` hi mice and rats which received ethanol and vinyl chloride compared to vinyl chloride alone. Certain malformations were observed among litters of mice, rats and rabbits which also received ethanol but their incidence was not statistically different than control litters. The effect of simultaneous ingestion of ethanol on the disposition of vinyl chloride in these animals seemed to enhance maternal toxicity to an extent greater than embryotoxicity. In summary, the results of these studies indicate that exposure of pregnant mice, rats and rabbits to vinyl chloride by inhalation was not teratogenic at the concentrations tested. Mice were more susceptible to the toxic effects of vinyl chloride than either of the other two species. Simul taneous exposure to vinyl chloride by inhalation and 15% ethanol in the drinking water resulted in toxic effects greater than those associated with exposure to vinyl chloride alone in the three species. Neither exposure to vinyl chloride alone or in combination with 15% ethanol in the drinking water caused a significant teratogenic response in mice, rats o rabbits. REFERENCES 1. Dawson, A. B. (1926) . A note on the staining of the skeleton of cleared specimens with alizarin red-S. Stain. Tech., 1_, 123-124. 2. Haseman, j. K. and Hoel, D. G. (1975). Tables of Gehan's generalized Wilcoxon test, with fixed point sensoring. J. Stat. Comp, and Sim., In press. 3. Hefner, R. E., Jr., Watanabe, P. G. and Gehring, p. J. (1975). Preliminary studies of the fate of inhaled vinyl chloride monomer in rats. Ann. N.Y. Acad. Sci. 246, 135-148. 4. Maltoni, C. (1975). The value of predictive experimental bioassays in occupational and environmental carcinogenesis. An example: vinyl chloride. Ambio, 4_, 18-23. 5. Maltoni, C. and Lefemine, G. (1974). Carcinogenicity bioassays of vinyl chloride. I. Research plan and early results. Environ. Res. l_t 387-4 05. 6. Keplinger, M. L., Goode, J. W., Gordon, D. E. and Calandra, J. C. (1975). Interim results of exposure of rats, hamsters and mice to vinyl chloride. Ann. N.Y. Acad. Sci. 246, 219-224. 7. Mastromatteo, E., Fisher, A. M., Christie, H. and Danziger, D. (1960). Acute inhalation toxicity of vinyl chloride to laboratory animals. Amer. Ind. Hyg. Assoc. J. 21., 394-397. 8. Schwetz, B. A. (1975). Manuscript in preparation. 9. Siegel, S. (1956). Non-Parametric Statistics for the Behavioral Sciences. McGraw-Hill Book Co., Inc., New York. 10. Steel, R.G.D. and Torrie, H. H. (1960). Principles and Procedures of Statistics, McGraw-Hill Book Co., Inc., New York. 11. Torkelson, T. R., Oyen, F. and Rowe, V. K. (1961). The toxicity of vinyl chloride as determined by repeated exposure of laboratory animals. Amer. Ind. Hyg. Assoc. J., 22, 354-361. 12. Viola, P. L., Bigotti, A. and Caputo, A. (1971). Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res., 31, 516-519. R&S 022843 i: V V it'? -f 4t\: , M*S*:t,- -i; $: *'i- Il 'I>4 i ti _ .' 1* :"' - {i. IP*! S^ ;* - -v, ,' TABLE 1 TERATOLOGY STUDIES WITH VINYL CHLORIDE Mice Rats Rabbits Mice, Rats, Rabbits ppm Vinyl chloride9 500 500 50 50 2,500 2,500 500 2,500 2,500 500 0 Ethanol9 0 15% 0 15% 0 15% 0 0 15% 0 0 *Mice and rats were exposed to vinyl chloride or filtered room air by inhalation 7 hrs daily on days 6-15 of gestation. Some of the mice and rats were given ethanol in their drinking water (15% v/v) on days 6-15 of gestation. Rabbits were exposed to vinyl chloride on days 6-18 of gestation. Some of the rabbits were given ethanol in their drinking water on days 6-18 of gestation. 1 i. i'^ "' vVf (, ch rf itK rIs>* ;r? i; r 33 fi CO rIOo 00 --* f TABLE 2 MATERNAL WEIGHT GAIN, LIVER WEIGHTS AND FOOD CONSUMPTION OF MICE EXPOSED TO VINYL CHLORIDE BY INHALATION ppm Vinyl chloride in air % Ethanol in drinking water3 Low Concentration 0 50 50 0 0 15 0 0 Number of dams Body weight on gestation day 6 Weight gain during gestation days 6-12 12-15 15-18 total Liver weight on gestation day 18 absolute. relative Food consumption during gestation days 6-15 21 30 + 2 3+3 6+3 7+2 16 + 4 20 31 + 2 16 26 g, mean + SD 31 + 2 29 + 2 4+2 5+1 7+4 17 + 6 0.4 + 4^, days 6-12 6 + 1 4+2d 12-18 15 3 7+3 ll7d total 20 + 3 2.75+0.26 2.76+0.40 2.37+0.52d 59.5+8.7 57.8+4.5 56.6+7.3 6 + 1 6+1 4+ 2d 2. 750.31 55 .5+5.5 6+1 500 0 19 29 + 3 5+4 12 + 3 17 + 4C TUo 15 7 30 + 3 l + 2d 8+6 10 + 7d 2.49+0.29 1. 78+0.36d 54.4+4.2 45 .8+5.1 5 + lc 3 + ld Mice were exposed to vinyl chloride or filtered room air by inhalation 7 hrs daily on days 6-15 of gestation. Some of the mice were given ethanol in their drinking water (15% v/v) on days 6-15 of gestation. ^3 mg liver/g body weight, c. ^Significantly different from control by an analysis of variance, p<0.05. Significantly different from vinyl chloride alone by an analysis of variance, p<0.05. SP2ZZQ S9U ^ MWHWlim I J--. V " ,* t ' TABLE 3 MATERNAL WEIGHT GAIN, LIVER WEIGHTS AND FOOD CONSUMPTION OF RATS EXPOSED TO VINYL CHLORIDE BY INHALATION ppm Vinyl chloride in air q % Ethanol in drinkinq water Number of dams Body weight on gestation day 6 oo ! OO 1 Low Concentration 500 0 High Concentration 2,500 "17^0-------- 0 15 28 258+26 31 19 <3* mean + SD 277+20d 288+27 16 274+19 16 272+14 Weight gain during gestation days 6-10^ 10-16 16-21 total 148 + ]1 125*19d 13 + 10 37 + 11 77 i ] 5 127+15 17 + 6 40 + 11 82 + 16 138+23 -l+12e 45 + 14 77 + 13 120+15e Liver weight on gestation day 21 absolute relative0 Food consumption during gestation day 6-15 14.811.79 15.00+1.14 36.5+4.1 37.1+2.6 21 + 2 22 + 2 14.271.38 15.55+1.23 16.52+1.50 34.4+3.3 37.82.6d 42 .1+2.4 22 + 2 21 + 2d 13 + 2 aRats were exposed to vinyl chloride or filtered room air by inhalation 7 hrs daily on days 6-15 of gestation. Some of the rats were given ethanol in their drinking water (15% v/v) on days 6-15 of gestation. ^Interim values for maternal weight gain were not available for the rats exposed to 500 ppm vinyl chloride, c mg liver/g body weight. ^Significantly different from control by an analysis of variance, p<0.05. eSignificantly different from vinyl chloride alone by an analysis of variance, p<0.05. 9t?8ZZ0 S9U PSPS* \\ft* * ~Jrr*r<'*' * r; - - ; Vtv'* * TABLE 4 MATERNAL HEIGHT CAIN, LIVER WEICIITS AND FOOD CONSUMPTION OP RABBITS EXPOSED TO VINVL CIILORIIJK By INHALATION ppn Vinyl chloride in aira 1 Ethanol in drinking water* Low Concentrat ion U 3W 0o II inh* Concentra t ion u------- ------- ----------------- r;3oo 0 0 15 Number of dams Body weight on gestation day 6 18 3.B510.23 20 11 kg, mean t SD 3.B210.2S 4.0810.21 4.3910.30 16 4.0210.47 Height gain during gestation days 6-12 L2-18 18-22 22-29 -0.0610.11 0.0110.21 0.0810.26 0.0110.16 -0.1210.00 0.0610.09 0.0210.08 0.0310.IS -0.1110.17 0.0S10.15 0.0710.IB 0.0510.17 -0.1210.03 -0.0310.07 0.0310.05 0.1310.04 -0.4110.17" -0.27l0.15d 0.1010.33 0.4210.36 Total 0.0510.19 0.0110.19 0.0610.27 0.0110.13 -0.1410.42 Liver weight on gestation day 29 absolute relative1* 96119 24.613.6 B9i)4 23.212.9 102116 24.712.7 122125 27.715.9 116130 30.016.3 Pood consumption during gestation days 6-1B 98130 76129 91 136 89126 1519 aRabblts were exposed to vinyl chloride or filtered room air by inhalation 7 hrs daily on days 6-18 of gestation. Some of the rabbits were given ethanol in their drinking water (151 v/v) on days 6-1B of gestation. bg liver/kg body weight. Significantly difterent from control by an analysis of variance, p<0.05. Significantly different from vinyl chloride alone by an analysis of variance, p<0.05. TAW.i; onsiwvATiotu; maim; at tiii: timk or rr.rAiov\n ;;i:cti<>n or mici: KxrosKi) to VINYI. ClIMMliOK HY INIIAIJVTfoM l|im Vinyl chloride in air \ Klhanot in drinking water" Number of litters Implantation Rites/damb I.IVt* (lltMBl'D/l it tel'*' X, Neuorptifins/implantat ions 1, Litters with resorptIoiih lotteis totally rc*;orhcd Hosnrpt ions/! itl *m with r<u;itrpt iimr. Sex ratio, M:F fictul body wo 1 (flit, gC Fetal crown-rump length, minc t, maternal deaths/ trented dams 1 Pregnancy*' `6-----U->-w---:.-h--ii-c6(>0h..l.i...il..in..n. O0 21 70 12*7 1 2M 1 *___ 10 n *4 10*4 1 I *4 10*4 IS (40/201) ft (111/7110 II (10/177) 67 (M/21) l 55 (11/70) 0 6*> (I I/Mi) 2 2,0 (40/M) rn:',n 1.00*0.11 21*021.9 1.6 ( I li/I 1) 50 s SO 1,02-0.10 24,2*0.11** 1,7 (10/U) 4 It : S 2 0.04*0.1 4t? 7 2,4 M . 5C 0 (0/37) 57 (21/37) 0 (0/27) 74 (20/27) 0 (0/28) 67 (16/20) Jl igh Conc?id mUoii 566' 0 "SOU" 15 26 19 7 14*2 11*2 10*6 I2'2 1 1 * 2U 8 6l' ' (76/151) 1 1 ( 1 )/24) f 19 (M/69) 1 (16/26) 76 (19/19) 06 (6/7) n (1 2 r (7 *i/ 1 6) 2.7 ( t 1/19) 54 ;4f. 92:4B .07M1.0G 0. 99*0.1ld ! 3,7 i 1 .2 23 .fill.0 2.2 U 1/6) 64 : 36 0.7B10.15 21.211,5 l (0/10) 17 19/29)f 1 (20/32) 72 (21/29) 13 (4/30) 31 (9/29)^ Mice were cx|>uscd to vinyl chloride or r i 1 t*rcd i onn air by inhalation 7 Itrs daily on days 6-15 of gestation. the mice were given ethanol in Lheir drinking water (15t v/v) on days? 6-15 of gestation. bHcnn f SO, cHe*n of litter means 1 SI). dSignlficantly different from control by an analysis of variance, p<0-05, Significantly different from vinyl chlorldn alone by an analysis of variance, p<0.05. ^Significantly different from control by the Fisher exact probability test, p<0.05. ^Signifleantly different from vinyl chloride alone by the Fisher 'xnct probability test* p<0.05. ^Masted on the presence of feturavi and/or resorption niton observed by gross examina ion at the time of Cesarean section. Some of ' i* TAMlrK 6 our.KnvATiows hade at the tihi: of cesarean sf.ction of rats exposed to VINYL CHLORIDE IIY INHALATION ppm Vinyl chloride in air0 t Ethanol in drinking water'1 oo biv Concentration 0 ft High Concentration 2750 2,500 0 0 15 f Number of litters Corpora lutca/dam*' 29 1 !> 11 it 1.1 21' 19 1412 16 15! 2*' 16 1412 Implantation si tos/chim*1 1212 it? 12*2 14*2 12 1Z Pregnancy Wastage** 3<2 0.41l`` 1 f 1 2*1 2*2 Live fetusos/iittor^ 12 Z 1212 12*2 1312 1212 Resorptions/implantations \t Litters with resorptions 1 (4/342) 14 (4/2B) 3 (11/J98) 29 (9/31) 4 (9/23B) 32 (6/19) 3 (6/220) 25 (4/16) 4 17/195) 25 (4/16) Litters totally roHorbod 00 0 00 Rcsorptions/littors with resorptions 1.0 (4/4) Sox ratio, MiF 52i48 Petal body weight, g' 5.6710.29 Fetal crown-rump lenqtli, mmc 42.GJL2 1.2 (11/91 50:50 5.44 10. IB*1 43.610.B*1 1.5 (9/6) 49:51 5.59*0.27 43. 6*1.5 1.5 (6/4) 53:47 5.6210.29 43.311.1 1.8 (7/4) 51 j 49 5.3410.32 42.410.9 I, maternal deaths/ treated dams \ Pregnancy* 0 (0/29) 96 (2B/29) o (0/n; 94 (31/31) 0 (0/20) 95 (19/20) 6 (1/17) 100(17/17) 0 (0/17) 94 (16/17) Rats wore cx|>08td to vinyl chloride or rlltcrod room air hy inhalation 7 hrs daily on days 6-15 ( or qcst.itIon. Some of the rats were qiven ethanol in their drinking water (151 v/v) on days 6-15 of gestation, bMcan 1 SO. Mean of litter means 1 SO. ^Siqnificanlly different from control hy an analysis of variance, p<0.05. eSigniricantly different from vinyl chloride alone by an analysis of variance, p<0.05. ^Daoed on the presence of fetuses and/or renorption sites observed hy gross examination at the time of Cesarean section. Ml f j i D B W o rKoS oo fa <o f i - V-*# i TAIU.I: 7 OUSKUVATIOfin MADE AT TIME Of CKSARKAN SECTION OF HATS EXPOSED TO VINYL CHUilUOi: !IY TNIIAIoATIUN , rr*n vinyl chloride in air X Ethanol in drinking water'1 Number of litters Corpora lutea/damb Implantation sites/dam11 Pregnancy wastage*1 Live futusns/1 i ttcr*1 X. RcftorpMons/implanti'itions X, Litters with resorptions Litters totally rosorhed Kesorptions/1ittcra with resorptions Sex ratio, MiF Fetal body weight. gc Fetal crown-rump length, mm*1 X, R/.teinal deaths/ treated dams t Pregnancy^ Isow Cnuccntr.ition 0 *>oo 00 1H 19 911 sn*1 911 8tld 0,4*1 1M fltl 712'1 6 (10/162) 9 (14/1501 44 o/im 32 (6/19) 01 1.2 (10/8) 53:47 JS.2314.82 91.014,2 2.3 (14/6) 50:50 34.1314.17 92.615*0 0 (0/1 ft> 100 UB/18) 0 (0/20) 95 (19/20) 0 0 11 10l2 0*2 211 613 22 (19/6A) 64 (7/11) 2 High Concentration 2,500 27500 0 15 5 16 10*7 1012 814 9l2 2i3 111 614 414 24 (10/4 2) S3 '79/149)' 80 (4/5) 88 (14/16) 17 2.7 (19/7) 61:39 36.4614.82 92.6*4.7 2.5 (10/4) 50:50 33.7714 .48 87.115.2 5.6 (79/14) 43:57 32.4815.88 87.716.3 0 (0/11) 100 (11/11) 14 (1/7) 86 (6/7) 16 (3/19) 95 (18/19) 30 fi (/) o Nro> ao (71 o "Rabbits wore exposed to vinyl chloride or filtered room air by inhalation 7 hrs daily on days 6-18 of gestation. Some of the rabbits wore given ethanol .in their drinking water (l*>t. v/v) on days 6-1B of gestation. bMcan 1 SO. CMoan of litter means t SO. Significantly different from control by an analysis of variance, p<0.05. siqnificently different from vinyl chloride alone by the Fisticr exact probability tost, p<0.0S. Based on the presence of fetuses and/or resorption sitoB observed by gross examination at the time of Cesarean section. 4 \ * . jJi* w }' 1 a 51 3l Qroii Aw/'jligi 0 0' 00 00 00 00 00 0.4 (1) 1 (1) 0 0 1 (it 1 (It 10 (It 10 (31 0.4 (1) 0.4(1) s (It ) (1) 0 0.4(1) a i (1) 0 0 0 0 0 0 0 0 2 (It 21 (11 0.4 (1) 7 (1) 0 0 1 (21 1 (2) 1 (2) (3) 0 0 0 0 0 0 1 (2) I (2) 0 0 1 (1) 10 i2> 0 0 0 0 0 0 l (2) 5 (it 1 (2) S (1) 0 0 2 (It 20 Ml 0 0 2 (1) 20 Ml 0 0 7 (4) 40 (2t 3 0 0 0 Snail thynu* lUAorfhJitt AiJncy tteaorrhi<7C. Uvr Hero fr hiqo* thynu Ltvcr hcaitoma SVlctM_ Ana^.i\ ict Skull-del jyd OftClf lemon -uoncfcuiipvdital atlaiVertcbrac-forkcd nimrq cervical centra r L - delayed cation of cervi cal areheft f jtorntiA-vftfufted delayed oiufl* CJtion S ftterncora me 00 00 00 00 0 * (1) 0 s (1) 0 s (4) 0 10 (3) 00 00 4 (2) 7 (It 0 0 0 0 0 0 0 0 9 (1)1 (2) IS (71 17 (7) 40 (41) 100 (14)d 0 0.7(1) 0 s (1) 4 (It 5 (U> SO (> 10 () 4 111 * (12) 35 (7) 40 (ft) 34 (251 SO (7)d o. (l) 7 (1) 2 ID 21 1)1 0.4 ID 1 m S 111 10 (2) 4 (C) ]f (S)d 00 00 0 0 00 00 3 () 1 (7) >0 (4) IS (S) 7 (IS) 4 1*1 SO (10) 35(7) 00 00 1 (2) 14 (2) 1) (30) S7 (l)d 44 ((7) 100 (M|d I (4) u (Jt 0 0 2 (2) 1 (3) 2 t2) 4 (1) 0 3 2 (2) 1 (2) 0 0 3 (2) 10 (2) 1 Cl 5 C) 0 0 1 (1) S (it 0 0 0 3 3 3 0 3 0 0 1) (21) U (14) 1 (D 12 ()) 3 ('ll 31 ID 4 (12) 31 (3) 0 0 0 0 so (43) 73 o*! SI (UtC 130 (SI1 s (7) 21 (4) 1 (O S3 (A? 1 (4) 21 Ml 11 (4| 20 C) U UJd 00 C Jt 14 Ml 40 HI*1 0 4 <2t 0 20 Ml 1 (2) 31 111) 10 Cl , to U)J 0 0 2 ID 19 (5) 1 (4) 12 (3) 0 0 0 0 9 (19) 42 ()* s (D 40 (2)d 14 M9I 10 1114 M2) 4) 124 l 42 (D* 10o (SI4 l (D 10 (2) 7 (4) 40 III4 hiec wore eaponed to vinyl chloride or filtered toon air by inhalation 7 nra duly on t-1S of wiutic" 1'W of the aiCtf *vrc qiven ethanol in their JniUilhf u|e; (111 v/w) on day* l*ll of iiv^t tUpn. b r*!ctueoai L*1 it ten. icantly lut**rnnt frnn control by the *iMii!i0<J wilccaon teat, p<C.3S. ^li.iai! leant l> different fm vinyl cnlonde alone by the Mdified rttleo*en teat, (--O.n'., C(*d,Oi>i <difiej uiicoten teat. " ' >jpr*W ( y. 30 a (/) N3 IO 00 CJI f.j.m ViA'/t (hl'<ri'lc in jtr4 tt^ih'll in 'irmKin't witer' timber of fvtupv* banner of litter* 32* 1*7 IS 21 22* - 214 1* IS u* IS Percent Affected (number effected! Cross /.-:> ill*'-! Omphalocele Tall-less Hunt r L f L r L S'ft T*. 'I** *."* (1 Mierii'thdl.m i dilated ureter uni l it**r j1 bilateral Soull kidney Pctcch i a 1 he.-.'* r r ha-ie 4 idney Mcmbrmd'ic, liver llcmorrhj'jo, k itlncy Short trunk! ectopic ovonei Ik :l*?t il ,\nfm i! ms Skill - lalv/'s-J ossification - unfuied Sib* - shortened r L r L r L r L T L T r L r L P L r i r - crooked r t * missing r L - extra P L - spurs r i Vertobrae - missing r L - missinq ccrvicol centre r L Sternum - delayed ossification p L 0 l III 0 2 (11 00 0a 00 0e 0 0 2 (2) 11 (2) 0 0 0 0 0 0 9 0 0 0 0 0 0 0 l (l) 2 (1) 1 (1) 2 IU 0 9 10 U2I 1) (41 0 0 1 (11 1 (1) 0 0 0.4 (1) S (1) 1 (3) S (1) 0.4 (1) S (M 0 0 0 0 0 0 0,3 (11 4 (1) O.s (1) 4 (1) 0 0 00 00 4 (3) 10 (3) 2 (2) S (11 0 0 IS (U) *4 <)' 12 () a me 0 0 90 00 0 l (1) 0 s (l) 00 90 1 (11 3 (l) 0 0 2 (1) 4 (1) 2 (2) 12 (3) 2 (1) 4 (11 2 (1) 4m 0 0 9 0 0 0 0 0 14 (3S1 1 (121 0 1 9 9 0 9 0 0 0 0 1 (2) 4 ID 9 0 12 (211 Si (1*1 0 0 0 0 0 0 0 0 0 9 9 (24) 12 llilc 0 0 0,3 (1) 4 111 1 (91 11 (21 2 (7) IS (Si 2 (41 l* () 14 '211 4 (SI St (111 31 (>*" S3 (tl) 3 (41 *0 (1*1 u uic 1 (21 S ill 0 0 0.4 (l) 0 5 (l) 0 2 (4) O.S(l) 10 (21 s (11 0.4 (1) O.S(l) S (1) 4 (1) 14 (32) 12 (2*> 41 (131 to mi 1 (3) S (1) 0 0 3 i 4) 23 (4) 1 (21 12 (2) 0 0 1 (2) 4 (1) 2 (4) 10 (21 4 '71 1* (31 21 ihf, 'i* (11 2 (3) 12 (21 7 (1SI 4 (*) S3 (10| SO H) * (21) * mi SI (13) 44 (71 21 Id 'll m 11 *21 34 (0) unfused - fused Other - missing fibula - missing scapula p* r L p L r L P 1, 0 1 (2) 0 4 (I) 00 00 0 0,2 (1) 0 2 (1) 0, 0 0A 00 00 3 (1) 32 (SI 0.4 (l) sm 2 (4) IS (3) 0.9(1) t lllc 9 0 0 0 0.4 (!) S (11 0 0 00 00 l (2) 12 (2| 9.3 (1) 4 (U 0 0 0 0 o.s 111 4 111 *Kats rtf* esposed to vinyl chloride or filtered room dir by inhjf.ii.nn 7 *i'* .miy on days 4-1S ot i*iuupn. Son* of the mi wore qiven ethanol in mcir UrmMn-i (lit v/v) on Jjyi -U of gestation, *r-fetuseti t,.Utters. c4ienifie*ntly different fre* control by the modified wtlcoson test, fra.oS. Significantly different from vinyl cnlonde alone by the mcdifieu w Uo-in test. f*9,3S, r-' $$ >*< p->-\ $ 'i \?`t \ .* 4i k! -I i -. i '&' w #.} ;*iwtVVv'4 > `! V-*>'V;'*:i lw jv;, i -7?..V' .:'rl ' t INCIDENCE or ANOMALIES amoni litters or PABBIT3 exposed to VINYL CHLORIDE BY INHALATION ppm vinyl chloride in air 1 Ethanol in drinking water Number of fetuses Number of litters Cross Anomalies Cleft palate Exencephaly r* L Crooked tail Soft Tissue Anomalies I kidneys Dilated renal pelvis Dilated cerebral ventricle Enlarged right atrium 2 left sub-clavian arteries Skeletal Anomalies Sternum - delayed ossification *5 - delayed ossification 6 Low Concentration ----------5--------------------OT5---------- 00 ----------3 0 ------- 27573 0 152 136 13 18 69 32 94 Percent affec ted (number affected) 0 0 1 (1) 6 (1) 0 0 0 0 0 0 i :i) i (1) 00 :> 0 30 00 \ 0 00 0 2 (I) 0 Cl 00 00 o0 00 00 00 00 00 30 30 00 Q3 9 10 (1) 0 25 (1) 0 0 00 00 00 28 (43) 77 (14) 22 (33) 72 (13) 33 (521c 94 (17) 11 <15)e 50 (9) 20 (14) 44 (4) 10 (7) 44 (4) 16 (5) 75 (3) 9 (3) 75 (3) 2,110 15 TO 9 1 ID i: id 0 0 0 0 0 0 5 *2) i: ;d 3 0 3 (2) 11 (1) 4 (1) i: iii 24 (17) 67 (6) 9 (6) 22 (2) *Rabbits were exposed to vinyl chloride or filtered room air by inhalation 7 hrs daily on days 6-16 of gestation. Some of the rabbits were given ethanol in their drinking water <151 v/v) on days 6-13 of gestation. bF>fetusest L-litters cSignificantly different from control by the modified Wilcoxon test, p<0.05. `"'TjU*'" '* " JO (0 o fO M 00 CJI t ("amts . TAMI.i: I I UUMMAKY - VIIIYI. C'MIjOIIIIIK TlIUATllUKlY ilTIIIII KS Vinyl chloride, 7 hr/day Gustation day* of treatment 151 Kthanol in drinking water Maternal deaths % I'rugnancy No. litters examined Maternal weight gain Maternal food consumption 5& ppm 6-15 Mice '500" PIXM G- 15 0" 6-15 no yes no you yt:u no no yen yen no -- -- dec -- 20 14 19 5 21 - - dec. dec. dec. -- -- dec. dec, dec. d<*c. Maternal liver weight - absolute - relative kkiplantatton silos/dam W Hesorptions I.itiers totally rosorbod (.ittor size Fetal body weight 1`eLal crown-iump length External anomalies -- -- -- 0/20 -- -- inc. -- dec. dec. -- 2/16 -- i nc, 0/10 dec. dec. dec. dec. --- dec. dec. dec. __ -- -- ~ -2/7 0/21 dee, -- dec. dec. dec. dec. ---- Viscera) anomalies Skeletal anomalies -- -- inc. inc. 500 i>i*m G-I5 no no -- 21 dec. -- K.ilil Tonfi W3*- --a-- G-l15 G-15 no yes you yes no no -- -- -- 16 1 G 19 -- dec * -- ih*t:, dec. doc. __ -- -- 0/11 -- dec. i nt.'. -- --- i iu: . i nc. -- -- 0/16 -- -- i. -- -- .. i nc. i nc * -- -- ---- n/io 0/10 -- -- dec, dec. dec. -- -- -- -- --- inc. 500 ppm G-IU K.ilihi ts 2500 -EC-- 6- 18 0 6-18 no no yes ye* no yes yes no -- -- -- -- LU 4 9 14 -- -- -- " dec. -- dec. dec * *- due, -- 1/10 due. -- "" -- -- -- -- -- "" *- " -- -- " - inc* ... l/s 7/16 2/14 -- -- -- "" "~ "" *" -- - -- ---- f R&S 022854 *-- - no change; dec. - decreaso; Inc. increase, '`in la ted ureter. -iV-s;; IS'! . TrrnT-^rTrrr ysr..-: ^ 1'*. -