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The Effects of Maternal!/ inhaled Vinyl Chloride on Embryonal and Fetal Dcvdooment in Mice, Rats, and Rabbits J. A John, F. A. Smith, B. K. J. Leong, and B. A. Schweiz Toxicology Research Laboratory, Health and Environmental Research, Dow Chemical U.S.A, Midland, Michigan 4S640 Reprinted from Tovicoloovan^ Applied PhahmacOlooy, Vol. 39, No. 3, March 1977 All Rights Reserved by Academic Press, New York and London Printed (n England ASI 00022829 \h > i s.a so i'!;.!} n ruA'tMA( (: 'x ,y 39. 497-5 i 3 (1977) The Effects of Maternally Inhaled Vinyl Chloride on Embryonal and Fetal Development in Mice, Rats, and Rabbits'-12 J. A Jon\. F. A. Smith. R. K. J. Itonc;.j \no B. A. Schwltz To.\ icnlnpv Rmctircl; 1 a!\:riitory. Health attd rnrira/ntic/ital Research, Dow Chemical U.S.4. Midland. \hchipan. -1S640 Rc< cired June 17, 1976; accepted September 20, 1976 The Fll'ccls of Maternally Inhaled Vinyl Chloride on Embryonal and Feta! Development in Mice, Rats, and Rabbits. John, J. A,, Smith, F. A., Lro\G, R. K. J., \nd Scttvvui/, B. A. (1977). Toxicol. Appl. Pharmacol. 39, 497-513. These studies evaluated the effects of inhaled vinyl chloride on mouse, rat. and rabbit embryonal and fetal development. Groups of preg nant CF-1 mice. Sprague-Davley rats and New Zealand white rabbits were exposed to 500 ppm of vinyl chloride 7 hr daily during the period of major organogenesis. Subsequently, other groups of mice were similarly exposed to 50 ppm of vinyl chloride and rats and rabbits were exposed to 2500 ppm of vinyl chloride. VVhilc_irmt.einal toxicity was observed, vinyl chloride alone did not cause significant embryonal or fetal toxicity and was not tcratogenie jn any_orj'_hTspectes aTTiTcImmiixaironST^icd. 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 cmbryotoxicity. Vinyl chloride is widely used in the preparation of polyvinyl chloride resin, as a co polymer 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 Mastromattco el 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 Torkelson el a!. (1961). Repeated exposure for 6 months to 200 ppm resulted in histologic changes in the ccntrilobular area of the livers of rabbits, but not in rats, guinea pigs, or dogs. In a study reported by Viola ct al. (1971), rats were exposed to 30.000 ppm of vinyl chloride vapor for 12 months. Findings on 1 The majority of this study was supported by the companies sponsoring research on vinyl chloride and was administered by The Manufacturing Chemists Association, - This work was reported, in part, at the 14th Annual Meeting of the Society of Toxicology, March 10, 1975, Williamsburg, Virginia. Present address- International Research and Development Corporation, Mattawan, Michigan. Com ris:ht o 1977 by AlmJoiiiil ITl-ss, lnc. All righis ol'icproducMun in .in;. Iti'iii reserved. Pi in led m (Jreof Hntom 497 ISSN 004I-008X AS 1 00022830 JOHN r.T AJ.. these rr.t^ were icponed to include severe chronic hepatitic, interstitial pneumonia, as v.eii as tumors of the >kin. limes, and hones. Maitoni and Lefemine (1974) reported tire onomenic e!feels of repeated exposure to inhaled vinyl chloride in rats and mice. Angiosarcomas. /ymbiil eland carcinomas, and nephroblastomas developed in rats exposed to concenmations of \iryi ehionde ranging from 50-10.000 ppm. 4 hr/dav. 5 da\s week for 12 months and subsequently maintained and observed until death. Pulmonmy adenomas, mammary carcinomas, and liver angiosarcomas were observed among mice exposal to the same range of concentrations for 7 months. Similarly, )\ ephr.ee" <7 ai. t i 975) reported neoplasms in mice and the tentative diagnosis of tumors m hamster- and mis exposed to 50. 200, or 2500 ppm of vinyl chloride. Tiie carcinogenic poten'ial of its ha led \ iny I chloride has also been studied by exposing annual- in :Hrm ; Maitoni. 1975). but observations to determine effects which are more in late with a da-sica! teratologic study were not made. Reports on the embryotoxic potential of vinyl chloride m laboratory animals have not been found in the literature. Thu-, 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 path.way for vinyl chloride is blocked by ethanol (Hefner et ul,, 1975), it 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 chloride-exposed animals were given 15 % ethanol in their drinking water during the days of exposure to vinyl chloride. The teratogenic potential of 1 5 % ethanol in the drinking water in mice, rats, and rabbits was previously studied in this laboratory and is summarized in Table 11 of this report (unpublished data. The Dow Chemical Co,). METHODS Animals and test material. Female CF-1 mice4 weighing 25 to 30 g, Spraguc-Dawley rats5 weighing approximately 250 g. and New Zealand white rabbits6 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 to be Day Oof pregnancy for mice and rats, respectively. The day of mating was considered to be Day 0 for rabbits. Between daily exposures, animals were housed in wire-bottom cages in a room con trolled for temperature, humidity, and light cycle. Commercial laboratory animal food7 and water were available. Food consumption was measured at 3-day intervals for mice and rats and at 2-day intervals for rabbits.8 Exposure of bred animals was conducted in stainless steel chambers of 3.7 m3 volume under dynamic airflow conditions. The atmosphere of vinyl chloride was generated by 4 Mice were obtained from Carworth, Portage. Michigan. ? Rats were obtained from Spartan Research Animals, Inc., Haslett, Michigan. 6 Rabbits were obtained from Langshaws Rabbitry, Augusta, Michigan. 5 Ralston Purina Co., St. Louis, Missouri. 8 Ethanol consumption was measured during a separate study in which animals were given only 1592 ethanol in the drinking water; these results will be reported separately (unpublished data, The Dow Chemical Company). Asr 0022831 INHAI.l D \!NYL CHLORIDE LMRRYOTOXICITY 499 diluting gaseous vinyl chloride with filtered room air at a rate calculated to give the dedred concentration. Samples of inhibited vinyl chloride monomer (chlorocthylenc)9 were used for the exposures. The actual concentration was measured with an infrared spectrophotometer (Pcrkin-Elmcr I2A or Miran I) with a multipath gas cell. Experimental design. In the initial experiment, groups of 30-40 bred mice, 20-35 bred rats, and 15-20 bred rabbits were exposed to 500 ppm of vinyl chloride for 7 hr daily on Days 6-15 (mice and rats) or 6-18 (rabbits) of gestation. Subsequently, additional groups of mice were exposed to 50 ppm of vinyl chloride. Additional groups of rats and rabbits were exposed to 2500 ppm of vinyl chloride. As summarized in TABLE 1 Teratologic Studies with Vinyl Chloride11 Vinyl chloride (ppm) Ethanol (%) M ice Rats Rabbits Mice, rats , and rabbits 500 500 50 50 2500 2500 500 2500 2500 500 0 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 hr 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-- J 5 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. Tabic 1. some of the animals which were exposed to vinyl chloride were also given 15% ethanol in their drinking water on Days 6-15 (mice and rats) or Days 6-18 (rabbits) of gestation. Maternal and fetal observations. All animals were observed daily throughout preg nancy and 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 Days 18 and 21 of gestation, respectively. Pregnant rabbits were sacrificed on Day 29 of gestation. The uterine horns were ex teriorized through a midline incision in the abdominal wall and the number and position of live. dead, and resorbed fetuses were noted. After being weighed, measured (crownrump length), and sexed (mice and rats), the fetuses were examined for external a,no- ' Vinyl chloride was obtained from IVfathcson Gas Products, Joliet, Illinois. 500 JOHN' ET AL. malic-.. One-third of each litter wax 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. Ail fetuses were then eviscerated, pre served in alcohol, and subsequently cleared and stained with alizarin red-S (Dawson. 1926) for examination of skeletal anomalies. Statistical evaluation. Tiie Fisher exact probability lest (Siegel, 1956) was used to evaluate the incidence of resorptions among liners. Maternal and fetal body weights and body measurements and maternal liver weights were analyzed statistically by an analysis of variance and the Dunnett test (Steel and Torric. 1960). The incidence of fetal ano malies was analyzed by the Wilcoxon test as modified by Hascman and Hoel (1974). Conti oh. 1 he group of animals which was exposed only to vinyl chloride served as the conttol for those animals which were exposed to vinyl chloride in combination with !5"J ethanol in the drinking water. The controls for animals exposed to vinyl chloride alone were exposed concurrently to filtered room air. RESULTS Maternal toxicity. Among mice exposed to 500 ppm of vinyl chloride by inhalation, there was a deciease m maternal weight gain and food consumption during gestation and in the absolute liver weight at the time of cesarean section compared to control values (Table 2). These effects were not observed among mice exposed to 50 ppm of vinvl chloride. Mice exposed to a combination of 500 ppm of vinyl chloride by in halation and 15 % ethanol in their drinking water also showed a decrease in weight gain during gestation and in liver weight (absolute and relative) on Day 18 of gestation. Ethanol in combination with 50 ppm of vinyl chloride also resulted in a decrease in maternal weight gain during gestation and a decrease in the 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. Except for an apparent decrease in maternal weight gain in rats (Table 3) and a de crease in food consumption for rabbits (Table 4), no signs of toxicity were observed in the adult rats or rabbits during exposure to 500 ppm of vinyl chloride. The apparent decrease in maternal weight gain among rats exposed to 500 ppm is most likely due to the lower body weight of the control animals on Day 6 and, subsequently, a higher weight gain of these animals during the later days of gestation. Among rats exposed to 2500 ppm, both the absolute and relative liver weights were significantly increased on Day 21 of gestation, but maternal weight gain was no difletcni 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 significantly increased among rats exposed to 2500 ppm of vinyl chloride and 15%ethanol. Maternal weight gain for this group of rats was significantly decreased during gestation. Food consumption during the exposure period was further decreased among these rats which were exposed to vinyl chloride in combination with ethanol in the drinking water. No effect on maternal weight gain, liver weight, or food consumption was observed among rabbits exposed to 2500 ppm of vinyl chloride by inhalation. A decrease in maternal weight gain was observed among rabbits during exposure to 2500 ppm of vinyl chloride in combination with !5% ethanol (Days 6-18 of gestation), but total AS I 00022833 V IN Y I. CUf.ORID!: l-.MHRVOTOXICn V 4 ABLL 2 Maifrnal Weigh r Gain, Livir Weights, and Loon Consume! ion or Mich Exposed to Vinyl Ciii.okihi. ns Imiai amov* Low concenlralion'' 0 50 00 50 15 High concent] alion'1 0 500 ;00 0 0 15 Number of dams Body weight on gestation Day 6`` Weight gain during gestation Days 6-18' Liver weight on gestation Day 18` Absolute Relative7^ Food consumption during gestation Days 6-15r 21 20 2 16 4 2.75 0.26 59.5 + 8.7 6 1 20 31 5; 2 17 6 2.76 0.40 57.8 4.5 6 1 16 3! 2 11 1. : T 2.37 0.52`` 56.6 7.3 4 3; 2J 26 29 -\- 2 20 l 3 2.75 0.31 55.5 5.5 6 i 19 29 3 17 p 41' 2.49 0.29*' 54.4 4.2 5 L 50 _ 3 10 - 1'' 1.78 t; 0. 45.8 J. 5. 3 -1; L " Mice were exposed to vinyl chloride or filtered room air by inhalation 7 hr 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. k Top row, vinyl chloride in drinking water (ppm); bottom row, percentage ethanol in drinking water. r Grams, mean + SL>. d Significantly different from vinyl chloride alone by an analysis of variance, /> < 0.05. c Significantly different from control by an analysts of variance, /> < 0.05. / Milligrams of liver per gram of body weight. 0>3 tX3 tss CO CO fe. Oi o , .1-1 NHOf jo 1'ABLH i Maternal Writ,hi Gain, Livi n Whkjii is, and Food ConsL'MpikjN or Rais 1 2<r< >sj D IT) VlNYl . Cnt.niRIOT BY lM!At \ I ION'' Number of dams Body weight on gestation Day 6r Weight gain during gestation Days 6-21r Liver weight on gestation Day 21*' Absolute Relati\e/ Food consumption during gestation Days 6-15*' Low concentrat ion'" 0 5*K) 00 28 258 26 148 11 14.81 1.79 36.5 4.1 21+2 31 27 7;!; 20'' 125 + I9'1 15.00 + 1.(4 37.1 v 2 6 22 -r 2 0 0 i19 288 f; 27 127 15 14.27 1.38 54.4 f 3.3 22 A 2 1 ligh coneenlrttron'1 ~y 500 0 2500 15 16 274 1- 19 138 1 23 15.55 + I.2.F 37.8 f- 2.6-' 21 16 272 r 14 120 1- 15' I16.52 i IA0 42.1 r 2 4*' i 3 2` " Rats were exposed to vinyl chloride or filtered room air by inhalation 7 hr daily on Days 6-15 of gestation. Some of the rats were given ethanol m their drinking water (15%, v/v) on Days 6 -15 of gestation. h Top row, vinyl chloride in air (ppm); bottom row, percentage ethanol in drinking water. c Grams, mean SD. d Significantly different from control by an analysis of variance, p < 0.05. v Significantly different from vinyl chloride alone by an analysis of variance, p < 0.05. f Milligrams of liver per gram of body weight. AS I 0 0 0 2 2 8 3 5 Cill.ORlDi. I.M1JRYOI OXIC1TY AS 1 0 0 0 2 2 8 3 6 TABLE 4 Maternal Weight Gain, I.i\i;r Weights, and Food Consume lion or Rahhiis Exeom-.d io Vinyl. Cm oridi-: uy Inil-m.Aitov' Low concentration'' 0 500 00 High concentration'' 0 2500 2500 0 0 \5 Number of dams Body weight on gestation Day 6C Weight gain during gestation Days 6 -291' Liver weight on geslation Day 29c Absolute Relative1' Food consumption during geslation Days 6-18 (g) 18 3.85 0.23 0.05 0.19 96 19 24.6 3.6 98 30 20 3.82 0.25 0.01 + 0.19 89 -l- 14 23.2 + 2.9 76 2W II 4.08 -t- 0.21 0.06 -t- 0.27 102 -1; 16 24.7 -t- 2.7 91 36 5 4.39 -1- 0.301' 0.01 -1-0.13 122 -t- 25 27.7 -i- 5 9 89 ;> 26 16 4.02 i- 0.47 -0.14 t 0.42 1 16 -I- 30 30.0 T 6 3 15 9r Rabbits were exposed to vinyl chloride or filtered room ;tir by inhalation 7 hr daily on Days 6 -IS of gestation. Some oi'the rabbits were gisen ethanol in their drinking water (15%, v/v) on Days (5-18 of gestation. h Top row, vinyl chloride in air (ppm); bottom row, percentage ethanol in drinking water. c Kilograms, mean SD. J Significantly different from control by an analysis of variance, p < 0.05. t' Grams oflivcr per kilogram of body weight. r Significantly different from vinyl chloride alone by an analysis of variance, p < 0.05. 'o-/I jon\' ;:r al. weight gum wa.-, not different from that among rabbits exposed to 2500 ppm of vinyl chloride ulune (Table 4). hoed consumption was .significantly decreased among the rabbits expo.-ed to 2500 ppm phis elhano1. ()hs*.: i nunic a; ihe time of u'sorcan section. Observations made at the time of cesarean section of mice. rats, and rabbits are presented in Tables 5 7. Maternal deaths \.e;e observed among mice exposed to 500 ppm of vinyl chloride alone and in com bination with ethanol (Tabic 5), There was an increase in the incidence of resorptions and the fetal body weights were lower them m controls for the 500-ppm vinyl chloride group. Litter si/e was also reduced. These effects were augmented among mice exposed to 500 ppm of vim, 1 chloride in combination with 15 % ethanol. Two litters in this group were totally resorbed. In addition, the number of implantation sites per dam, fetal crown-rump length, and percentage pregnancy were significantly lower in the ethanol group as compaied to mice receiving 500 ppm of sins 1 chloride alone. Among mice exposed to 50 ppm. the fetal crown rump length was significantly greater than among controls; a decrease, however, in fetal body weight and crown-rump length was observed in the 50-ppm vinyl chloride plus ethanol group. The incidence of resorptions was not significant!} greater among mice treated with 50 ppm of vinyl chloride in combination with ethanol, although two litters were totally resorbed. One maternal death was observed among rats exposed to 2500 ppm of vinyl chloride bv 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 than among the control rats. Percentage pregnacy was unaffected among rats exposed to vinyl chloride alone or in combination with 15% ethanol in the drinking water, Petal body weight and crown rump length were significantly reduced among rats exposed to 2500 ppm of vinyl chloride in combination with ethanol. A significant reduction in feta! body weight was also observed among rats exposed to 500 ppm of v inv 1 chloride alone, but not among those exposed to 2500 ppm. Significant decreases in the number of corpora lutea per dam w ere observed among rats exposed to 500 ppm of vinyl chloride alone and among those exposed to 2500 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 2500 ppm of vinyl chloride. Since the number of corpora lutea is established prior to Day 6 of gestation, these differences are not con sidered to be 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 (2500 ppm) plus ethanol group, in which seven litters were totally resorbed (Table 7). Exposure of rabbits to cither 500 or 2500 ppm of 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 of vinyl chloride alone, but not among those exposed to 2500 ppm of vinyl chloride alone or in combination with 15% ethanol in the drinking water. Since the decrease in litter size was associated with a decrease in the number of corpora lutea which is established prior to Day 6 of gestation, this effect is probably not due to exposure to vinyl chloride. Pregnancy wastage in rabbits was unaffected by exposure to vinyl chloride. No differences in fetal body weight or crown- rump length were observed in any of the exposed groups of rabbits. AS I 00022837 l Aiii.i: ? Ousi-.kv.vi ions Madi. at i m; Timv: or Ci sari/an Su i m\ ni Mu i. l-xnnxn) mliwi ( momni uv Inhuamu INHAU'.D V IN V I. Cm.ORIDl- t-.MURVOl'ONICIT'i AS I 0 0 0 2 2 8 3 8 Number of litters implantation sites,'dam'' Live fetuses.litter6 Implantations resorhed ("(,) Litters with resorptions ( Litters totally resorbed Resorptions/1 iltcrs with resorptions Sex ratio, M : L Fetal body weight (gV Fetal crown-rump length (irun)' Maternal deaths/trealed dams (%) Percentage pregnancy Low concentration 0 50 50 0 0 15 21 |a a 10 -1; 4 15 (40/261) 67 (14/21) 1 2.9 (40/14) 50:50 ! .00 0.11 23.0 1.9 0 (0/37) 57 (21/37) 20 12 h 4 ' 11 4 8 (IH.-238) 55 (1 1 -20) 0 1.6 (18 If) 50; 50 1.02 0.10 24.2 0.S,f 0(0.'27) 74 (20/27) f6 11 :4 10 + 4 11 (19472) 69 (11/16) ) i.7 do, m 48:52 0.84 0.14' 22 4 1.5- 0 (0;28) 57 (16/28) i Left concent! at inn 0 500 500 0 0 15 26 14 L 2 12 V 2 7(26/351) 58 (15-261 0 1.7 (26/15) 54:46 1.07 ): 0.06 "il 7 i j a 0(O'30) 88 (28'32) 19 i 3 .t; 2 1 1 J- 2J 13 (33 '248 r 79 (15.19) 0 2.2(33 15) 52:48 0.99 ;h 0 IP' 23.6 r 1.0 17 (5/291 72 (21 29) 10 l 6` 8 i; 6f 19 (13 69) 86 (6/7 > "> 2 2 113 6) 64:36 0.78 + O.I5r 21 2 J; 1,5` 13 (4/30) 31 (9,29V' u Mice were exposed to vinyl chloride orfillci ctf room air by inhalation 7 hr daily on Days 6 15 of gestation. Smite of the mice were given ethanol in their tit inking water (15'(. v/v) on Days 6 - 15 o! gestation. how and high concent rations; lop low, \ inv! chlmidc ;.-t air (ppm): hot (out nut, percent.me ethanol in drinking water. 6 Mean SD, c Significantly dilleienl from vinyl chloi ide alone by an analysis of vat iance, p / 0.05. J Significantly different from control by an analysis of variance, i> < 0.05. c Significantly diilerent front control by the l-'isher exact probability test, p < 0.05 f Mean of litters + SD. " [i;|scd on the presence of fetuses and/or resorption sites observed by gross examination at the time of ( es.irean Action. Significantly different front vinyl chloride alone by the lisher exact probability test, p < 0.05. 1 Si 0o4 TA15LK 6 Ohm rvmkins Maui, ai iih, (isirort i wktan Si chik m R a i s Lxi'o.si d io Vim i (`mi okiim: hv Inhai \iiuv1 Number of liners Corpora lulea. dam'1 Implantatior sites/dam" Pregnancy wastage"-1' Live fcluses/liuer" Implantations rcsorbeti {%) Litters with resorptions (%) Litters totally resorbed Resorptions/litters with resorptions Sex ratio, M:F Fctal body weight (g)r Fetal crown-rump length (mm)' Maternal deaths/treated dams Percentage pregnancy" Low concent ration 0 500 00 28 15 d 12 2 -1 -b 2 12 2 1 (4/342) 14 (4/28) 0 1.0 (4/4) 52; 48 5.67 0.29 42.6 1.2 0(0/29) 96 (28/29) 51 13 X 2` 13 j 2 0.4 f; 11 1 2 _=; 2 3 (11/398) 29 (9/31) 0 1.2 (11,9) 50; 50 5.44 0.38*' 43.6 0.8*' 0(0/33) 94 (31/33) 0 0 19 14 4; 2 12 x 2 11! 12 J; 2 4 (9,'238) 32 (6/19) 0 1.5 (9.6) 49; 51 5.59 h 0.27 43.6 1.5 0(0/10) 95 (19,20) I lig.h eoncenti ation 2500 0 L. 15-1 2' 14 ! 2 2! i 15 a 2 5 (6/220) 25(4 16) 0 1.5 (6/4) 53:47 5.62 0.29 43.3 r 1.1 6(1/17) 100(17/17) 2500 15 16 14 2"' 1 2 -1; 2 1. s ]n ! a 4 ( / i 95 i 25(4 16) 0 1 8 17 4) 51 ; 49 5.34 -j: 0 52' 42.4 0.9J 0(0/17) 94 (16; 1 7) Rats wei c exposed to vinyl chloride or filtered room air by inhalation 7 hr daily on Days 6-15 of gestation. Some of the rats wcjc given ethanol in thetr drinking water (15 %, v/v) on Hays 6-15 of gestation. Low ami high concentrations: top row, v.nyl chloride in -air (ppm); bottom row, peieenbuie ethanol in drinking water. 6 Mean SIX ` Significantly different front control by an analysis of variance, p < 0.05. * Significantly different from vinyl chloride alone by an analysis of vat iance, /> : U.t)5. r The number of corpora In lea minus the number of implants. / Mean of litters + SIX 9 Based on the presence of fetuses and/or resorption sites observed by gross examination at the time of Cesarean section. ASI 00022839 TAHLL 7 Ohservai ions Made at hie Timi: oi Ci sarlan Sici ion oe Rmshiis Lxpmm-d 10 Vjnyi ('ttitminr hy Imiai .uiun" Number of litters Corpora lutca/dam1' Implantation sitcs/danC Pregnancy wastage''*1' Live let uses,diner6 implantations rcsorbed (%) Litters with resorptions (%) Litters totally rcsorbed Rcsorptions/litters with resorptions Sex ratio, M: F Fetal body weight (g)J Fetal crown-rump length (mm)7 Maternal deaths/treated dams (%) Percentage pregnancy" Low concentration 0 500 00 18 9 1 9 1 0,4 1 8 i: 1 6(10,162) 44 (8/18) 0 1.2(10/8) 53:47 35.23 4.82 91.0 4.2 0(0/18} 100(18/18) 19 8 L 1" 8 11 1 J: 1 7 <: 2" 9 (14/150) 32 (6/19) I 2.3 (14/6) 50:50 34.13 4.17 92.6 5.0 0 (0/20) 95 (19/20) 0 0 1! It! f 2 82 2 i 6 j. 3 22(19 88) 64(7/1 1} 2 2.7 (19,7) 61 :39 36.46 4.82 92.6 3: 4.7 0(0/11) 100 (1 l/l 1) Nigh concent! at ion 25t)t) 0 5 10 i- 7 84 "> t 6 v4 24 (It) 42) 80(4 5) 1 2.5 (If), f) 50:50 33.77 4,48 87.1 :-5.2 14(1/7) 86 (6/7) 2500 15 16 10 2 9 i: 2 1 h1 1 i -t 53(79 149L 88(14 16) 7 5.6 (79* 14) 43:57 32.48 5.88 87.7 -|: 6.3 16 (3/19) 95 (18 19) " Rabbits were exposed to vinyl ehloride or filtered room air by inhalation 7 hr daily on Days 6 IS of gestation. Some of (he rabbits were given ethanol in their drinking water(15%, v/v)on Days 6-IS of gestation. Low and high concent rat ions: top row, vinyl chloride in airtppm); bottom row, percentage ethanol in drinking water. b Mean SD. ` Significantly different from control by an analysis of variance, /> < 0.05. 11 The number of corpora lutea minus the number of implants. *' Significantly different from vinyt chloride atone by the Fisher exact probability test, p < 0.05. s Mean of litters SD 9 Based on tile presence of fetuses and/or resorption sites observed by gross examination at the time ofccsaiean section. ASI 00022840 508 JOHX EV AL. Incidence of anomalies. The incidence of anomalies among litters of mice, rats, and rabbits exposed to vinyl chloride by inhalation is indicated in Tables S. 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 litte; s of mice exposed to 500 ppm of viny 1 chloride in combination TABLE 8 KciDixn: or Asomu.ifs vmong Littprs or Mice Exposfd ro Vinyl Chloride uy Inhalation0 ------------- Gross anomalies Soft tissue .inomalicx Skeletal anomalies Bones of the skull'' Groxx anomalies LxenccphaK Anopthalmia Hct; palate Sofi tissue anomalies Small tinmus Skeletal anomalies Sternebrac Unfuscd Delayed ossification No. 5 sternebra missing Ribs Fslra Spurs Vertebrae Forked atlas Missing cers ical centra Delayed ossification of ccr\ ical arches Skull Delayed ossification Unfused occipital Lev. concentration Hi gh concentration 0 i) 22! CO) '4 120) 221 CO) 147 (20) a 0 ! (101 50 0 - - -- ' 50 0 15 0 -- 500 0 ---- -------- [Number examined, fetuses (litters)] 220 (20) 153(14) 325 (26) 215 (19) 75 CO) 50 (14) 107 (26) 73 (19) 220 120) 153 (14) 325 (26) 215(19) 145 ft1)) 103 (14) 217 (26) 142 (19) [reiuxex .ilFoctod (" ,,) (litters atfecicd, ",)] 00 1 (8) ] (10) 00 00 1 (10) 2 Cl) 0 1 (5) 500 15 56 (5) 19 (5) 56 (5) 37 (5) 2 (20) 2(20) 4 (40) 0 0 4 (7) 0 00 3 (20) 7 (50) 0 4 (30) 4 (35) 0.4 (5) 0 0 9 (35) 0 3 (25) 4 (35) 0 5 (30) 5 140) I (10) 0 0 8 (37) 0.7(5) 13 (57)`` 44 (100)' 3 (21) 0.6 (7) 2 (21) 4 (36) 0 1 (14) 40 (lOOf 24 (50)*' 2 (19) I (12) 0 3 (31) 4 (31) 0 0 0 13(54) 1 (12) 9 (42)" 6 (42)' 1(10) 3 (32) 3(21) 0 1 (10) 0 30 (58)-1 5(21) 34 (80)' 43 (100)' 7 (40)r 14 (60)1' 14(80) 4(20) 38(60f 5 (40)' 70 (100)c 11 (20) Mice wc/c t'\pcd in v/jn I chloride or filtered room for by in lull inn 7 hr djilv on d:i>.s of gesution. Some of the mice were given ethanol in their drinking waiter (15 v 'v) on day s (>-15 of' gestation. Low and high concentrations: top row. s;iw \ chloride :r ,j,> < ppm', bottom row percentage ethao'*' m cb tr.hmt* wate- ^ 1 11111 i 1 *1 i m m, i.o 'i. t , i' j,n u\j, c, . b ni'i d "i; m /, j "j pj-noc. /.p;' trends o'. \he tout popu- ... \ . a. ii'.j'-o ' it i e.tpi mder oi t Me fet uses are decapitated during the soft tissue examination. Among Inlets of mice w hich were exposed to 50 ppm of ' in> 1 chloride, one litter consisted of only one fetus, which was decapitated* during the soft tissue examination ' Significantly dnierent from v myl chloride aloite b> the modified Wilcoxon test, p < 0,05. a Sigmficantis thherem from control b> the modilied Wilcoxon test, p - 0.05. " p 0,057, modified Wilcoxon test. v\ ith 15 % ethanol exhibited anopthalmia. The incidence of cleft palate in this group was slightly higher than that observed among mice receiving vinyl chloride alone, No soft tissue anomalies occurred at an incidence significantly greater than control in mice. Two fetuses from one litter among mice exposed to 50 ppm of vinyl chloride plus ethanol exhibited a small thymus. No skeletal anomalies were observed at an incidence significantly greater than in controls among mice exposed to 50 ppm of vinyl chloride. Significant increases in the incidence of delayed ossification of sternebrac AS I 00022841 !Mr\!!D \!\Yk rilLOUini. I X'BRYOTOYlCn V 509 ' \o. 5) and bones of'the skull Acre observed among liners of mice exposed to 500 ppm of vinyl chloride. The incidence ofun.fi.iscd centers of ossification of sternebrae was also significantly higher in this group, Among mice exposed to vinyl chloride in combi- TABLE 9 Incidence oi Anomaiiis xmoxc; Litikks of Rats Exposed to Vinyl Chloride bx' Inhalation" Low concentration Higlt concentration --- --- ----- ---------- Gross anomalies Soft lissue anomalies Skeletal anomalies'' Bones of the skull' Gross anomalies Omphalocele Soft tissue anomalies Micropthalmia Dilated meter (unilateral or bilateral) Small kidney Skeletal anomalies Slerncbrae Unfused Ribs Spurs Vertebrae Missing cmieul centra Skull Delayed ossification Unfused . ---- 0 500 0 0 00 ------................. ..... .......... 2500 0 ... . ... - 2500 15 .. ^ [Number examined, fetuses (litters)] 359 C.8) 5S7C51) 229 (i9) 214(16) 188 (16) 113 (28) 129(31) 76 (19) 73(16) 63 (16) 537(28) 387 (31) 229 (19) 214(16) 188 (16) 225 (28) 259 (31) 153 (19) 141 (16) 125(16) [Fetuses affected ( %,) (litters affected, ",))] 0 ! (3) 0 4 (5) 0 0.5 (6) 0 0 0 0 2 (6) 2 (7) 0 2 (6) 0 5 GO) 0 27 (50% 0 5 (19)" 2(6) o 1 (4) 0.3 (4) 16 (61) 0 1(6) 9 (52)J 2(16) 12(61) 0 3 (32) 14 (68) 7 (53) IS (58) 53(90) 0.5 ((,)" 12(69) 4 (50) 6 (31)J 3 (12)J 1 (12) 35 (69)" 21 (Sir 3 (25) 2(12) " Rats were exposed to vinyl chloride or filtered room air by inhalation 7 hr 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. Low and high Concentrations: top row, vinyl chloride in air (ppm); bottom row, percentage ethanol in drinking water. `Among litters of the low concentration control rats, two fetuses were misplaced and were not available for examination for skeletal anomalies. " Calculations of the ir dencc of anomalies of boner of the skull arc based on approximately twothirds of the total population of fetuses. The remainder of the fetuses are decapitated during the soft tissue examination. * Significantly different from control by the modified Wilcoxon test, p < 0.05. r Significantly different from vinyl chloride alone by the modified Wilcoxon test, p < 0.05. nation with 15% ethanol, several skeletal anomalies occurred at an incidence signi ficantly greater than among mice exposed to vinyl chloride alone. Among litters of mice exposed to 50 ppm of vinyl chloride plus ethanol, increases in the incidence of delayed ossification of bones of the skull and stcrnebrac (Nos. 4-6) were observed upon skeletal examination. The incidences of unfused occipital, unfused sternebrac (Nos. 5 and 6), 510 JOHN HP AL. and forked ;nkis were also significantly increased in the 50-ppm plus ethanol grout). Among inters of mice exposed to 500 ppm plus ethanol, increases in the incidence of delayed ossification, of nones of the skull, sternebrae (Nos. 2 6). and arches of the cervical xeNebrae wcie observed. Sbmificunt increases in the incidence of lumbar `purs, mosinsi cent;:: of the cerMca! wtehiae, tmfuscc! sternebrae. and missing fifth s'enicbra.e were observed ;n tin's group a.s well. The incidence of extra ribs was slight!}', b:u not significantly, increased compared to One controls. in rats, no gross anomalies occurred at an incidence significantly greater titan among coruroi animals tTnNe 9) Among inters of rats exposed to 2500 ppm. the incidence of EADLL 10 i.NCii'r'so; m- Anomm.ils vwcwg l.nrras of Rabbits Exposed to Vinyl Chloride by Inh-vla nos" bow' concentration'' 0 500 00 High concentration''' 0 2500 2500 0 00 Gross anomalies Soft tissue anomalies Skeletal anomalies Gross anomalies Cleft palate Soft tissue anomalies Dilated renal pch is Dilated cerebral ventricle Enlarged right atrium of heart Skeletal anomalies Sternebrae Delayed ossification No. 5 [Number examined, fetuses (litters)] 152 (IS) ! 56 (18) 69 (9) 32 (4) 70 (9) 50 (IS) 47(18) 24 (9) 10(4) 25 (9) 152(IS) 136 (18) 69 (9) 32 (4) 70 (9) [Fetuses affected (%) (litters affected, %)] 0 0 0 0 1 (11) 0 0 0 0 8 (11) 0 0 0 10(25) 0 0 0 0 0 8(1!) 28 (77) 38 (94)'' 20 (44) 16(75) 24 (67) " Rabbits were exposed to \ myl chloride or filtered room air by inhalation 7 hr daily on Days 6-18 of gestation. Some of the rabbits were given ethanol in their drinking water (15%, v/v) on Days 6 -vt 8 t. f gestation, h Top row, vinyl chloride in air (ppm); bottom row, percentage ethanol in drinking water. `` Significantly different fmm contiol by the modified Wilcoxon test, p < 0.05. dilated ureter was significantly higher than among control litters. The incidence of dilated ureter was significantly lower, however, among litters of rats exposed to 2500 ppm of vinyl chloride in combination with 15% ethanol. Among litters of rats exposed to vinyl chloride, on!}' minor skeletal variations were observed at an incidence higher than that of controls, The incidence of lumbar spurs was increased among litters exposed to 500 ppm. No increases in the occurrence of skeletal anomalies were observed among litters of rats exposed to 2500 ppm. 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 centra of the cervical vertebrae were, however, significantly increased among AS I 00022843 IMIM.IU) MNVI CHLORIC)!'- LMBRSOIOXICITY 511 litters of rats exposed to 25(10 ppm of vinyl chloride m combination with 15 % ethanol in ihe drinkmc water. In rabbits, no gross anomalies were observed at an incidence greater than among control litters, although a cleft palate was observed in one fetus among litters of rabbits exposed to 2500 ppm of vmv I chloride plus ethanol (Table 10). Dilated renal pelvis was also observed in two fetuses from one litter among rabbits exposed to 2500 ppm plus ethanol. Two fetuses from this group also exhibited an enlarged right atrium of the heart. Among litters of rabbits exposed to 500 ppm, the incidence of delayed ossification of the fifth sternebra was significantly higher than that of control litters. Delayed ossification of sternebrae did not occur at a significantly higher incidence than control among litters of'rabbits exposed to 2500 ppm of vinyl chloride alone or in combination with 15% ethanol in the drinking water. DISCUSSION The results of these studies indcatc 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 1 1, Less maternal toxicity was observed among rats and rabbits than among mice during exposure or at the time of cesarean section. Among rats, one maternal death and an increase in liver weight were observed at 2500 ppm and a decrease in maternal weight gain was observed at 500 ppm. Among rabbits, one maternal death was observed at 2500 ppm and there was a decrease in food con sumption at 500 ppm. In comparison, 500 ppm was quite toxic to pregnant mice, as evidenced by the significantly decreased weight gain and food consumption and by the occurrence of a number of maternal deaths. Exposure to viny! chloride alone was not consistently embryotoxic in the three species studied. Among mice, at 500 ppm, the incidence of resorptions was significantly higher (13%) than among the concurrent controls (7%). Since the incidence of re sorptions among the mice which served as controls for the 50-ppm group was 15% and the incidence among groups of control mice from recent studies in our laboratory was 10% (193/1895), the apparent increase at 500 ppm was probably due to the unusually low incidence of resorptions among their concurrent control group. Resorptions among exposed rats and rabbits occurred at a frequency comparable to controls. Some de creases in fetal body weight and crown-rump length were observed in rats and mice, but not in rabbits. A teratogenic response to maternally inhaled vinyl chloride was not observed in mice, rats, or rabbits. With the exception of unilateral and bilateral dilated ureter among litters of rats exposed to 2500 ppm of vinyl chloride, no externa! 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 varia tions; no major skeletal malformations were found at an incidence significantly greater than in the control groups. Ingestion of 15 % ethanol in the drinking water enhanced some of the effects of in haled vinyl chloride. In each of the three species tested, maternal weight gain and food consumption were lower than among animals exposed to vinyl chloride alone. The percentage resorptions was slightly but not significantly increased among mice exposed ASI 22844 TABLE- I I Summary- -Vinyi Cm uitim-: Ti-:kat<jy<x.!c S i uoirs "iv r j Mtur 50 0 15 Mice'1 500 0 15 Ral 0 500 2500 15 0 0 15 0 500 15 0 Rabbits'* 2500 0 15 0 15 Gestation days of treatment Maternal deaths Percentage pregnancy Number of litters examined Maternal weight gain Maternal food consumption Maternal liver weight Absolute Relative Implantation siles/dam Percentage resorptions Litters totally rcsorbed Litter size Fetal body weight Fetal crown-rump length External anomalies Visceral anomalies Skeletal anomalies 6 -15 No No __h --- 20 16 -- Dec -- Dec -- -- -- -- 0/20 -- -- fnc .-- -- -- Dec --- -- 2/16 -- DecDec __ ___ Inc 6 - 15 Yes Yes -- - Dee 19 7 Dec Dec Dec- Dec Dec -- -- 1 nc` 0/19 DecDec -- ___ ___ -- Dec Dee Dec 2/7 DecDec Dec _. __ Inc 6 -! 5 No 21 Dec .- -- 0/21 _. DecDee _ __ Inc 6 15 No 31 Dee _. _- 0/31 __ Dec fnc _ _ -- 6 15 Yes No __ - 16 Dec 16 Dec Dec hic Inc __ _ 0/16 __ ,_ _ Inc ._ 0/16 _ DecDec 1 IlC1' -- 6 -15 No . 19 Dec Inc 0,19 Dec Inc 6 -18 No 19 Dec Dec 1/19 Dec __ -- 6 -18 Yes Yes 5 16 Dec 6-18 No 14 Dec Inc _ l;5 7'16 2i 14 .. _ __ _ __ _ __ . - -- " Top row, vinyl chloride in air (ppm); bottom row, percentage ethanol in drinking wafer. b --, No change; Dec, decrease; Inc, increase as compared to control values. c This apparent increase was due to a lower than normal incidence of resorptions among the control group; see Discussion for details. " Dilated ureter. AS I 0 0 0 2 2 8 4 5 i\n\im \ i\m. rm.oRmi i.mpryotoxicity 513 to \ ms 1 chloride in combination with ! 5 ethanol in the drinking water. A statistically significant increase in the percentage of resorptions was observed among rabbits exposed to s ins i chloride plus ethanol. Fein! hods1 measurements were lower among litters ol mice and rats which received ethanol and smyl chloride compared to vinyl chloride alone. The combination of ethanol and vinyl chloride did not cause a teratogenic icsponsc m the three species tested, although higher incidences of some skeletal vari ations were obsersed among litters of mice and rats. Certain malformations were observed among litters of mice, rats, and rabbits which also received ethanol, but their incidence was not statistically different than among litters of animals exposed to vinyl chloride alone. The effect of simultaneous ingestion of ethanol on the disposition of vinyl chloride m 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 rats or rabbits. Simultaneous 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. Fxposurc to vinyl chloride alone or in combination with 15% ethanol in the drinking water did not cause a significant tera togenic response in mice, rats, or rabbits. ACKNOWLEDG MFNTS The authors are grateful to Mr. K. D. Nitschke, Ms. H. D. losct, and Ms. M. F. Balmer for their assistance in all aspects of this study and to T. R. Torkelson for advice and assistance in the prepartion of this report. REFERENCES Dawson. A. B. (1926), A note on the staining of the skeleton of cleared specimens with ali/arin red-S. Slain. Tcchnnl. 1. 123-124. Hasf.man, .1. K.., and Hon.. D. G. (1974). Tables of Gehan's generalized Wilcoxon test with fixed point sensoring. J. S/at. Comp. Simul. 3, 117-135. Unwin. R- E., Jr.. Watanahi:, P. G.. and Gemrinc;, P. J. (1975). Preliminary studies of the fate of inhaled vinyl chloride monomer in rats. Aim. /V. Y. Acad. Sci. 246, 135-148. Kcplinger, M. L., Goodf, 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, Mauoni. C. (1975). The value of predictive experimental bioassays in occupational and environmental carcinogenesis. An example: vinyl chloride. Amhio 4. 18-23. Mai toni, C, and Lf.i'emine, G. (1974). Carcinogenicity bioassays of vinyl chloride. I. Research plan and early results. Enciron. Res. 7, 387-405. MASiRoMATrro, E.. Fw, A. M., Christie, H., and Danziger, D. (1960). Acute inhalation toxicity of vinyl ehuj, ide lo laboratory animals. Anwr. hid. Hyg. Assoc. J. 21. 394-397. Sit-xna.. S. (1956). Non-Paramctric Statistics for the Behaciond Sciences. McGraw-Hill, New York. Sti-xl, R. G. D., and Porrif;, H. H. (1960). Principles and Procedures of Statistics. McGrawHill, New York. Forkli son, T. R,, Oyen, F., and Rowe, V. K. (1961). The toxicity of vinyl chloride as determined bv repeated exposure of laboratory animals. Amer. hid. Iivy. Assoc. J. 22. 354361. Vioi a, P. L., Rjgotti, A., and Caputo, A. (1971). Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res. 31, 516-519. AS I 00022846