Document 93nneNOvRk0voXDkQJ6bBzQae

\J isy /Y HEALTH SAFETY AND ENVIRONMENTAL AFFAIRS T. A. LINCOLN, M.D. Corporate Medical Director P2594 (203) 794-5212 A. A. Lang R. W. Holland DATE 9/6/83 R ECEIVED SEP 6 1983 A & LANG. M.D. I noticed this letter in the August 20, 1983 Lancet. You might find the comments on latency interesting. T. A. Lincoln OLD RIDGEBURY ROAD, DANBURY, CT 06817 ucc 061026 456 THE LANCET, AUGUST 20.1983 exchange, about 10% will be acutely ill and need treatment in an intensive care area.8 The claims for plasma exchange need constant critical evaluation, perhaps through an apherests register.9 R! Ciw Blood Bank Groainccfl-DfcntRt, Graunirn, Hinterlanda l1- 0. DaS C. Th , SMIT SlBINGA ANGIOSARCOMA AS A MODEL FOR COMPARATIVE CARCINOGENESIS Sir,--By pooling data from several sources we have been able to contrast the latent periods for angiosarcoma of the liver induced by vinyl chloride monomer (VCM)and 'Thorotrast' (thorium dioxide). The average and minimal latent periods are probably much shorter for VCM cases than for thorotrast cases, although the hepatic neoplasms induced by these two agents are histologically the same.1 The thorotrast-induced angiosarcomas we analysed were those reported from West Germany,*2 I3the UK,' Denmark,4 and the United States.' Those attributed to VCM have been gathered by three ofus (J. S., R. S., and E. D.), from personal contacts in twelve countries. The earliest case-repons involving thorotrast and VCM were published in 1947 and 1974,' respectively. Exposure to thorotrast occurred primarily in the period 1928-55, while exposures to VCM were heaviest in the 1940s, 1950s, and Cumulative density function* of latent periods for hepatic angiosarcoma induced in males by VCM and thorotrast. 1960s. For neither agent have all cases yet occurred (table), nor are denominator data generally available. For these reasons the distributions in the figure are not definitive, but the first two decades of the experience are now sufficiently well defined to venture that angiosarcomas induced by VCM begin to appear much sooner after initial exposure than do cases caused by thorotrast. Medians for males are, respectively, 22 and 29 years, and we note that a recent report on thorotrast-induced cases in Japan gives a latent period of 33*5 years.* Because of the large relative risks 8, DaaPCSmitSibmgaCTh PJatma exchange programme in a regional blood bank with a reference to Guillam-BuT* syndrome. Rtttra dm Lab 198), 13: 47-74 9 Hamblin TJ, An apheretn refiner. Lower 1982; >r 550 I Popper H, Thoma* LB, Telle* NC, et al. Development ofhepatic angiosarcoma in man induced by vinyl chloride, thorotrast, and aracnic. Am J Pathol 1978; 92: 349-74. 2. van Kaick G,LorenaD, Mutb H,ct al Malignancies m German ihorotrasipatientsand estimated tissue doae. Health Phvt 1978; IS: 127-36 3 Barnet PJ, Langland* AO. Anthony PP, et al Angiosarcoma of the liver: marker tumour for the late effects of thornran m Great Britain Br J Canter 1980, 41: 446-5) 4 Faber M, Malignancies in Danish theratrau patient*. Htalth Pkvt 1978; JS: 153-58 5 FalkH,Telle*NC.IthakKG,rial Eptdemiolagyof(horrast`inducedangiourcQma in the United State*. Em Ret 1979; IS: 65-7). 6 MacMahon E, Murphy R$, Bate* Ml Endothelial-cel) sarcoma of liver following ihormrm miction*. AmJ Pathol 1947; 23: 585-611. DISTRIBUTIONS OF YEAR OF DEATH BY AGENT AND SEX Ycar-of-dcatb 1955-59 60-64 65-69 70-74 75-79 80-81 Total* Thorotrast Male 2 8 19 33 32 7 101 Female 2 6 6 7 2 24 VCM (male) 2 4 9 21 45 14 95 reported for cohorts having thorotrast and VCM exposures2'46 a* nd the low incidence ofthis tumour, it seems reasonable to treat a case of angiosarcoma as if the given exposure were the cause. Differences between the series in demographic composition seem clearly not to be responsible for much of the difference in latent period, and one might expect any difference between workers (VCM) and patients (thorotrast) to be reflected in a longer latent period for the healthy workers in terms ofeither onset or death. The exposure is different for the two agents, being continuous from the date ofthe injection ofthorotrast and intermittent from date offirst exposure to VCM. Also, we sec no way of quantitating the two exposures in the same units. The limited data available on radistioninducedsolidtumours suggest that latent period does not depend on dose.10 *Some pan of the difference in average latent period could derive from differences tn "stage ofepidemic" or in completeness of ascertainment tn the later decades, but these differences could hardly contribute in any important way to the differences seen in minimal latent period, especially since the thorotrast cases derive from cohorts followed up from the time oftreatment. The minimum latent period for leukaemia following exposure to thorotrast is appreciably longer (8 years) than that associated with X-ray or gamma radiation (2-4 years).11 We believe that the distributions m the figure may well reflea differences in the mechanisms of chemical and radiation carcinogenesis, and that other comparisons of this nature would be ofinterest. Cohort analyses ofVCM workers and thorotrast patients arc clearly indicated as the effeas ofthe exposures run their course, and animal experimentation with these compounds might provide some understanding of the differences in their carcinogenic action. Since both VCM and thorotrast are multipotential carcinogens,14'12 m* ore analytical studies oflatency should carefully consider all causes of death, as well as levels of exposure. National Cancm Institute, National I minute* ofHatah, Bethewfe, Maryland 20205, USA Health and Safety Executive, London National Institute for Occupation! Safety and Health, Cincinnati, Otuo FimcjunatitiiMt, Copenhagen, Denmark Center* for Diacsoe Control, Atlanta, Georgia Deutsche* Krebfoncbungzeotmm, Heidelberg, Vest Germany Imperial Chennai Indiutnev, Welwyn Garden City R, Spirtas G. Beebe P, Baxter E. Dacey M. Faber H. Falk G, van Kaick J, Stafford 7 Creech jL Jr, Johnson MN. Angiosarcoma of liver in the manufacture of polwinvl chloride. 7 OcrMed 1974; 1C; 150-51. 8, Yamada $, Hoaoda 5. Taieno H. et al. Survey of ihorotrast-roociated Uvet cancer* in Japan. J NmI Canter hat 1982; 78: 31-35 9. Heath CWJr, Falk H, Creech JL Jr CharacrerunesofcasrsofangiOurcoma ofI be liver among vinyl chionde workers in the United State*. Am NY AtadSa 197 5,246: 231-56. 10 Land CL, Toktanagi L, Induction period. In* Botce JD Jr, Fraumeni JF Jr, eds Katfaatn cartmngeneto Epidemiology and biological significance New York. Raven Pima (in press). 11 National Academy at Science*. Committee on the Biological Efficcis of Ionizing Radiant: Tbe effects on populations ofexposure to low1 levels of ionizing radial ion 1980; Wmbmgton, DC National Academy Press, 1980, 331-57 12. Lelbnch WK, MarsteWer HJ Vinyl chloride-associated disease In. Frick P, von Harnack G-A, Kortdiek K, Martini GA, Prader A, eds Advances in internal medicine and pcdtatric*. Berlin: Spnnger-Verlag, 1981 1 -- 110 UCC 061027 C A cjs&s- GAMUT D-89 BONE EROSION OF MULTIPLE TERMINAL PHALANGEAL TUFTS (ACRO-OSTEOLYSIS) COMMON 1. Arteriosclerosis obliterans 2. Burn, thermal or electrical 3. Diabetic gangrene 4. Frostbite 5. Hyperparathyroidism, primary or secondary 6. Neurotrophic disease (see Gamut D-88) 7. Psoriasis 8. Raynaud's disease 9. Rheumatoid arthritis ` *10. Scleroderma, dermatomyositis 11. Trauma UNCOMMON 1. Brachydactyly B 2. Buerger's disease 3. Clubbing of fingers (see Gamut D-79) 4. Congenital (familial) acro-osteolysis (eg, HajduCheney S.) 5. Congenital indifference to pain 6. Disseminated lipogranulomatosis 7. Drug therapy (eg, dilantin, phenobarbital, ergot) 8. Ectodermal dysplasia *9. Epidermolysis bullosa *10. Gout 11. Leprosy (Continued) ucc 061028 BONE GAMUT D-89 12. Lesch-Nyhan S. (mental defective finger biting) 13. Osteomalacia (eg, .r.alabsorption syndromes) 14. Osteopetrosis 15. Pachydermoperiostosis 16. Pityriasis rubra 17. Plantar warts 18. Polyvinyl chloride osteolysis 19. Porphyria 20. Progeria; Werner's S. 21. Pseudoxanthoma elasticum 22. Pyknodysostosis 23. Reticulohistiocytoma (lipoid dermatoarthritis) *24. Rothmund's S. 25. Sarcoidosis 26. Sjogren's syndrome 27. Streeter's congenital amniotic bands 28. Syringomyelia 29. Tabes dorsalis *30. Thromboangiitis obliterans *May be associated with calcification, References: 1. Greenfield GB: Radiology of Bone Diseases. Philadelphia, JB Uppincott Co, 1969, p 304. 2. Moss AA, Mainzer F: Osteopetrosis: an unusual cause of terminaltuft erosion. Radiology 97:631-632,1970. ucc 061029 GAMUT D-89 BONE SUBGAMUT D-89A ACRO-OSTEOLYSIS CONFINED TO pNE DIGIT 1. Angiomatous malformation 2. Carcinoma of nail bed 3. Epidermoid cyst 4. Fibroma 5. Giant cell tumor of tendon sheath 6. Glomus tumor 7. Infection (eg, whitlow, osteomyelitis) 8. Metastasis; lymphoma 9. Neurofibroma UCC 081030 HEALTH SAFETY ANO ENVIRONMENTAL AFFAIRS A. A. LANG. M.D. DATE Assistant Corporate Medical Director P2593 (203) 794-5215 ^ NtU/ J# ^f/jli+e ^7^77pZff'6f7 V'S- (h&rtcg-, leltvy *)/>& */ce i #?</*&*e ^ by >/- $ - if#/?. u?s/) $ / & X> C/ Yy G'f^c e* / #(_# - Am*J, t'W ^ I -* u, />/ , - 33 7 a 9?r) ^y 5?// /? t-`#y f -C j-Jatrisposrd. OLD RIDGEBURY ROAD, DANBURY, CT06817 Uc 61Q31 3' V/i VINYL CHLORIDE (Teratogenesis) References Anderson, D; et a); Environmental Health Perspectives, Vol. 21 , 1977; pp* 71-78 Haas, J.F.; et al; Journal of Occupational Medicine, Vol. 21, No. 9, 1979; PP- 607~6l3 Mutation Research Vol. 41, John, J.A.; et al ; Toxicology and Applied Pharmacology, Vol. 39, 1977; pp- 497-513 John, J.A.; et al; Teratology, Vol. 17, 1978; pp. 48a Mirkova, E.; et al; Khigiena a Zdraveopazvane, Vol. 23, No. 5, 1977; pp. 440-443 Schwetz, B.A.; et al; Toxicology and Applied Pharmacology, Vol. 33, 1975; pp. 134 Ungvary, Gy.; et al; Toxicology, Vol. 11, 1978; pp. 45-54 Ungvary, Gy.; et al; Eges2segtudomany, Vol. 21, 1977; pp. 363-369 f*/*'-' ~ J c' ucc 061032 htnironmmitil Jfcuhh Voi 21, pp. 71-78. 1977 Dominant Lethal Studies with the Halogenated Olefins Vinyl Chloride and Vinyiidene Dichloride in Male CD-I Mice by Diana Anderson,* M. C. E. Hodge,* and I. F. H. Purchase* The mula^fnic activity of vinyl chloride < VC) and vinyiidene dichloride (VDC) al three exposure levels was assessed in fertile male CD-I mice with the dominant lethal test. Each compound was assessed in a separate study. Male mice were exposed by inhalation to VC at 3000, 10,000, and 30,000 ppm and to VDC at 10, 30, and 50 ppm for 6 hr/day for 5 days. By comparison w ith control males exposed to air, no mutagenic effects on any maturation stage of spermatogenesis in treated males were detected. There was no significant increase in the number of postimplantational early fetal deaths as shown by the number of females with one or more early deaths or the number of early deaths/pregnancy or the number of early deaths/total implants/pregnancy. There was no evidence of pre-implantatlonal egg tosses as indicated by the total implants/pregnant female. There was also no reduction in fertility. (The reduction in fertility at 50 ppm VDC was unproven). The lack of effect was not due to the insensitivity of the sy stem used, since both the VC and VDC study a mutagenic effect was clearly demonstrated in male mice dosed IP with the positive control compounds cyclophosphamide (CTX) and/or ethylmethane sulfonate (EMS). During dosing these animals were housed under similar exposure conditions to those animals exposed to the test substances but with a flow uf air through the exposure chambers. Thus, neither VC nor VDC is mutagenic in the mouse at the stated exposure levels as measured by the dominant lethal test. Introduction VC used in the manufacture of poly(vinyl chloride) has been found to cause tumors in rats (/) and man (2). It has also been shown to produce chromosome breaks in exposed workers (J-(5) and causes mutations in Salmonella lypliininrium (7. 8). Another chlorinated monomer, VDC is also known to cause mutation in Salmonella typhimurium (7,8). We. therefore, carried out dominant lethal studies to determine if there were any mutagenic effects of *linpcn;tl ( hemic.d InduMites I td., Central Toxicology I ahoiatoiy. Aldeilcy Park nr, Maeelcsliold, Cheshire SKIO 4`IJ. Knpiund. this type in mice after VC and VDC exposure at three levels. Al the same time negative control animals exposed to air and positive control animals also exposed to air and given EMS and/or CTX were assayed, Materials and Methods Chemicals VC was obtained from Air Products Ltd., Worsley, Walkden, Lancs., U.K. VDC was obtained from ICI Ltd., Mond Division, Runcorn, Cheshire, U.K. EMS was obtained from Koch-Light Ltd., Colnbrook. Bucks, U.K.. and CTX (Endoxana) from Ward Blenkinsop Ltd., London, U.K. December 1977 71 t Animals CD-I mice (Chiulcs River, Mansion. Kent) were used thmughout (he experiment. Uiulosoil females weie 8-10 weeks old when mated and male mice immediately after dosing were 10-12 weeks old. Males were caged individually and females in pairs. I hey received food and Wider <ul lihirinn. Dosing of Male Mice Dose levels for VC and VDC were selected on the basis of preliminary toxicity studies. A dose of 30.000 ppm of VC was found to he in the toxic range and this was chosen as the highest exposure level since it was desirable that the maximum toler ated dose or higher should be used. Other levels of 10.000 and 3000 ppm were also used. The required concentrations of VC were generated by mixing known volumes of VC and compressed air using rotameters as indicators. VDC was much more toxic to the mice. Exposure levels of 50. 30, and 10 ppm were selected. The required concentrations of VDC were generated by a controlled Fluidfeed/atomizer technique (9). The method involves continuously passing a known volume of the com pound through a concentric jet atomizer, where it is vaporized by a calculated volume of dry clean air. The volume of air required as calculated from Eq. (I). Air flow (l./min) = Syringe size (ml/cm) x density (g/ml) x 24 x 10* Injection rate (min/cm) x ppm x mol. wt. Ol During dosing the mice were housed individually in chambers made of stainless steel and glass with an internal capacity ol three lines. Mosl of the negative conliol animals and all ani mals dosed with CTX or EMS were housed under identical conditions during the dosing period hut with a How of air through the chambers. Those dosed with CTX were injected IP on day 5 of ex posure and those dosed with EMS were given an oral dose by gavage for 5 consecutive days. Both of these substances were prepared as aqueous solu tions immediately before use. Some of the animals in the negative control group controlling the VDC study were housed under normal conditions. Before test mating began, groups of mice were treated as shown in Table I. In the VDC study, in order to obtain a group with sufficient animal numbers some of the animals which were infertile in week 0 were included in group 4. Of the 8 survivors. 5 did not mate in the fertility study and one had a higher than average number of early deaths. However some of these males mated in the main study. One male surviving 50 ppm for 2 days died in week 2 of the study. Of the 17 animals, three in the CTX treatment group also did not mate in the fertility study. Fifty animals were included in the negative control group to pro vide a better data base against which to compare the treated groups. There were no statistical differ ences between males exposed to air and housed under normal conditions. Mating Fertility Teatinn. Male mice were caged with virgin female mice, 1 male and 2 female mice in Group VC Study Group 1 Group 2 Group t Group 4 Group 5 CilOlIp <S V|)(` Study Group \ (titMip 2 (tioup } Group 4 < 111 >U| * 72 Table t. Treatment group*. No. survivors/ no. animals treated (mice* T reatment .--- ---- -- 20/20 18/20 19/20 9/20 15/15 25/25 MI/SO 20/211 nv2 2/20 17/17 Air (negative control) 3000 ppm VC, 6 hr/day. 5 dav* 10.000 ppm VC. 6 hr/day. 5 davs 30,000 ppm VC. 6 hr/dav, 5 davN 200 mg CTX in water/kg body weight, once by IP injection on day 5. 200 mg I MS in watcr/kg body weight orally, oncc/day. 3 days |5 sir. 15 noim;il continuin', (ne'eutive cnnlinll 10 ppm V|>< . fy hr/day. 3 davs to ppm VIM'. /i lir/il;iy. 5 d.ivs 50 ppm VDC. s hr/duy. 2 d;iv' 75 ppm VIW. ft hr/d;iy for 1 d.iy. then ^0 ppm VIX , f> hr/day. lor 1 day 200 mg ( I X in watci/kg body weight, once by IP injection on day 5 Environmental Health Perspectives ucc 0SA034 i.kIi cage AIIci 5 days 11iu Icmiilcs were Itnns- lencd ti> uthei cages. I ho female mice were killed is d.iys alter tlisi mtioducing them In the males and esamiiied I'm pregnancies. I he 106 males whieli suivivcd dosing in the VC siudy and 11.1 males in I he' VI)C study and were successful in fertilizing al leasi I lemale in their cage were selected lor eoniimiaiion m the espeiiment l.xiH'iiincii/ii/ Milling. I wo virgin lemale mice 8-10 weeks old were put into each ol` the 100 and 111 cages m which the males were individually housed. After 5 days the lemales were removed and ichoused m pairs. A week after the initial introduc tion the males were caged with another two virgin females and again left for 5 days. This process was icpeated until the treated male mice had been mated at weekly intervals for 8 weeks with virgin females. The males were then killed and not examined fui (her. No attempts were made to establish whether or when mating had occurred. Instead it was assumed that most matings leading to fertiliza tion would occur 2 or 1 days alter introducing female mice to the cages containing males. I cmalc mice W'cic killed I.' days allct the as sumed date of fertilization, t o.. 15 or 16 days after caging females with males. Assessment l lien of killed mice were examined for live im plantations. early deaths, and late deaths Statistics I'hc data have been statistically analyzed as re ported previously (10). Results Mating weeks after treatment are represented m Tables 2-13 by numbers 1-8 and the mating week before treatment is represented by week 0. As sessment of females which became pregnant during the fertility test yielded the data for week 0 but only data from those animals that survived treatment have been included in weeks 0-8. Tables 2-7 relate to the VC study and Tables 8-13 relate to the VDC study. Table 2. Number and percentage of male mke which survived treatment successfully mating at each week. Week O' 1 T 1 4 5 ft 7 K "P * 0 (Mil. Group air (negative control) No, of r/t mice 20 20 :o 70 20 19 20 20 . 20 100 too 100 100 100 9S,() 100 100 100 Citoup 2. VC, l.(MK) ppm (ft hr x 5) No, of 7, mice IK 1(H) IX 100 IX too 18 MKI IX 100 18 l(K) IX 1(H) IX 1(H) IX 100 Group 1. vc. 10.0(H) ppm (6 hr x 5i No. of rf* mice 19 MX) IX 100 19 100 It too 19 100 It KM) 19 100 19 100 19 100 Gioup 4, vc. .10.(XX) ppm (ft hr x 5) No ,of 7, mice 9 MX) 7 77.X 9 MX) t 100 9 1(H) 7 77 X 9 MH) 9 100 S XX.9 Group 5. C1X, 200 mg/ke tp No. of 7, mice |S UK) U 91.1 14 91..1 14 9.1,1 15 |(H) 15 MX) 14 MX) 14 91.1 ii t:.t Group ft. KMS. 200 mg/kg (oral) x J No, or 7, mice 25 H10 1" 12.2 21 t:,o 2i t:.o 25 MKI 25 MKI 24 MK) 25 MX) 25 MKI Tilhie 3. Number of mail'd females becoming pregnant. Week Group 1. air (negative control) Citoup 2. VC. l.ooo ppm 16 hr v 5) No. No. No. No. pregnant muted pregnant mated 0 15 40 10 V. 1 u 40 ,11 v. 1ft 40 Vi V* 1 u 40 10 1ft A ix 40 14 1ft 5 is 40 14 is ft 1ft 40 11 16 7 17 40 n 1ft K .17 40 12 16 "/> II.(KM V 0.01. Group .1. VC. 111,000 ppm (ft hr x 5) No. No pregnant mated 28 IX 12 V> ,11 IX 14 IX .1.1 IX in IX 16 ,1X .17 .IX 11 17 Group 4, vc. .10,(XX) ppm (ft hr X 51 No. No. pregnant mated is IX 11 18 |5 18 Ift 18 17 IX 12 18 IX 18 IX 18 14 IX Group 5, (. 1 X. 200 mg/kg IP No. No. pi eluant mated 2S K) 2ft 10 25 10 2ft to 10 10 28 10 2ft 2X n 10 :i 2X Group ft. IMS. 200 nig/kg (oral) x 5 No, No. pregnant mated 17 5(1 4" SO 11* 50 40 <0 4ft 5(| 47 50 47 4K 41 50 4.5 50 December 1977 73 Wl^k 0 1 A 4 S ft 7 X > - (Mil V o.o5 Week 0 1 ? 3 4 5 ft 7 X > <0.01. Week 0 1 2 * 4 S 6 7 X */> < 0.01. Week "p < 0.05. V <0.01. 74 Group 1. air (negative control) 11 0 i: 9 i: x 12.5 i: 4 15 7 11 7 I1 N 12.7 I iihit1 4. Menu lulul implants pef pieummt female. Gioup 2, VC 3.000 ppm 16 In -c 5) 12 ft 11 6 t: 7 . 12 12 l 12,7 II.1) lift i: i Gtuup 3, VC. It).IKK) ppm (ft lu < M 12.0 13-4 1 <0 12,4 11.7 14.1 12.5 11.8 12 6 Gump 4. vc. 1(MH)0 ppm (ft lit < G ICO Ift 1 1\ 7 11 2 10.0" 12 X 12.ft 12,0 12 1 (. tump 5. CTX. 200 mg/kg If II 1 x X" 10 V 10,9 i: ft 12 * 12 5 n.s 12 < Gump ft. EMS. 200 mg/kg liu.ill X 5 1 I.X 4.0" X 4" 12 5 12 7 i: 7 12.1 12.4 12 * Tnhlc 5. Number of pregnant females with one or more eiirl) deaths (LI)). Group l. air (negative control) Group 2, VC. 3,000 ppm (6 hr x 5) Group 3. VC. 10,000 ppm (6 hr x 5) Gump 4. VC . 30,(XX) ppm (6 hr x 5) Gump 5, Cl X. 200 mg/kg IP Gump ft. HMS. 200 mg/kg (oral) x 5 0 a 1 ED 0 | ED o 1 ED 0 31 1 ED 0 18 17 21 9 16 22 10 5 22 15 20 16 17 10 21 65 1 19 17 18 18 13 20 ft 9 2 18 16 16 14 19 15 9 ft 8 16 22 17 17 20 13 9 7 14 17 18 18 16 14 22 3 9 12 17 19 II 22 21 15 7 II 13 18 19 13 18 21 16 8 10 10 13 24 14 18 15 16 8 6 13 3 1 ED 0 y 21 25" 1 23" 5 18 15 16 19 16 25 13 24 12 22 10 22 3 1 ED 26 3 26" 25 27 22 23 19 23 Table 6. Mean number of early deaths per pregnancy Group 1, air (negative control) 0.77 0 Xft 0.K3 0.91 0.89 1.06 0.97 0.76 1.03 Group 2. VC, 3.IKK) ppm (6 hr x 5) 0.33 0.91 1,00 0.73 0.79 0.71 0.9) 0.87 o.xx Group 3. VC. 10.000 ppm (6 hr x 5l Q.M 1.00 1.15 0.88 0.61 1.03 0.75 0.68 1.00 Group 4, VC. 30,000 ppm (6 hr x 5) 0.40 0.45 0.93 0.93 0.69 1.00 1.39 0.89 0.71 Group 5, CTX. 200 mg/kg IP 0.1ft 4 27" 4.84" 1.54 1.13 Ml 0.65 0.82 0.70 Tabic 7. Early deaths as a percentage toul implants per pregnant female* Group 1. air (negative control) 6.5 6.6 6.4 7.5 7.2 7.8 8.2 6.4 8.0 Group 2. VC. 3.000 ppm (6 hr x 5) 2.7 6.7 7.9 59 6.5 5.5 7.6 7.3 7.2 Group 3, VC. 10.000 ppm (6 hr x 5) 4.4 7.5 9.6 7.0 45 7.3 6.0 5.7 81 Group 4, VC. 30,000 ppm (6 hr x 5) 3.! 2.7 6.9 7.8 6.5 7.9 II.I 74 5.9 Group 5, CTX. 200 mg/kg IP 1.4* 49.1* 44.6* 14.1 9.0 9.1 5.2 7.0 5.7 Group ft. FMS, 200 mg/kg (oral) x 5 0.59 3.50" 2.58* 1.45 0.85 0.72 0.85 0.63 0.73 Group ft. EM$. 200 mg/kg (oral) x 5 5.1 77.78* 31.5* 11.6 6.7 5.7 7.0 5.1 5.9 Environmental Health Perspectives * 1 1 1 1 f 1 ; 1 t \ . ) I t j t , I * * i \ 4 1 / ' ; s* -'^n ?h1* ' t*- > 5. i :/ '0 I uMv K. Niitnlw i nittl |h`11 int.i^r nl in.lie mUr wlikli sni \ivi'il ImiImk hI nihiI'.sliill): inulln^ Ml rtH.it week. Week 0 1 2 \ 4 ft 1 K "t> n.ooi. V < 0 01. 'P < 0 05. Group 1 (pooled negative control) No of mice 7 <0 . VI MV 50 44 47 4*7 49 4H 1(H) 9K 9ft 94 4K 94 XX 92 92 Group 2. VDC. 10 ppm <6 hr x 5) No. or mice 20 20 20 20 20 20 20 20 19 Of too 9S uni 100 ioo UK) too `X) 95 Group 5, VD<\ W ppm (ft hr x 5) No of mice IH IS m IX 17 17 17 17 r/t UK* 100 94 X9 X9 w m 100 XX Group 4, VDC, 50 ppm (6 hr x 5* No of mice Of X" ix k* 75 7* k6 7" 4.1 7" 41 7" 57 7" 45 Ml T 57 Group 5, CTX. 200 mg/kg 1C No, of nocc % 17* 7ft 17* 65 16 94 17 94 15 IOO 17 100 17 kk 16* 75 19 K6 Table 9. Number at mated females becoming pregnant. Week 0 l T 1 4 5 6 7 X Group 1 (pooled negative control) No No. pregnant mated 71 100 k2 100 75 100 k2 too k5 9k ko 94 75 9k 77 9k 7? % *P< 001. * /> < 0 001 *p < 0.05. Group 2, VDC, 10 ppm (6 hr x 5) No. No. pregnant mated 50 40 40 56 40 14 40 56 40 34 40 11 40 11 40 2k 39 Group 3, VDC. 30 ppm (6 hr x 51 No. No. pregnant mated 25 36 VI 3ft 32 1ft 26 36 27 36 26 34 12 34 28 34 26 34 Group 4, VDC. 50 ppm (6 hr x 5) No- No. pregnant mated 5* 16 7" Ift 7* 14 5B 14 6* 14 6* 14 5* 14 7 13 7 14 Group 5, CTX. 200 mg/kg IP No. No. pregnant mated 17* 14 20* 14 25 33 28 34 26 32 29 34 25 34 19* 31 28 39 Week 0 1 1 4 5 ft ,7 X 7> x. 0.01. V < o.ooi. '!> -- 0.05 December 1977 Table 10. Mean total implants per pregnant female. Group 1 pooled (negative control) 12.2 12.3 12.3 12.0 12.0 11.8 12,5 12.6 12.3 Group 2, VDC. 10 ppm (6 hr x 5) II 0" 13 1 11.7 12.1 12.1 II 7 12.4 13,0 13.6 Group 3, VDC. 30 ppm (6 hr x 5i 12.0 I2.k 12.1 13.2 13.0 12.5 12.1 12.5 12.4 Group 4, VDC, 50 ppm (6 hr x 5) 11.4 11.7 11.0 13.4 12.7 10.7 12.6 12.7 12.6 Group 5, CTX, 200 mg/kg IP II.J 9.2* 9.5* 110* 12.1 12.0 II.111.7* 11.6 75 ..V + -- .. Wi-vk 0 1 2 \ 4 S ft 7 X "p 0 001, V ' o.oi. table U. NuiiiIht of pregnant females with oih- or outre early deaths || |>). (.KHip 1 (pooled negative control) (I - 1 l 1) 4(1 11 41 41 19 . 1ft 4* 17 10 4 S 4! 10 40 IS 41 14 17 1ft turnip 2, vdc. It) ppm (6 hr v M t) -- 1 ID 14 16 IX 14 ix ix IX 16 17 19 1' 19 14 19 |5 ix 1 * IS Gioup 3, V1X . Ml ppm (6 hr x 3) 0 -II 1) III IS 19 14 17 IS |S II 11 Ift 11 15 17 15 D 15 1 7 ( !| lH|p 4, VDC M) ppm (ft hi * G 0 1 t [J 14 ft 1 54 41 :4 11 :i 5: 14 Group 5, crx. 200 mg/kg ip 0 -- 1 KD 9X <r 20 2" "> i T' 21 12 14 12 17 12 13 10 9 15 3 Table 12. Mean number nf early deaths jkt pregnanes. Week 0 1 -} \ 4 ft 7 X 'ft - 0.001 Group l (pooled negative control) 0 0 72 0.69 0 7ft 0 79 0 7X tl XX 0.65 0,X5 Group 2. VDC. 10 ppm (ft hr x 5) o.xo 0.72 0 73 0.6X 0.X3 0 ftX 0.91 0.X5 0.75 Group 3, VDC. 30 ppm 16 hr x 5) n.x4 0.61 0.56 0.46 1.00 0,73 0.94 0.57 0.XJ Gioup 4. VDC. M) ppm (ft hr x 5) 2 00 0 29 0.71 o.xo 1 00 0.67 I 00 0.57 0.71 fable 1J, Early deaths as a percent*** of total implants per pregnant female. Group 5, CTX. 200 mg/kg IP 1 06 4,004 04* 2 3^" 0.X5 0.X6 0.64 0.74 Q 30 Week 0 1 2 3 4 S ft 7 X l> < 0,001. Group 1 (pooled negative control) 4K 5,7 5,5 64 ft.9 ft ft 70 49 7.0 Group 2S VDC. 10 ppm (ft hr x 3) 10.1 53 6.9 6.5 7.1 61 7,6 6.6 5.4 Group 3. VDC. 30 ppm (6 hr x 5) 7.3 5.1 4.4 3.6 7.4 6.2 X.3 4.5 7.1 Gioup 4. VDC. 30 ppm (ft hr x 3) 17.5 37 53 10.3 5.6 75 4.3 3.X Group 5, CTX, 200 mg/kg IP 91 45 6" 41 4' 2 1 .X" 6,9 69 36 ft 1 :4 Fertility Sitrccwfnl Mating Frcquency. The numbers of males successfully mating at each week are shown in Tables 2 and 8. Numbers remained high during the experiments. No statistically significant differences in the mating frequency were found be tween VC treatment groups ami (he control at any week by using a chi-squaied test. I here was. how ever, a significant difference between the KMStreated group and the negative control group in week I. In the VDC study, the milling frequency w;is high in the two groups exposed to the lowest doses of VDC in all weeks by comparison with the negative control group. The mating frequency, however, was statistically significantly lower in the high exposure group in weeks 0-8 and the positive control group in the weeks 0. I, and 7. This effect in the VDC highest exposure group was, however, probably due to infertility of the males used. /Ycgiiriiiri; Frctiuvnvij. I he numbers of females in each group which became pregnant at each week of mating arc shown in Tables 3 and 9. In the VC study, statistical differences between treated groups and the negative control group were found only in the liMS-treated group at weeks I and 76 Environmental Health Perspectives i i * V t t f t > t 1 t t , / * t H . ^ '4 . " ucc 061038 1 l'> using a iIn-square Icsi. In llie; Vl)( study theie were significant differences in ihc Inchest VDC exposure jiroitp nt weeks (>--6 and tlie positive control proup tn weeks 0, 1, and 7. Again this w;is pi nimbly due u> infertility ofthe males. I hese results indicate that VC al lhe tincc ex posure levels anil VDC al leaM al expoxmes ol' 10 anil 30 ppm iliil noi cause a icilueliun in Icililily. Any reduced fertility at 50 ppm VDC was unpioven. Total Implantations I'he mean total number of implants per pregnant female in each group is shown in Tables 4 ami 10. I he mean values were adjusted to take account of the unequal number of pregnant females per male and were compared statistically by using an analysis of variance and a f-test, In the VC study statistically significant differences were evident in week I in the C'TX-treated group and weeks I and 2 in the IMS-treated group. A significant differenee (/> - 0.05) in week 4 was also found between the group exposed to the highest dose of VC (Group 4) and the negative control group. In the VDC study the CTX positive control group was statistically significantly different from the negative control groups in weeks I. 2, 3, 6. and 7. Only the VDC group to be exposed to 10 ppm showed a significant difference front the negative control group in the pre-experimental nontreatment week. Thus there was no indication of a preintplantation loss of eggs in either study except in week 4 after 30,000 ppm of VC. Early Deaths mill IMS treatment In the VDC study only the CTX positive control group was significantly dif ferent from the negative control group in weeks I. ....... .. 3 i fable 121. I'.artii Deaths as a Percentage of Total Inil>lants )>er Pregnant Female. Again, it was necessary to stabili/.c the variance prior to analysis. In the VC study CTX and EMS treatment giotips were significantly different from the negative contiol groups in weeks I and 2. whereas VC-treatcd groups were not (Table 7). The CTX treatment group also showed a significant difference in the week before treatment. In the VDC study only the CTX treatment group was significantly different from the negative control group in weeks I, 2, and 3 ffable 13). Thus with all these different methods of analysis of the data of early deaths no statistically significant differences from the negative control groups were seen in the VC or VDC treated groups. Late Deaths In each study late deaths were randomly distrib uted throughout all the groups and did not appear to be treatment-related. Conjoined Placentae Conjoined placentae were seen in this strain. They were the result of very close implantation sites and were not monozygotic twins 02). In the experiments they were classified as double implan tations. Since they occurred with equal frequency in all groups they did not appear to be correlated with treatment. T he data for early deaths have been presented in vai ions ways. Number of Pregnant Female* with One or More F.arhj Deatha. In the VC study. CTX and IMS treatment caused increases in the number of pregnancies with early deaths (Table 5). The effect was significant (chi-square) in weeks I and 2 for the C'TX-treated group and in week 2 for the EMSticated group. No differences from the negative control group were seen in the VC-treated group. Similarly in the VDC study there were only sig nificant differences in the CTX positive control group in weeks I. 2. and 3 (Table II). The Mean Number of Furl;/ Deaths per Pregnancy. A large number of low or zero values were encountered, so il was necessary to siabili/e the variance prior to analysis. There were no statis tically significant increases in early deaths after VC treatment ( table 6). However, differences were evident in weeks I and 2 with CTX treatment and Discussion The best indication of mutagenic activity of a substance in the dominant lethal test is an increase in the number of post-implantationai foetal early deaths 03). From the data for early deaths there nas no evidence in either study of a mutagenic eflect with VC or VDC at the administered exposure Tevels. I his did not appear to be a result of luck of sensitivity of the animals used, since there was a marked response to CTX and EMS. High doses of CTX and EMS were used in our studies to obtain a highly significant positive result. However, the dominant lethal study in our hands 04) is at least as sensitive as (hat reported elsewhere. We have shown ethyl methane sulfonate on previous occasions to give a positive result with a single IP dose of 150 mg/kg body weight, which is comparable to that reported previously 05) for the same mouse strain. December 1977 77 i-oi icaxonx described emtier (/(/, //). dillcient evaluation methods were used foi the early deaths' data and much of the data at the studies were sub jected to various methods of statistical analysis. I'rc-implantation egg losses, while representing some of the mutagenic effect, aie not as important as postiiuplantational losses, for they could also arise due to other than genetic factors (13). Preimplantational egg losses have been studied by comparing values of total implants m females mated with Healed males and those mated with control males, as suggested by Epstein (16). rather than counting corpora lutea. Thcie was no prcimplantalional egg loss by compatison of VC- or VOC-treated groups with the negative control group, except in the highest VC exposure group in week 4 (/> < 0,05). When a treatment group is sig nificantly different from a negative control group there is generally a uniform reduction in implants, whereas in week 4 after VC treatment the low result was tine to a large extent to the result from one female. Without this female, mean values would not have been significantly different from the negative control group. Therefore, this result is not con sidered biologically significant. Yet another reason is that there was no corresponding incicase in early deaths at this time. Late deaths also are not considered as important as early deaths in (lie assessment of the mutagenic potential of a test substance (13). Late deaths were excluded from analysis to incicase test sensitivity. Ciropp and Kolbus (17) have shown, however, that trisomies in mouse foetuses cause death and elimi nation before term, it might be aigued that sampling for late deaths at a later stage of pregnancy might lead to different results lot late deaths. However, the randomness of their distribution in both studies would suggest otherwise. There was no reduction in fertility as measured by the mating and pregnancy frequency at any week at the exposure levels of VC. suggesting no antifer tility effect. The same was true for the VDC groups exposed at 10 and 30 ppm. How-ever, the decreased fertility in the group exposed to 50 ppm was proba bly due to the infertility of the males which had to be used to establish a group of sufficient sue to perform the experiment. A fertility study was undertaken prior to the expcrinicnt also for reasons described earlier (10. II). I coin these initial fertility data the background dominant lethality of all groups was determined primarily to ascertain that there were no initial dif ferences between groups. Mutagenic effects of vinyl chloride have been ob served in laboratory tests and in exposed workers. fiTthis study where exceptionally high doses oT VC wete used, which would never be encounteied by workers, no mutational effect in the genii cells was observed.' A possiblc'cxplahaium is thal the active metabolites did not reach the genu cells. Similarly the lower but more toxic levels of VDC did not produce getm cells mutations. It can be concluded, therefore, that VC and VDC do not cause dominant lethal miilalioiisT in male"t IJ-I mice at .HjfRTT 10.01)0. and 31),000 ppm and It), Ml, and SO ppm, respectively.* 1 * * * 5 I he authors would like to thank the Inhalation Section loi exposing the males to VC and VDC. Mis S, Palmer lot her technical assistance, and Mr, T Weight and Mr S, H Hits for then statistical evaluation of the data All poi tunnel mentioned ate at this iabofnioiy except Mr. S. H. Ellis, who is at 1CI Pharmaceuticals Division. Aldetley Park REFERENCES 1 Maltoni. C.. et al. Vinyl chloride caivinogcncsis--current icsults and perspectives. Med. lavaro 65: 421 0974). 2. Cieeeh. J. 1... and Johnson. M. N Angiosarcoma of the liver in the manufacture of polyvinyl chioude. J. Oceup, Med. 16; 150M974). 5 Ducal mart* A.. Hirschom, E-. and SehkolT. I, J. Vinyl chioude exposure and human ehiomosome aberrations Mu* union Res. 51: IM U97M. 4. hunes-Cravioto, K. et al. Chromosome aboirations in workers exposed to \in\l chloride, t ancei I; 459<I975/. 5 Puichase. 1, E M . Kichaidson, C R., and Anderson. D, Chtomosomal effects m penphci.il lymphocytes. Proe. Roy Sot Med. (l.ondoni 69 290 (1976) A. S/entest. 1.. et al. High rate oi chiomosomal abeiration in PVC woikcrs. Mutation Res 57 5|* (1976). 7. Hartsch. H,, Marseille, (. and Montesano. K. Human, rat and mouse liver-mediated mutagenicity of vutyl chlondc in .S, t\(ihtwnruf(n strains Int, J. C anter 15: 429 0975). H. Buiisch. H.,ctal. '1 issue-mediated mutagenicity ol vinylideno chlonde .ind 2-chloiobuladiene in Salmonella t\i>hwuuntni Nature 255: Ml (1975) 9. ti.ige. J. C . 1 oxicity of epichkirhydrin vapiiui Brit J Ind, Med. 16: II 0959). It). Anderson. D.. McGicgor. D. B., Purchase, I. I- H Dom inant lethal studies with paratpui and dimi.it in male ( D-l mice Mutation Res, 40- 549 0976). \\. Anderson. D.. Hodge. M, C. E.. Purchase. 1, K H. Vinyl chloride: dominant lethal studies in male CD-I mice. Mutaiion Res. 40: 559 tl976i. 12. Bateman. A, J, Dichorial. one-egg twins in the mouse. Na ture IK7: 559 < I9WM, 15. Bateman, A J.. and bpsiein. S S. In, Chemical Mutagens. Principles and Methods for Their Detection. A Hollander, Pd.. Plenum Press, New York-1,ondon. I97| pp. *4|.*6H. 14. Anderson. D., ct al. Dominant lethal test results with known mutagens in two laboratories. Mutation Res. 45: 251 (1977) IV Ray. V A., .mil ((vncsk, M f Smite pnin.uy considctations in (he mtcrpictatum ofjfic dumtu.uif kl)ul assay 1 nviron, Health Peispect. 6 25 IJ97.5). 16. Epstein, S. S, The use of the dominant lethal test to detect genetic activity of environmental chemicals. Environ. Health Peispcct, 6 25 (1975) 17. Gropp. A,, and Kolbus, U, Kscnccphaiy in the s\ ndrome of trisomy no. 12 of the foetal mouse. Nature 249: 145 (1974), 78 Environmental Health Perspectives Risks to the Offspring from Parental Occupational Exposures Ioanna F. Haas, M.D., and David Schottenfeld, M.D. Risks to the offspring of workers with occupational chemical exposures may derive from mutagenic, terato genic or carcinogenic effects of industrial agents to which the parents are exposed. Evidence for impaired pregnan cies and hazards to the offspring of working populations with chemical exposures is. however, very limited. Perhaps the best documented example is increased spontaneous abortion rates in female operating room personnel who have first trimester exposure to waste anesthetic gases. Evi dence is reviewed for hazards to the offspring resulting from parental occupational exposure to vinyl chloride, benzene, chloroprene, radiation and petroleum-derived hydrocarbons. It is essential in investigating the role of occupational factors that other environmental and behav ioral factors with maior effects on pregnancy outcome be accounted for. These include smoking, alcohol, and drug exposures. An approach to surveillance for chromosomal abnormalities in offspring of occupationally exposed parents is outlined. L/erived from the Creek word "repat," meaning a mar vel, prodigy or monster, the word "teratogenic" was used by 1857 to mean the "production of monstrous for mations or births."' The term has been refined to refer to the biological science dealing with "the causes, mecha nisms, and manifestations of developmental deviations of either structural or functional nature An agent may act as a teratogen when administered in a number of ways, whether to the male or female before mating, to the female during pregnancy, or to the fetus directly ' Agents in the environment may produce alterations in the ge nome. mytations, and by that mechanism lead to abnor malities of development in the offspring Viral, drug or chemical agents acting through common pathways may produce indistinguishable results The outcome of such mutations may theoretically be fetal defects or predis- from Cornell Umvrrwtv Medtcal Collfyr AfKl Sloan Kputung Cancer Center. Hon 40. 127^ York Ave New York NY 10021 Supjxyted m part by a contract the Amertcan Petroleum Institute position to neoplasia. Agents which alter the rate of growth of the fetus or are lethal to the fetus without pro ducing specific anatomic or functional anomalies are bet ter termed developmental toxins than teratogens. Cancer in the offspring of individuals exposed to a car cinogen may theoretically be the result of any of three types of exposure: prezygotic, by alteration of parental germ cells; transplacental, by passage of carcinogenic substances across the placental barrier; or postnatal, by contamination of the environment with carcinogenic sub stances, pnmanly through ingestion (including substances excreted in human breast milk) and through inhalation. Evidence for risks to the offspring of humans occupa tionally exposed to potentially hazardous substances is reviewed m the present work. The term trans-generational carcinogenesis is used to describe the occurrence of cancers in the offspring which can be attributed to paren tal exposures. This term is proposed to encompass not on ly the established process of transplacental carcino genesis. by which a substance administered to the mother during pregnancy results in cancer in the offspring, but also the controversial issue of preconception alterations in genetic matter resulting in increased teratogenic or cancer risk in the offspring The latter effect could theo retically result from each of three distinct mechanisms: chromosome breakage, point mutation, or abnormalities in gametogenesis or fertilization. Mutagenic and Teratogenic Characteristics of an Agent and Trans-Generational Carcinogenesis Mutagenesis, teratogenesis and carcinogenesis are re lated phenomena, but the nature of their relationship is complex and occasionally controversial It has been argued that the "same c hemical that causes abortion in the early stages may produce malformations during organ development and neoplasia when exposed later m preg nancy Therefore, screening tor transplacental hazards should include, whenever possible, the entire range of fetal response, including cancers that may develop some time after birth "4 Teratogenic, mutagenic and carcino genic activities are all demonstrable tor a number ot com- Journal of Occupational Medicine/Vol. 21, No 9/SeptemOer 1979 607 ucc 061041 pounds, lint transpl.ii riil.il < ,ir< inogcmi |W)lenli,il h,is MK'ly been deimiii'.lf.ited lor known teratogens and mu tagens teratogenic effects may reflect a variety ot ac tions involving the mother, the placenta or the feto-plarenlal unit These effects ran be indirect and may not in volve immediate action of the agent on the target fetal tissue' transplacental carcinogens may act directly on the fetal tissues which are inherently tnore vulnerable be cause of the high rate of cell division, high proportion of undifferentiated cells and immaturity of immunosurveillance mechanisms To appreciate potentiaf danger from an environmental exposure to offspring of the exposed organism, the out come of all exposed pregnancies should be considered. Early fetal wastage commonly results from abnormal fetal development. At the other extreme, transplacentally-mduced tumors hardly ever ap|>oar in rodent species until the animal reaches maturity. In the only docu mented example of chemical transplacental carcino genesis in humans, vaginal adenocarcinoma following diethylstilbestrol (DES) exposure, the tumor occurs decades following exposure Thus, since transplacentally-induced tumors, unlike malformations, are rarely present at birth, one must also observe the offspring into adulthood to see the full spectrum of resulting tumors. Environmental Threats to Pregnancy Outcome Recognized threats to pregnancy outcome are not un commonly encountered m the environment and must be excluded when new problems are suspected. Threats are posed by biologic, chemical and physical agents, and may derive from medical therapeutic interventions, drug and substance abuse, and environmental or occupational exposures to chemical agents. Alcohol. -- The most widespread documented threats to the fetus come not from the maternal physical environ ment but from the use of tobacco and alcohol during pregnancy. Only recently has the risk of alcohol ingestion in pregnancy been evaluated and the concept of the fetal alcohol syndrome refined.' Of infants born to recognized chronic alcoholic mothers, 83 3% had birth weights under the tenth percentile, compared with 2,3% in a compara ble non-alcoholic population.* In addition to intrauterine growth retardation, the infants had retarded post-natal growth and intellectual development. A characteristic facies, with short palpebral fissures, hypoplastic philtrum, thin vermilion line of the upper lip and retrognathia, has also been described. In addition to these features of the fetal alcohol syn drome, congenital malformations of various types occur with excess frequency. Recognition of an associated in crease in the occurrence of congenital malformations ironically reemphasizes ancient observations and ad monitions against alcohol use during pregnancy * The ad verse effects on the fetus of maternal alcohol abuse dur ing pregnancy were further quantified by Ouellette et a!.10 who classified women at the first prenatal visit into four categories according to alcohol consumption and in dependently evaluated pregnancy outcome Compared to those born to abstinent or moderate drinkers, infants born to heavy drinkers had twice the risk of having an ab normality at birth The frequency of congenital abnormal ities in the offspring of heavy drinkers was extremely high--t2% wlu'ii minor abnormalities were included, 17% it only nia|or anomalies were considered Mullipl" abnormalities were present in 20% of the offspring of heavy alcohol users Maior, minor and multiple abnor malities occurred significantly less often among abstinent or moderate drinkers Animal models, including chicks, rats and guinea pigs, have been developed for evaluating the effects of ethanol alone on offspring, and support the view that ethanol itself is a harmful agent, and that its teratogenic potential is common to several species " Smoking. -- Another factor influencing pregnancy out come, maternal smoking habits, should be addressed m efforts to tie environmental exposures of the parents to the survival and integrity of the fetus. Kline et a!" suggest that the odds of spontaneous abortion among women who smoke are 1 8 times those of nonsmokers. This association remains statistically significant after maternal age, and number and outcome of previous pregnancies are taken into account. The manner in which smoking affects fetal survival is controversial. Smoking results in lowered birth weight, which may or may not be related to the increased fre quency of spontaneous abortion.1114 Lowered birth weight may result from impaired maternal nutrition or fetal anoxia, neither of which produces an excess of chromosomal abnormalities in the conceptus. Alternate explanations for an association between smoking and spontaneous abortion have been proposed. Since spon taneous abortion serves as a means of selectively termi nating abnormal conceptions and since 95% of abnormal pregnancies are believed to terminate this way, an associ ation between an environmental factor and increased spontaneous abortion should always evoke suspicion of a teratogenic phenomenon.'5 Smoking increases the risk of spontaneous abortions by a factor of 1.8, yet the percent age of abortuses which are chromosomally abnormal is close to that expected. The absolute risk of a chromosom ally abnormal fetus appears, therefore, to be higher in smoking mothers. The relationship of karyotypic abnormalities in spon taneously aborted products of conception to maternal smoking habits is complicated by variation in the propor tion of chromosomally abnormal fetuses with increasing maternal age and by the fact that a large proportion of smokers are younger women.1* An increase in the rate of chromosomally abnormal conceptions might be masked in a proportionate ratio analysis if there was also an in creased loss of conceptions without demonstrable cyto genetic abnormalities. Evidence for an increase m congenital malformations in children of smoking mothers is limited and conflicting. In a cohort of births occurring in the first week of March, 1958, in England, Scotland and Wales, Frednck and col leagues" identified a 60% increased risk of congenital heart disease among children of women who had smoked at least one cigarette per day after the fourth month of pregnancy (7 3 vs, 47 cases per 1000 live births). This ef fect remained statistically significant after adjustment for maternal age, parity and social class. An investigation of congenital defects of all systems registered in South Wales from 1964 to 1966 which took into account mater nal age. parity, social class, area of residence and date of delivery did not associate maternal smoking habits with 608 Risks to Offspring from Parental Occupational Exposures/Haas and Schottenfeld UCC 061042 (<niKiMiil.il m.illium.ilions in .my sysinm '* It is possible III,it imuli-ruti* mi rouses m relative risk might I hive gone undetected Analysis of data from a mailt'd survey of a large num ber of professional women in medicine identified a rela tive risk of up to 1 7 for spontaneous abortion and a risk of congenital abnormalities of up to 2 5 in heavy smokers after the effects of age, parity and exposure to anesthetic gases were considered 1,1 Drug fx/rosure. -- In utero ex(>osure to diethylstilbestrol (DtS\ with the subsequent development of vaginal adenocarcinoma, is the prototype for transplacental on cogenesis in humans. Appreciation of the causal role played by in utero exposure to DES followed hard u|>on the report of seven cases of the hithertofore exceedingly rare tumor, adenocarcinoma ol the vagina, presenting in young individuals, 14 to 22 years of age The syndrome appears lo involve not only cases of vaginal adenocar cinoma, but a spectrum of abnormalities of the vagina and cervix. Only when exposure to DES occurred during the first four months of gestation were neoplasms or ab normalities observed. The DES vaginal adenosis-adenocarcinoma relation ship is the only documented example in humans of cancer in the offspring attributable to parental environ mental chemical exposure The therapeutic agent was given in a high dose, commonly more than 10 grams in the first half of pregnancy DES, a synthetic nonsteroidal estrogen analogue, was iikely to have a direct effect on the development of the target tissue at a crucial point in embryogenesis. Since the risk to the offspring appears limited to exposures which occur during the first four months of gestation, direct exposure of the fetus is likely to be essential to the pathogenic mechanism. The long latent period, with exposure in utero and emergence of clinical sequelae in adolescence, probably reflects the im portance of pubertalendogenous estrogens as promoting factors, and is a reminder of the need for an extended period of observation following suspect trans-generational carcinogenic exposures. The association between DES and vaginal adenocarcinomas, as important as it is. does not serve as a model for preconception parental oc cupational exposures and trans-generational carcinogen esis. Teratogenic effects of a multiplicity of drugs are now documented or suspected and these will not be discussed here further except to reiterate the need to remove the potentially confounding effects of such agents in investi gations of other possible causes of impaired fetal devel opment " Given the current status of our understanding, it is essential that in the search for exogenous causes of teratogenesis and abnormal pregnancy outcomes, the poten tial confounding impacts of maternal smoking, drinking, and medication intake be taken into account. Radiation. -- If preconception radiation exposure in creases cancer risk in the offspring, it would heighten con cern over other agents which produce chromosomal dam age. Chromosomal, aberrations attributed to vinyl chlo ride and benzene exposure, for example, are reminiscent of those produced by radiation. While the genetic consequences of irradiation from the atomic bombings might be expected to produce some lethal mutations in the offspring of survivors, this effect has lioen difficult to demonstrate ( ohort studies of olt- spimg of survivors ihesilieil by radiation exi>osure level did not show excess mortality in the children of the high exposure group. Neither were excess congenital malfor mations, increased infant mortality, nor impaired survival during the first ten years of life detected, regardless of ex posure level to either parent. Thus, although animal exixmencc strongly suggests that such effects should be manifest in offspring of individuals exposed to ionizing ir radiation, studies of lapanese atomic, bomb survivors have not demonstrated measurable effects." No substantial increase in leukemia risk has been de tected among offspring of survivors Analyses having a 90% chance of detecting a four-fold increase in risk of leukemia among children of heavily exposed survivors wen* negative.' Neither tiki age-at-onset of observed leukemia cases nor type of leukemia in offspring differ by level of parental radiation exposure. Despite the docu mented persistence of chromosomal aberrations in, the somatic cells of adults exposed to the atomic bomb, no measurable impact on mortality, congenital malforma tions or leukemogenesis has been- detected in their off spring." In addition to radiation exposures related to the atomic bombings, diagnostic and therapeutic medical radiation of the parents has been investigated to assess risks t the offspring. The magnitude of leukemia risk associated with previous radiation was estimated, after adjustment for maternal age and pregnancy history, to be 1.73 times that of mothers who had not been x-rayed. Despite the sugges tion of greater risk in women with higher x-ray exposure, no clear-cut dose-response gradient was demonstrated. The relative risk of feukemia in the child associated with preconception diagnostic radiation of the father (1.31) was not statistically different from unity. When both mother and father had histories of preconception diag nostic x-rays, the order of magnitude'! the relative risk, 1.49, was about the same as that for children whose mothers alone had received radiation." Exposure to medical radiation has been widespread. By the early 1960's, approximately 35% of a group of mothers of healthy children reported having received diagnostic radiation prior to conceivipg. Similarly, about 22% of fathers of the same children reported having had some sort of diagnostic radiation at some time prior to the child's conception. Self-reporting considerably under estimates the extent of x-ray exposures." The tendency to substantially underreport x-ray exposure points up the dif ficulty of excluding differences in prior radiation expo sure as a basis for observed differences in chromosomal aberrations Neither is it reasonable to automatically pre sume that medical radiation exposures have been equally present in the study and comparison groups, especially if these groups have not been matched by age or calendar period of observations. Occupationai Exposures and Pregnancy Outcome Vinyl Chloride.--Chromosomal abnormalities may oc cur more often than expected in persons exposed to vinyl chloride, especially following intense exposures of long duration. The import of such abnormalities for reproduc tive outcome remains ill-defined. Chromosomal aberrations were reported in 1975 in Journal of Occupational Medicine/Vol. 21, No. 9/September 1979 609 ucc 061043 Swedish vinyl chloride workers. Abnormalities appeared in (ells drawn from seven m,lies with oirup.ilionn! cxpo- Pregnancy outcome in the wives of men exposed to vinyl i liloride monomer (VCM) has been said to be less sure histories ot nine to 29 years (mean 1 b.fa years) at vinyl chloride concentrations which had declined to 20-JO ppm by 1974.'* The group exposed to vinyl chloride had aber rations in a total of 9 52% of all cells, compared to 1.94% for cells of three unexposed controls. Differences in the frequency of chromatid and isochromatid breaks ac favorable than that experienced by wives of a group of polymerization and polyvinyl chloride (PVC) fabrication workers,11 14 One comparison group, the polymerization workers, was believed not to have been exposed to VCM, wheras PVC workers had low VCM exposure. Data on pregnancies and pregnancy outcomes in the wives were counted for most of the disparity. Considerable variation was exhibited, with cells considered abnormal ranging from 1 5% to 19% in the exposed workers There was no apparent relationship between duration of exposure to vinyl chloride and the proportion of cells which were ab normal. Age. radiation exposure or other environmental factors were not evaluated, nor was it clear on what basis individuals had been selected for study. In New York State, chromosomal aberrations were re ported in somatic cells of 11 men who had worked in a polyvinyl chloride polymerization plant and who were be lieved to have experienced intense intermittent exposures to vinyl chloride in concentrations of over 500 ppm, as well as chronic, but unquantitated exposure for four to 28 years (mean, 15 years). Control samples were drawn from four males in the same factory who were not known to have had vinyl chloride exposure, and from six males from outside the factory. Complex chromosomal aberra tions (rings, dicentrics, fragments) occurred significantly more frequently in the exposed than in the control group, a result which was properly interpreted with caution. The authors point out the absence of age-matched controls and the substantial age difference between study group and controls. The frequency of chromosome breaks and gaps was unexpectedly high in both control and study groups.10 No relationship could be defined between dose derived from interviews and questionnaires administered to male workers. Participation rates for the groups queried ranged from 62% to 77%. Fetal death was de fined as any known conception which did not result in a live birth and rates were adjusted by paternal age. Mater nal age was not known, but was presumed to correlate closely with paternal age. Analysis of questionnaire re sponses suggested that the number tff fetal deaths per 100 conceptions was. higher in the VCM-exposed group than in the comparison group. This difference was reported on ly for the period following vinyl chloride exposure. Ad justments removing women who were chronic aborters eliminated statistically significant differences. While the authors felt that these observations were likely to reflect a real difference in pregnancy outcome not attributable to either interviewer or patient recall bias, the conclusions were based on indirect sources of information and could not take into account the multiplicity of maternal factors known to affect pregnancy outcome. The study design precluded documenting in even the crudest manner the validity of pregnancy histories. Without, such adjustments and validation, the inferences made by Infante and col leagues" 14 cannot be sustained and little light is shed on the possible association of abnormal pregnancy outcome with paternal occupational exposure to VCM, Studies of pregnancy outcome (i.e., spontaneous abor or duration of exposure and frequency of chromosomal aberrations Investigators reporting from the United Kingdom studied 56 men who had had chronic exposure to vinyl chloride monomer in the course of manufacturing poly vinyl chloride and compared them with 24 unexposed in dividuals." Vinyl chloride exposure of men employed in its manufacture could not be easily quantitated. Individ uals with exposures to radiation or with recent viral infec tions or prolonged drug treatment were excluded. Samples were coded and read blindly. A higher propor tion of cells with chromosomal aberrations, the majority of which were breakages, was found in vinyl chloride-ex posed workers than in controls Unstable and stable chromosomal aberrations and breaks were all signifi cantly more common in cells from exposed workers Not all investigators have identified such changes No differences were found in frequency of chromosomal aberrations between a group of 209 employees of a vinyl chloride plant (occupational exposures averaging 48 3 months) and a group of 295 individuals undergoing pre employment physicals," No relationship was established between duration of vinyl chloride exposure and degree of chromosomal damage, (he absence of blind evalu ation in this study, as well as inclusion of individuals with minimal exposures in the exposed group, makes interpre tation difficult. This difficulty is compounded by the sub stantial difference in mean age of study and control group members. tion. late fetal death [stillbirth], low birth weight, neonatal death) in other settings have shown the profound and sub tle effects of confounding variables such as race, socioeconomic status, maternal age, birth order, parity, smoking and alcohol exposure during pregnancy, mater nal infections, and previous pregnancy outcomes. These effects may readily reverse the direction of the relation ship between a suspect antecedent factor and the out come of pregnancy " Future efforts to document a rela tionship between impaired pregnancy outcome and occu pational exposures of fathers must validate information on pregnancies and take into `account known con founding factors. Chloroprene. -- Structural similarities between vinyl chloride and chloroprene have raised questions about long-term hazards resulting from chloroprene exposure. Chloroprene is mutagenic in certain systems and possibly carcinogenic The possibility that it has induced excessive miscarriages in the wives of male workers and led to chromosomal aberrations has been asserted but not well documented '* Benzene -- Benzene also may cause chromosomal aberrations following heavy occupational exposures. Twenty males working in a factory in which benzene had been used as a solvent were Studied for chromosomal aberrations Members of this group had one to 20 years of benzene exposure; 14 were known to have previously been neutropenic Chromosomal abnormalities were re ported in 2 5% of all cells from exposed workers, com- 610 Risks to Of(spring from Parental Occupational Exposures/Haas and Schottenfeld UCC 061044 IMInl lit I l) III I 1% IlH I IMlIluls Most III ill!' I'M OS' W.IS due lo i hmmosom.il .iix'fMlMnn iif Hu- unsl.ihlo ly|M's In .iililiiiiin iii tin1 i-xu-ss ill .ilnMmn.il mill m tin* I'xposi'il yroup. the number of unstable alterations per abnormal cell was higher in the exposed group than in the control* " In a separate investigation, ten workers in an Italian rotogravure plant who had had sequential Exposures, first to benzene and later to toluene and xylene, were studied, 1 hese workers had been subjected to concentrations of benzene ranging Irom 125 ppm to 532 ppm or higher. 1 he maximal allowable concentration at the time of these ex posures had been 25 ppm Another group of 24 workers had primarily toluene exposures except for trace contamination with xylene These 34 workers were matched with healthy controls of similar age and sex, who had been drawn from the general population and who had no history of benzene or radiation exposure, A signifi cantly higher proportion of abnormal cells was found m the benzene-exposed individuals than in their matched controls. In the group with toluene exposure, no such ex cess was present. When the benzene and toluene groups were compared to each other, an excess of chromosome changes was present in the benzene group. Most of the excess was attributable to unstable chromosomal altera tions.'* These studies and others" support the impression that exposure to benzene, particularly exposures inteqse enough fo7esult~in acute toxicity, can be followed by a measurable excess of chromosomal aberrations which may pefsisFTdr years. If similar effects occur in germ cells of exposed individuals, the consequences in fetal wastage, congenital anomalies and even neoplasms might be manifest in their progeny. To date no excess of such abnormalities has been reported in offspring of benzeneexposed individuals. Anesthetic Cases. -- Occupational exposures of female operating room personnel to waste anesthetic gas es have been held responsible for decreased fertility, in creased rate of spontaneous abortion, low birth weight and possibly impaired development in the offspring. An excess of spontaneous abortions in wives of men pro fessionally exposed to anesthetic gases has been suggest ed but not confirmed. Among operating room nurses, 29.7% of pregnancies terminated with spontaneous abortion, compared to 8.8% among control nurses. A similar excess of spon taneous abortion was observed for female physician anesthesiologists compared to female physicians in other specialties.*" Spontaneous abortions occurred two weeks earlier on the average among the operating room ex posure groups than among controls, at an average of eight rather than ten weeks of gestation Another survey suggested that for female nurse-anesthetists who had worked during pregnancy, the frequency of birth defects in the offspring was 16 4% compared to 5 7% for pregnancies in which the mother had not worked.*1 Studies from Finland and the United Kingdom also sug gested a deleterious effect on pregnancy outcome from maternal exposure to operating room environments An increase in frequency of early spontaneous abortions, as well as low birth weight, has been reported for Finnish fe male operating room staff41 Among female physicians in I Mgl.iml ami Wales, gie.iler iiHidemo ll iongenil.il ilefei Is, lower birth weights and higher stillbirth rales, but not biglier spontaneous abortion rates, were reported from pregnancies of women holding anesthesiology appointments than from those of other women physi cians *' The effects of paternal exposure to anesthetic gases are uncertain Congenital anomalies were reported to be increased by 25% in the offspring of male anesthesiolo gists compared lo offspring of male members of the American Academy of Pediatrics, but no difference was reported for spontaneous abortion rates among wives of male operating room personnel compared to controls ** Questionnaires completed by 5119 married male physi cians in the United Kingdom suggested an increase in minor but not in maior congenital anomalies, and no dif ference in frequency of infertility, spontaneous abortion or cancer in the offspring of male anesthesiologists com pared to control physicians.*' Hydrocarbons. -- Two studies offer conflicting evi dence with respect to the risk of cancer m children of men whose work might lead them to be exposed to petroleum-derived hydrocarbons Fabia and Thuy** re viewed death certificates in Quebec during 1965 to 1970 to identify children who had died from malignant disease while under five years of age For 386 of 402 such children, birth certificates were also found in the Quebec population register A control group of 772 children (two for each cancer death) was selected using the birth registration record preceding and following that of each case in the official files. This effectively matched for season, calendar period, and province of birth. Occupa tion of the father at the time of birth was taken from the birth certificate. An industrial hygienist independently grouped the fathers into three levels of probable exposure to petroleum-derived hydrocarbons. Occupation of the father was unknown for 30 cases and 56 controls. The dis tribution of occupation of the father at birth differed for cases and controls. Most of this difference was the result of an excess among cases of paternal occupations con sidered to be hydrocarbon-related. The relative odds of cancer in the children of men holding hydrocarbon-re lated jobs at the time of the child's birth were 31, an in crease which was unlikely to have occurred by chance. Most of the excess in exposed occupations was account ed for by motor vehicle mechanics, machinists, miners and painters. When similar analyses were conducted by the type of cancer in the child, the excess of hydrocarbonrelated occupations prevailed for the following group ings: leukemia-lymphoma, nervous system malignancy, other tumors. Fathers of four of five cases of Letterer-Siwe disease were in the hydrocarbon-related work groups. No such excess was present for children with Wilms' tumor. The differences observed could not be attributed to dif ferences in parental age or in geographic residence at the time of birth. The investigation was based on deaths in children under five, and it is possible that social class bias may have been introduced in that fashion. If, of those with a childhood neoplasm, children of more affluent parents are more likely than children of poorer parents to survive beyond age five, they would not be included in the study group. This would lead to a higher proportion of children with parents of lower socio-economic status in Journal of Occupational Medicine/Vol. 21, No. 9/September 1979 611 ucc 061045 tin* t ,iih it iIimIIi group iIIhI would ln.i' the distribution ol f.illiiT s ik i iiji.ition Moreover, il w.ts not possiblt- to i ik* linn lh.it l.ithrrx in th* so-c.illod "rxpospd" group did, in f.u t. h.ive i ont.ict with hydrocarbon-derived chemicals While not uleal, the death certificate probably is a valid source of diagnostic information for cancer deaths in young children fhe cancer groupings employed in the analysis - 'leukemias and lymphomas," "nervous system" and "others" -- were quite broad and included many histopathologic entities Oi ( upational assiHMtinei was not restrw ted to specific entities wtthm the broader categories. Because the epidemiologic features of the various histopathologic types of leukemias, lymphomas, and nervous system tumors are distinctive, a specific chemical exjiosure or class of exjiosures, if assumed to be of causal significance, would more likely be linked with specific histopathologic as well as organ system effects. A study with a similar design but different findings was conducted in Finland.*' Children under 15 years of age who developed cancer were identified from the Finnish Cancer Registry, 1959-1968. Of 1409 cancer cases so iden tified, the final series consisted of 852 pairs for whom birth records which included father's occupation were available. Each case was matched with a child whose birth date immediately preceded that of the case and who was bom in the same maternity welfare district. This procedure matched effectively by calendar period, season and domicile at the time of birth. Father's occupa tion was drawn not from birth registration records but from records of the free, nationwide antenatal care system in operation in Finland. Father's occupation was classified by likelihood of hydrocarbon exposure in a manner which was shown to be comparable with that employed in the study done m Quebec No excess risk for hydrocarbon-exposed fathers was detected for any class of neoplasms either for children whose cancers occurred under five or under 15 years of age. The two studies differed primarily in the source of in formation on father's occupation. For the Finnish study, that information was drawn from antenatal records com piled for the most part in the first trimester of gestation. This may be a better index of exposure at the time of con ception than information recorded at the time of birth in the birth certificate itself. Although a large number of pairs were discarded in Finland because father's occupa tion was not recorded, these came from a circumscribed calendar period. Since a matched pairs design was main tained throughout the analysis, removal of these cases should not influence the result. The Finnish study used in cident cancer cases and looked at age groups up to 15 years at the time of cancer diagnosis. The Quebec group, identified only cancer deaths up to five years of age Variation in terminology of occupational classification might also contribute to the differences, although efforts were made to establish their comparability At present, the contradictory findings of the two studies are not readily reconciled. Surveillance of Spontaneous Abortions as a Strategy of Environmental Monitoring The relationship between mutagenic, teratogenic and carcinogenic effects in the offspring of persons exposed to noxious environmental agents is a complex one SurvrTlI.inci* of spontaneous abortions may have a ihhuIkt of advantages as ,i prospei live means of monitoring for such effects since defective conceptions are aborted selectively As a result, studies of teratogenesrs focusing on spontaneous abortions may he con siderably more efficient than those conducted in new borns Because the frequency of abnormalities is higher in s[Kwlaneous abortions, (he sample size needed to demonstrate a c hange in risk is much smaller than that requiretf for a parallel query addressed to defects recog nized at birth, fhe magnitude of this difference can be dramatic. It chromosomal defects diagnosable on the ap pearance of the newborn are considered alone, the sam ple size required may be hundreds of times that required in an investigation examining prevalence of chromosomal anomalies in early abortion.** In addition to sample size conskkr.itions, study of stiontaneously aborted concejstions offers a lead time of at least six months over studies of live births. The abortion specimen can be studied with care and thoroughness, permitting detection of anomalies lethal to the fetus which might escape detection in studies of live births. The problem of power and sample size is only one of the issues which beleaguer investigators of associations between exposures to parents and outcomes of pregnan cy. Related factors are the timing of the exposure, mater nal or paternal, in relation to conception and/or gestation, and the specific measures of pregnancy outcome.** Pater nal exposures may act in two ways -- by contamination of the maternal environment resulting in secondary maternal exposures, or directly by affecting paternal ger minal tissue. Since spermatogenesis is a continuous pro cess, paternal exposures occurring shortly before concep tion are those requiring the most investigative attention Cumulative or delayed effects are more likely to be of im portance in maternal exposures. By focusing on early pregnancy wastage, particularly if the frequency of chromosomal abnormalities in the aborted product of conceptions can be determined, the objectives of study are better focused and achieved with reduced sample size, and the confounding effects of the maternal in utero environmental factors are minimized. In conclusion, while the potential clearly exists for teratogenesis and trans-generationaI carcinogenesis in the off spring, of workers exposed to mutagenic and carcinogenic agents, such effects have been difficult to demonstrate conclusively in humans. Future investigations must take into account a variety of environmental and behavioral factors which can affect fetal development if causal associations between occupational exposures and pregnancy outcome are to be identified. References 1 O t O The Compart Edition of the Oxford English Dictionary Oxford Oxford University Press, 1971, cites Dunglison Medical Lexicon, 1BS7 2 Wilson ic Environment and Btrth Defects New York Academic Press. W I StapfesRE Definition of teratogenesis and teratogen, in Methods for Deles non of fnvironmental Agents that Produce Congenital De fects, I H Shepard et al (Eds) Proceedings of the Cuadaloupe Con ference Amsterdam North Holland Publishing Company. 1975 4 Eauroeni IF, tr The susceptibility of the fetus and child to chemical pollutants Chemicals in human teratogenesis and transplarental carcinogenesis Pediatrics S3 807-012. 1974 612 Risks to Offspring from Parental Occupational Exposures/Haas and Schottenteld ucc 061046 ' I M'.itflii |A .iimI kotm I1 J*i.ihtgi,ni-\t'* <mm ogi-m-M1* A study ot iH.ilMmd|<* Wi h r.Hhoi III l J I, MN*l l> Km |M An ov-\ n*w of cnl.il < brum.il(.in mugimeMs M.tfu/ogy H M 1 UV 1**71 7 ( l.wrrn %K .iimI Amtfh DW I he fet.il ahohol syndrome N fog/ ) 298 l%M%7, 1978 8 lillel.mil < N the offspring ol .i)roiw>li< mothers Ann Ni Acad Sm 197 l(>7 169, I972 9 I ones ki .mil Smith OW KiMngmUnn ot the let.il nltohol syn dromi* mi Mii\ ml,mi y I .UN et 2 'Itf liml, 1971 10 ()uel|e|lr IM, Kosett ( II r Kosm.mJ'N, .mil Werner l Adverse el- lt-< Is on oilspring nl iimtern.il .ill nhol .iImim* iluring pregnant y N / nul / Med 797 5211 r>HI. 1977 M Kromck |H feratogeisK ottetts ot ethyl .dcohol administered lo pregnant lime Am / ObsfCf Cvneco/ 124 676680, 1978 12 Kline |. Stein /A. Susscr M and Wnrlmrton 17 Smoking A risk ln< tin lor v|Hinl.miM>ir, alxMlmn N Inal ) Mini 297 791-7%, 1977 1 l Kush I) .iik) K.*s I MiiCrrn.il smoking A reassossinent of the as sociation with |x*nn'it<il mortality Am / Ipidcmiol 96 181*1%, 1972, 14 Mrynt MW. Iona* US. and Tonasoa JN Perinatal events as* mm rated with m.ilcmiil smoking during pregnancy Am j Epidemiol 103 464 476. 1976 15 Stein /, Stm<TM, and Warhurton D Spontaneous abortion as a screening device I lie ?ffe< t ol (out survival on the incidence of birth defects Arn / tpnbmiol 102 275-290, 197S 16 Alherman I. Creasy M Elliott M, et al Maternal factors associated with fetal chromosomal anomalies in spontaneous abor tion* Br I Obutet Gynaecol 83 621-627, 1976 17 Freriru k }, Alberman ED. and Goldstein H: Possible teratogenic elfet I ol ugatette smoking Nature 231 529-5 VO, 1971 18 Richard* IDC: Congenital malformations and environmental in fluence in pregnancy. Br ! Prev Soc Med 23 218-225, 1%9 19 Himmelhorger DU. Brown RW, )r. and Cohen fc'N Cigarette smoking during pregnancy and the recurrence of spontaneous abor tion and congenital abnormality Am I Epidemiol 108 470-479, 1978 20 Horhst Al and Scully RE Adenocarcinoma of the vagina in adolesi ence A report of 7 cases including 6 clear-cell carcinomas (so called me'onephroma*) Cancer 25 745-757, 1970 21 HerbM AL. Ulfelder H, and Poskanzer DC Adenocarcinoma of fhe vagina Association of maternal stilbestrol therapy with tumor ap pearance in young women N Engl I Med 284 878*881, 1971 22 Herbst AL, Kurzman R|, Scully Rfc and Poskanzer DC Clearcell iidenocan moma of the genital tract m young females N Engl) Med 287 1259-1284, 1972 21 Herb*t AL. Poskan/er DC, Kobboy SI, et al Prenatal exposure to stilbestcol A prospective comparison ot exposed female offspring with unpxposrd controls N Engl / Med 292 J34-J39, 1975 24 Hemonen OP Birth Defects and Drugs m Pregnancy Littleton. Ma*s PSC Publ Co. 1977 25 Kato H. Srhull W|. and Neel IV A cohort-type study of survival m the t hildrrn of parents exposed to atomic bombings Am / Hum Genet IB 119 171. 1 %f> 2<> Hoshmo L Kaio H, Finch SC and Hrubec Z leukemia in off spring of atomic bomb survivors Blotxl 30 719-729, 1967 27 Graham S, levin ML, Lil'enfeld AM, et al: Preconception, in trauterine. ami postnatal irradiation as related to leukemia Nat/ (sneer Mst Monogt 19 147-171. V*66 2H Grtihani S. levin Ml. Lilienfeld AM. et al Methodological prob lems and design of the Instate leukemia survey Ann NY Acad Sci 107 V*7 Mi9 |`H t 29 I itnr> t lavMito I, I.uhImm! H, I mihlrli |, r| ,ll ( Iiiiiiihim hiii> ,i)M-irtilion*> in worker* ex|>oM'd to vinyl r hlornb' (letter) (anu9 1 459, 1975 U OiK.ilnMn A Hum horn K, and Selikoti l| Vinyl rhloridi* |k>miii` .nul human (bromovome alierrations Mufat 31 16I-M41, 1975 11 Pun h.i%e II ft, KmIi-uiKom ( K, 4m<) Anderson D ( fuomosoni.il ami dominant letlial ettiM Is v>l vm> 1 < blonde Lancfl 2.410411 1975 12 Pm i.ino (>|. Hake Wl (.ay It, and Kilian Ul Vinyl chloride < ylogeneln 11 / On up Mod 19 527 5 tt), 1977 H Ini.tnte It W.igoner |k Mi Mu )iai*l Al. (9 al (iemMii risks oi vinyl < hfonde Lancet 1714-7(5. 197b 14 Infante PF. Wagoner |K, Mr Michael Al. el al- Genetic risks ot vinyl i hkinde Lancer 1 1289 1290. 197b 15 Haling |R and f manuel l lndu< ed abortion and subsequimt out< ome ol pregnam y in a smes of American women N Engl I Med 297 1241 1245, 1977 lb Infants* PF, Wagoner JK. and Young R) Chlorofireix* OliM-rva- tions of carcinogenesis ami mutagenesis, in Origins of Human (',an<(*r Book A Incidence of Cawer in fluinans, H It Hiatt, 1 D Watson, and \ A Wmsten (Vds) Coldspring Harbor Laboratory. 1977, i>p 205-217 37 Tough IM and Court Brown WM Chroniosome aberrations and exposure to ambient benzene / ancet 1684, 1975 38 Forni A, Pacifico E, and Limonta A Chromosome studies in worker* exposed to benzene or toluene or both Arch Environ Health 22 373-378. 1971 J9 Form AM. Cappellim A. Packfito E. and Vigliani FC Chromosome change* and their evolution in subtects with past exposure to benzene Arch Environ Health 23,385*391, 1971 40. Cohen EN. Sellvttle JW, and Brown BW- Anesthesia, pregnancy and miscarriage. A study of operating room nurses and anesthetists Anesthesio/ogy 35 343-347, 1971 41 Corbett TH, Cornell RG, Endres )L, and Liedmg K Birth defects among children of nurse-anesthetists Anesthesio/ogy 41 341-344, 1974 42 Rosenberg P and Kirves A Miscarriages among operating room theatre staff Acta Anaesthestol 5cand /Supp/J S3 37-42, 1973 43 Pharoah POD, Alberman fc, and Doyle P Outcome of preg nancy among women in anesthetic practice Lancet 1 34-lb. 1977 44 Cohen EN, Brown BW. (r, Bruce DL, et al Occupational disease among operating room personnel A national study Anesthesio/ogy 41 321-340. 1974 45 KmlMones KP, Newman 11), and Spence AA Anaesthetic prac tice and pregnancy Controlled survey of male anesthetists m the United Kingdom Lancet 2 807-809, 1975 4fc Fabia I and Thuv TD Occupation of father at time of birth or children dying of malignant diseases Br / Prev Soc Med 28 98*100 1974 47 Hakulmenen T, Salonen T. and Teppo L Cancer in the offspring of father* m hydrocarbon-related occupation* 8t / Prev 5oc Med 30136*140. 197b 48 Kline I, Stem Z, Strobmo B et al Surveillance of spontaneou* abortions Power n environmental monitoring; Am I Epidemiol 106 345-350 1977 49 Strobino I3R. Kline ), and Stein Z Chemical and physical ex posures of parents E ffects on human reproduction and offspring Farly Hum Develop 1 371-399 1978 journal of Occupatiori.il Medic.ne/Vol 21, No, 9/SeptemOer 1979 613 ucc 061047 y.ilation Research, 41 (1976) 131--142 Z ls*vir/North-Holland Biomedical Pre* CARCINOGENIC, MUTAGENIC AND TERATOGENIC RISKS ASSOCIATED WITH VINYL CHLORIDE PETER F. INFANTE, JOSEPH K. WAGONER and RICHARD J. WAXWEILER I in ,tmn of Surveillance. Hazard Evaluations and Field Studies, National Institute for occupational Safety and Health. Main Post Office Building. Cincinnati, (Shio 45202 (U.S.A.) tR'-ceived May 13th, 1976) Summary The data presented demonstrate clearly that vinyl chloride (VC) is related to significant excess of mortality from cancer of the liver, lung and brain among workers occupationally exposed to VC. The risk of dying from cancer of the iymohatic and hematopoietic system also appears to increase with an increase in latency. These cancer sites could have been predicted by the animal bioassay conducted by Maltoni. With regard to the liver, even the histopathologic type of cancer (angiosarcoma) was observed first in experimental animals. A study of cancer mortality among populations residing proximate to VC polymeriza tion facilities also demonstrated an increased risk of dying from CNS and lymphatic cancer. These latter findings raise cause for concern about out-plant emissions of VC, but without further study these cancers obviously cannot be interpreted as being related to out-plant exposure to VC. Various test systems now have elicited a positive mutagenic response to VC. Thus, our observations of a significant excess of fetal mortality among the wives of males, who were occupationally exposed to VC, raise public health concern that VC may be mutagenic in humas. With regard to the teratogenicity of VC, observations of a significant excess of children bom with birth defects were reported among populations residing proximate to VC polymerization facilities. Additional epidemiologic study is needed to determine whether a repeated pattern of excessive numbers of children born with birth defects can be observed in other communities with VC polymerization facilities. Introduction In 1930, the first adverse health effects of vinyl chloride (VC) were reported (22). Since then, numerous investigators have reported the toxic effects of VC on the central nervous, system, the liver, the bones of the fingers and the lungs [2,5,9--12,18.20,28]. More recently, the study of VC toxicity has broadened to assess the spectrum of carcinogenesis [2,15,16.21,26,29,30], mutagenesis [1,3,4,6,13,14,23,24], and teratogenesis [8J. These efforts were stimulated by the work of Viola et al. [29] who, in 1971, reported the induction of tumors of the skin, lungs and bones in rats exposed by inhalation to 30,000 ppm of VC over a period of twelve months. Widespread concern for the carcinogenic activity of VC, however, did not occur until early 1974, when Creech and Johnson [2] reported four deaths from angiosarcoma of the liver among workers employed in the manufacture of polyvinyl chloride (PVQ resins. It was later learned that Maltoni had previously demonstrated VC-in duced hepatic angiosarcomas, lung adenomas, brain neuroblastomas, lymphomas and various other tumors in mice, rats and hamsters [15,16]. With regard to mutagenicity, several investigators have induced mutations via microbial test systems [1,14,24], Also, VC metabolites have induced mutations in mammalian cells [6]. In addition, reports from several countries have demonstrated significant excesses of chromosomal aberrations among workers exposed to VC as contrasted with those not exposed [3,4,13,23], Because of these observations and of the widespread exposure to VC among both workers and the general population, the National Institute for Occupational Safety and Health (NIOSH) in the U.S.A. undertook an epidemiologic program to evaluate the magnitude and spectrum of VC toxicity to humans. This program was 4-fold in nature and sought to evaluate: (1) The site-specific risk of cancer among workers exposed to VC [30]. (2) The risk of some cancers among populations residing proximate to VC polymerization facilities [8], (3) The site specific risk of congenital anomalies among populations residing proximate to VC polymerization facilities [8]. (4) The risk of fetal wastage among the wives of workers occupationally exposed to VC [7]. Cancer risk among workers exposed to VC To assess the neoplastic risk of workers exposed to VC, a population con sisting of employes from four VC polymerizing facilitates was selected for cohort mortality study [30], Since occupationally induced cancers often take many years to become manifest, the study cohort was restricted to workers who had achieved five or more years of employment and for whom at least 10 years had lapsed since initial employment. Thus, the exposure period was five or more years and the latency period was 10 or more years. Follow-up for study cohort members was greater than 99%. Table I shows the total mortality experience among the study cohort. The expected numbers are based on United States mortality data applied to NIOSH's modified life-table method. For all malignant neoplasms, there were 35 observed vs 23.5 expected, the SMR was 149. This excess was significant the P< 0.05 level of confidence. For total mortality, 136 deaths were observed vs 126.3 expected. Selecting a cohort with at least five years work experience and 10 years latency eliminated the "healthy worker effect" (19) and so there were more total deaths than expected and this was mostly at the expense of cancer mortality. Table II shows cancer mortality experience by greater than 10 and 15 year latency periods. At the greater than 10 year latency period, only biliary and { j ] 3 \HLE i .ktality experie l : of death \ " rriitlitnant neoplaalB* ! Aft \ .n-malouunl reeptntoi < \`I Mthrr cauiet ICO, International Cl*. sMR, standardized me s^mHcalU at P < 0.0 !>\or cancer death v.:ih 15 years of , avgories of canc i-ancer of the 1; :i-wever, the SM 1 iu-se comparisc . k cupationally-in : mile It .NCFR mortalit .<> VINYLCHLORIDI - : malignancy . riuUianant neoplasm n jnd CN3 cancer * * -juratory system car and liver canee "-irtwtlc and bemai -1 "ihwg malignant n bvrved. . \ peeled. '*4'{ uandaidued r H MfraAt at /* < 04 "iftCdAt at / < Q. 133 T4BLE1 MORTALITY experience among cohort workers exposed to vinyl chloride of death 1CD code 4 Observed Exptcttd SMRb - .ibtnant neoplasms n-'nalitnam respiratory diseases rr" i. nr causes (140--204) (400-443) (470-437) (481) 36 67 2 36 23.6 64.7 3.4 4.0 40.7 148* 104 ITS 40 88 ICO. International Classification o( ! 5 standardized mortality ratio. r Significant af /' < 0.05. 7th revision, li\ er cancer deaths were significantly in excess; however, when the sub-cohort ttith 15 years of latency since initial employment was used, deaths from three t;.i-gories of cancer were significantly in excess. The excess in mortality from i.a:uer of the lymphatic and hematopoietic systems was not significant; i ever, the SMR increased from 159 to 176 with an increase in latency. T.iese comparisons show the importance of latency when looking for ocr.i nationally-induced cancers. TC'LE II C\\'CER MORTALITY BY INTERVAL SINCE INITIAL EXPOSURE AMONG WORKERS EXPOSED T j . YL CHLORIDE '> f f malignancy 10+ yn 19+ Min V neoplasms Cfji- and CNS cancer 'w :*iory system cancer ttiliirv tnd liver cancer L'f'Tphitic And hematopoietic system cancer ' ' 1 nrr malignant neoplasms . observed. . f'xpceivd. : > itandardixed mortality ratio ''.r.ifieant At /* < 0.06. 'i-nificiuit at /* < 0.01. Ob. * Exp. b SMR * ObsExp. SMR Ob*. Exp. SMR Obx. Exp. SMR Obs. Exp. SMR Obt. Exp. SMR 36 23.6 149" 3 0.9 329 12 7,7 166 7 0.6 1144 * 4 2+6 169 9 11.7 77 31 16.8 < 184 * 3 0.6 < 498 d 11 4.7 < 194 d 7 0.4 < 1806 * 3 1.7 < 176 7 8.4 < 83 ucc 061050 134 In laboratory studies, Maltoni has induced hepatic angiosarcomas, lung adenomas, brain neuroblastomas and lymphomas [16]. Therefore, the predictive value of animal bioassay studies can be demonstrated, not only by the observation of a significant excess of angiosarcoma of the liver among VC workers, but also by significant excesses of lung and brain tumors. Cancer risks in populations residing near VC polymerization facilities i ' 4 The risk of some cancers among adult populations residing in the only three Ohio communities with VC polymerization facilities was assessed [8]. All three communities had at least one facility in operation by 1954 and Painesville had a second plant in operation by 1967. The community population sizes varied from 12.000--24,000. Between 1960--70 the population had remained stable in Painesville and Ashtabula, but had increased by 30% in Avon Lake. As reported previously, for the three communities taken as a whole, there were no apparent differences in racial origin or family income as compared to the aver age for the State [8], As a result of previous findings [26], cancers of the central nervous system, lymphatic and hematopoietic systems, were selected for study. Because of possible error in death certificate data in terms of metastases from other primary sites, data for lung and liver cancers were not analyzed. Table III shows data for observed versus expected CNS cancer deaths in the white population by sex for the period 1958--73. North Ridgeville is the onhcommur.ity shown which does not have a PVC polymerization facility. It was included in the analyses of the cancer data because it is located contiguous to Avon Lake and because it had a high incidence of children bom with birth defects [8]. If North Ridgeville had not been included in the analyses of cancer mortality, little difference in the results would have been observed. Expected values are based on the occurrence in the balance of the counties over the same period of time. As shown in Table III, there was a significant excess of CNS cancer deaths in males. The excess was greatest in Painesville and North Ridgeville. With all communities combined, there were 27 CNS cancer deaths observed in males versus 14.1 expected; the SMR was 191The difference was significant at P< 0.01. In females, with all groups combined, there was only a slight excess. With sex groups combined, the excess was significant at P < 0.01. It may be note worthy that one father-daughter combination for CNS tumor deaths occurred in Painesville. The daughter died of a papillary ependymoma in 1963 at the age of 16. The father died two years later of a glioblastoma multiforme at the age of 58. It would be difficult to determine whether this observation is the result of genetic or environmental factors. Table III also shows deaths from lymphomas. Although the differences between observed and expected deaths in males were not significant, there was a consistent excess in each community. In females, there were excess lymphoma deaths in two of the four communities. With sex groups combined, a significant excess of lymphoma deaths was observed in Ashtabula. With sex groups and communities combined, the number of observed deaths was 61 versus 48.7 expected. The SMR was 125. This was significant at 1 4 i ; 1 ! 1 t i ii i 1 I t i i A !.K HI .. it VCD AND EXPE . Ks AND OLDER IN . .1.ITIES. 19SS-73 .. numlxri of c*r .. m u hich the ..,-1*14 - ,;iii .ilu untile* hv-U - ilk ,rti U Mele: ob*.. CNS 71 12 2t e; 27/1 Leu) 13.M a. i; 2; 24-1 - ilii * .k1 ,-kt* mHr -in ur Lv 18/: 12' 4 3' 37/ 'wauonil ClAt&ifiC ' 05, oi. ' ' P< 0.10. '*= shown in ' " ical. 1 `- rtli defects amt ]' occurrenc :."iunities wi: ' Ridgeville mitial anal Ridgeville I'jent analy - <|uent analy among coir  135 hi WSVK'ED AND EXPECTED DEATHS FOR THREE TYPES Of CANCER FOR RESIDENTS 45 U \ = S and OLDER in THE OHIO COMMUNITIES WITH VINYL CHLORIDE POLYMERIZATION MC..1TIE5. 1958-73 -vp, - fQ numbers of cancer deaths based on occurrence over the tame period of Drat in the haitnct of c jp'ie* m which the communities are located. Males Females Sexes combined Ob*. 'Exp. SMR Ob*./Exp- SMR Ob*./Exp. SMR ;r -'wJc --lumrn r Irivd '-U nr .le ' L-ks 1 ,. v \.. e* r 'n.njt,"* r.-- ...--d CNS cancer (191 -192i* T 6.1 12 3.8 2, 2.3 6 19 ns 316 d 87 316 b 27 14.1 191 c 6/ 3.8 2/ 2.8 1/ 1,8 2/ 1.5 11/ 9.9 Leukemia anti aleukemia (204-207)* 13 14* a 7j 1 36 2 32 88 113 28 63 11/ 8.6 7/ 6.8 5/ 2.2 0/ 1,9 24 28 6 84 23/19. S 158 71 56 133 111 128 103 227 0 118 13/ 9.9 14/ 6.6 3/ 4.1 8/ 3.4 38/24.0 24/23.3 15/13.9 6/ 5.8 2/ 5.1 47/48.1 131 212 1 73 235 lit1 103 10S 103 38 98 .. ,.,s i. < At , kr - ~ - .nr., r -c L' mphomai <20Q~-203)* 18 12.6 12 10.4 4 3,3 3 2,8 143 115 121 107 37,29.1 127 14/ 5.8 4/ 8.3 5/ 2.9 1/ 2.6 24/19.6 02. 01. ( 002 Classification of Diseases, 8th revision codes. 241 * 48 172 38 122 32/18.4 16/18.7 9/ 6.2 4/ 5.4 61/48.7 174 6 145 74 125 0 05 < P < 0.10. Data were then analyzed for leukemia and aleukemia mortali ty As shown in Table III, the observed versus expected mortality was virtually identical. Birth defects among populations residing near VC polymerization facilities T: e occurrence of birth defects was studied [8] for the same three Ohio ""'..^unities with VC polymerization facilities. Birth data for residents of "in Ridgeville were not combined with data for the three index communities i" tr.e initial analyses (Table IV) because the high incidence of birth defects in v i .n Ridgeville. which lies contiguous to Avon Lake, was identified in 'Livequent analyses [8]. Data for North Ridgeville, however, were included in y.biequent analyses (Table V) for specific birth malformations. The observaI'jns among community residents were compared to both the occurrence in ^ s. '*-* 3r: UCC 061052 136 TABLE IV RESIDENT BIRTHS. MALFORMATION RATE PER 1000 LIVE BIRTHS IN OHIO AJ*D IN THRU SELECTED COMMUNITIES AND OBSERVED VERSUS EXPECTED MEMBERS OF MALFORM*. TIONS IN EACH CITY. YEARS COMBINED. 1970--73 Malformations arc basotl on codas 740--759 of tha Intamatiooal Clarification of Dittatsa. Stb 196*. ') I Malformation* Area Births Rata/103 Number obeerved Number axpected * X* i Enure state of Ohio Ashtabula city PainesvUle city Avon Luke citv All three commuiuui combined 719,2*7 1900 13*1 73* 4019 10.1 17.4 1*1 30.3 1*2 729* 33 2b 1* 73 * Expected number! Are based on state rate per 1000 Uve births. b/><0.01. ' p < 0.001, _ 19.3 14.0 7.5 40.8 -- 9C7b l<E3b 7J* b 27.13 c t \ a the balance of the counties in which the communities are located and to the occurrence in the State for the period 1970--73. Between 1970--73, the nte for birth defects changed from 9.3 to 11.3 per 1000 live births for the balance of the counties, from 9.6 to 10.8 for the entire state, whereas, the rate for the three index cities combined changed from 17.0 to 22.5. Thus, the incidence of birth defects for children bom in the index cities was almost twice as great as the incidence in the balance of the counties or in the entire State, and the differences appear to be increasing with time. Table IV shows data for malformation rates in each index community versus the rate for the entire State. These differences were all highly significant. The rates ranged from 17.47 in Ashtabula to 20.33 in Avon Lake, as compared to 10.14 for the entire State. When the community experience was compared to the occurrence in the balance of the counties in which the communities were ` I 5 ' TABLE V OBSERVED. EXPECTED AND RELATIVE RISK FOR SPECIFIC CONGENITAL ANOMALIES IS INDEX AREAS INCLUDING N. RIDGEVILLE, 1970--73 * Da feet eaisiory Number of defects RR b AU (lefeeta <740--758. 758. 7591 e Central nervous system (740--749) Cleft palate and Up (749) GemtaJ organs (752) Clubfoot (754) All other defects Observed 109 17 10 1* 23 43 Expected 56.0 5.6 6.5 8.4 82 27.2 IM 3.01 1.53 1.90 2.79 1.5* I ... ..u-d, the differs occurrence in i . -hate, or for t . _,mtal anomalie : ,iiile V shows c and selected n , observed, the | . c NS, cleft lip a i>een reported l, :,J mortality amt -.nee vc had ..-n nis [1.6,14,24 : mosomal abei . . o-od to VC [ .. '-..ii'.al mutation; ... -,a;n the incici alive) among n \ nwrization ar idl'd for stud\ . . . ;cd from wo -idled as a grou a workers' wive i.r.vtlv through . r.t. Data for .ir.isted with d r.trols"), who r- c lively, the data in T: ruing to the ; ic death rates *' 'i i lively. Subs - . respective!; can be seei end's exposui > t ars of age a: i - T.ury VC exp< ' -bands less th. "i and prim rent. P< O.C ' .al mortality practice of p - where occ .. however, r located, the differences remained significant. Therefore, whether you compare ;he occurrence in the communities to the expected, based on the average for he State, or for the balance of the counties, the excess of children with congenital anomalies in the communities appears to be significant. Table V shows observed versus expected numbers and the relative risk for !OtaJ and selected malformations. Although an increase in most organ systems ujs observed, the greatest excess of severe defects included malformations of C'NS, cleft lip and palate, club foot and genital organs. These observations have been reported previously in more detail [8], Fetal mortality among wives of workers exposed to VC Since VC had elicited a positive mutagenic response via microbial test -\rkms [1,6,14,24] and also had been associated with significant excesses of .hrcmosomal aberrations in the lymphocytes of workers occupationally exposed to VC [3,4,13,23], concern was expressed that VC may induce terminal mutations. Thus, a questionnaire-interview survey was conducted to asci-rtain the incidence of fetal loss (defined as any product of conception not orn alive) among wives of workers exposed to VC [7a]. All current VC polymerization and polyvinyl chloride (PVC) fabrication workers were ..k: tided for study together with a similar number of current rubber workers kltvti.d from work areas relatively free from known toxic materials and matched as a group to the VC workers by age. No interviews were conducted wnn workers' wives and no data were obtained concerning maternal age, except ad rectly through paternal age. Group participation rates ranged from 62--77 percent. Data for die wives of VC polymerization workers (study group) were contrasted with data for the wives of PVC fabrication and rubber workers i controls"), who were known to have had very low or no VC exposure, respectively. The data in Table VI show the paternal age distribution for fetal deaths according to the husband's exposure. Prior to husband's exposure, the crude fetal death rates for the control and study group were 6.9 and 10.1%, respectively. Subsequent to husband's exposure, the crude rates were 8.8 and 16.5T, respectively. As can be seen in Table VI, the excess in fetal mortality subsequent to husband's exposure, was associated with younger-aged husbands. For husbands 30 years of age and older, the rates for the control group 17/142 (12.0%) and primary VC exposure group 9/69 (13%) were about the same; whereas, for husbands less than 30 years of age, the rates for the control group 7/131 (5.3" i and primary VC exposure group 14/70 (20.0%) were significantly different, P < 0.001, x3 = 10.52, with one degree of freedom (df). The excess f fetal mortality among wives of younger-aged husbands may be a reflection a practice of placing newly hired personnel because of little or no seniority, m jobs where occupational exposures to VC may have been worse. This hyponc-sit. however, needs further assessment in other working populations. Since parental age was positively correlated with fetal mortality in this population and m a previous study [25], fetal mortality rates for the study group were adjusted to the age-distribution of the control group. 13S TABLE VI PATERNAL ACE DISTRIBUTION FOR FETAL DEATHS ACCORDING TO HUSBAND S VC E.\pa SURE Paternal *ge group (years) "Controls** Primary VC Exposure 1 <20 20-24 25-29 30-34 >3S All ages crude rate Mean paternal age at conception (v*ar) Age*adiusied * rate >20 20-24 25--29 30-34 >35 All ages crude rate Mean paternal agt at concepucn (year) Age* adjusted * rate Pregnancies (N) Fetal deaths <N> Prior to husband's exposure 31 80 38 6 4 159 23 0 2 6.5 4 5.0 4 10.5 1 16.7 0 0.0 11 6.9 <6.9) Subsequent to husband's exposure 1 43 87 87 55 273 30 4 0 0.0 4 9.3 3 3.4 7 8.0 10 18.2 24 8.8 (8.8) Pregnancies lN> 7 44 56 27 14 148 26.4 0 22 48 36 33 139 30.2 Fetai dead* (N> r. - i 0 00 2 4.1 7 125 5 18 5 9 1 7.1 15 10.1 ' Ij ! 0 0.0 3 13 0 11 22.9 3 8,3 i 6 18.3 i 23 16.5 <]5.t' fi 1 Fetal mortality rates for primary VC exposure group are direct age-adiusted to the paternal agr-dutribv tion of the pregnancies in the control group (shown in parentheses). data in Tat . ,.f male worK , .-..res, the rat* .lively. Subs . - for the contr ...... -icnificant at .7. t\:* 4.84,df ,;ed changes i , liange from ( iiusband's e: h.025, x3 * S-' exposure a ] :is. after exp r reduction i) . rence in the - n in Table V . croup was i . whereas, su - were virtu; >!*.s mean ag< determine w . have weight . y VC expos r four or r .calculated . .e rate coul< M'ned for eac TABLE vn mean paternal, number of pregnancies and age-adjusted fetal death rates ACCORDING TO HUSBAND'S VC EXPOSURE "Controls'*1 Primary VC exposure b Number of families Mean paternal age at conception (years) Number of fetal deaths among wives Number of pregnancies Age-adjusted fetal deaths/100 pres. e Number of families Mean paternal age at conception (years) Number of feta) deaths among wives Number of pregnancies Age-adiusted fetal deatha/100 preg. c Prior to husband's exposure 98 23.0 11 159 6.9 70 26.4 15 141 6.1 Subsequent to husband's exposure 113 62 30.4 30.2 24 273 8.8 23 139 15.3 d 1 Rubber mb P\"C fabrication workers. ^ VC polv mrnrxtion workers. c Ratal age-.diusted to "control" group paternal age distribution. d Subsequent to husbands' exposure, the frequency of fetal deaths among wives was stgiuficantlv greater in the primary VC exposure group as compared to the "contrail" (P < 0.05) or to the frequency in tl Study group pnor to husband's exposure (P < 0.02} bv aae adiusted Chi-square testing. \ r.i I. OF pregna - VC EXPOSUI 'the primary VC :.-vu*nd's expos 'iliintJ'i exposu ^nd's ex post end's expoiug -'band's ex post '0*nd't exposur 139 The data in Table VII show fetal death rates per 100 pregnancies for the tv:\es of male workers in the control and study groups. Prior to their husband's eNposures, the rates were 6.9% and 6.1% for the control and study groups, respectively. Subsequent to husband's exposure, however, the rates were $,$% for the control group versus 15.8% for the study group. This difference was significant at the P< 0.05 level by Mantel-Haenszel Chi-square testing (17] 4.84, df = 1). Further, the before and after exposure comparisons indicated changes in rates from 6.9 to 8.8% for the control group as compared ;<j a change from 6.1 to 15.8% for the study group. The rates for before and 3f:er husband's exposure in the study group were significantly different 0.025, x:_5"8. df* 1). For the study group, it may be noted that before exposure age-adjustment reduced the crude rate from 10.1 to 6.1%; whereas, after exposure the crude rate was reduced from 16.5 to 15.8%. The greater reduction in rates for the before exposure age-adjustment resulted from a difference in the age distribution of the husbands in the two groups. As can bo seen in Table VII, prior to husband's exposure, the mean paternal age in the study group was 26.4 years as compared to only 23.0 years for the control group, whereas, subsequent to husband's exposure, the mean ages of the two groups were virtually the same, i.e., 30.4 years versus 30.2 years. In both situations mean age was a good measure of central tendency. To determine whether women who had chronically experienced abortions rr..".t have weighted the results in favor of a higher fetal mortality rate in the \ary VC exposure group, data for the pregnancies of women who had two, i rue or four or more spontaneous abortions were eliminated. The data were u.uj-, recalculated to determine whether or not the trend of a greater mis(.i.rnage rate could be maintained. As shown in Table VIII, the trend was maintained for each analysis. \bleviu M MBER OF PREGNANCIES AND AGE-ADJUSTED FETAL DEATH RATES ACCORDING TO HUSB.ND'S VC EXPOSURE EXCLUDING PREGNANCIES IN WOMEN WITH >2, 3 OR 4 FETAL deaths HaU't for the primary VC exposure croup are afe*adjusted to the Control croup Controls Prunary VC exposure Number of pregnancies Fetal death rate Number of pregnancies Fetal death rate 8tfore husband's exposure After husband's exposure ^2 Fetal deaths excluded ISA 2&S 5,8% 4.7% 126 111 1.7% 6.2% B- husband's exposure ' ' husband's exposure ^3 Fetal deaths excluded 159 265 6.9% 6.8% 141 120 3.1% 10.8% 'u husband's exposure A tt< r husband's exposure 2^4 Fetal deaths excluded 159 265 6.9% 6.3% 142 127 5,8% 11.8% 140 As reported previously [7a], additional analyses suggested that the significant excess in fetal mortality after the husband's exposure would not seem to be the result of bias from interviewers nor from respondents. Because of the highly volatile nature of vinyl chloride [27], carry-home exposure to the wife would seem unlikely. Therefore, the leading possibility for the mechanism involved would seem to be germ-cell damage in the male through direct VC exposure. References 1 Bknscn, H.. C. MriavieOe and R Moracuno. Human, rat and mou tivermediated mutagenicity of vjfiv) chloride in 5. lyphimurium strains. lot. J. Cancer, 15 (1975) 429--437. 2 Creech. J.L.. Jr. and M.N. Johnson. Angiosarcoma of liver in the manufacture of polyvinyl chloride, l. Occu? Med.. 16 <1974) 150--151. 3 Ducatman, A.. K. Hirichhorn and l.J. SeiOcofL Vinyl chloride exposure and human chromosome abtrra'.iom. Mutation Re*,. 31 (1975) 163--169. 4 Funes-Cravioto, F.. B. Lambert alic J. Lindsun, Chromosome abenauons in workers exposed to vinyl chloride, Lancet 1 (1975) 459- 3 G<?dnpk, P.. R. Muller and H. Beefctclsheimer. Morphology of lit et damage among polyvinyl chJoridv workers. Ann. NY Acad. ScL. 246 (1975) 278--294. 6 Huberrrun. E.* H. Barlach and L. Sachs. Mutation induction in Chinese hamster V79 ceils bv tee \ir\j chionde metabolites, chloroeihy lene oxide and 2^jhloroacetaidehyde, int. J. Caocs, 16 (1975| 63&--?44. 7a Infants. P.F.. J.K. Wagoner and A.J. McMichael. Genetic risks of vinyl ehloride. Lancet. 1 (1974) 734--"35. 7b If far:*. P.F.. J.K. Wagoner, A.J. McMichael. R.J. Waxweiler and H. Falk, Genetic risks of vinyl chloridr* Uictl. 1 (1978) 1289--1290. 8 Irnante. P.F., Oncogenic and mutagenic risks in communities with polyvinyl chloride producuoa LciliUes. Ann. NY Acad. Sci.. 271 (1976) 49--57. 9 Krarr.er. C.G. and J.E, MutchUr. The correlation of clinical and environmental measurements toe workers exposed to vinyl chloride. J- Amtr. Ind. Hyg. Assoc.. 33 (1972) 19--30. 10 Lafiy#, C.E., S. Juhe, G. Stein and G. Veltman, Further results in polyvinyl chloride productive u jrk^rs. Ann. NY Acad. Set., 246 (1975) 18--21. 11 Lister, D.. L.A. Greenberg and w.ft. Adams, Effects of single and repeated exposures of huouns and rats to vmvl chloride, J. Amtr Ind H* g. AtSOC** 24 (1963) 265--275. 12 LJis. R.. H. Anderson. W.J, NicooLson. 5. Daum. A.S. Fiachbeui and IJ. Sclikoff. Prevalence of disease among vinyl chloride and polyvinyl chloride worker*. Ann. NY Acad. Sci, 246 (1975) 22--41. 13 Loken, E. and E. Thiis*Evenaen, Preliminary report on the medical examination of 286 employees*; th* P\fC plant. Norsk Hydro a,*,. Porsgmnn Fabrikker. Forsgnmn. Norway, unpublished* 14 Lopneno, X., R. Barrie and S, BaronceUL Evaluation of the genetic effects induced by vinyl thlondr monomer (VCM) under mammalian metabolic aetivotioo. studies in vitro and in vivo. Mutation Re*. 40 (1976) 85-95, 15 Mritoni. C.. Preliminary report on the carcinogenicity bloassays of vinyl chloride, U-S* Dept. Leant Informal Fact-Finding Hearing on Possible Hneeds of Vinyl Chloride Manufacture and Uv, *ati- ingtoo. D.C,, February 1974. 16 Mritoni. C. and G. Lefemioe, Carcinogenicity biongtayt of vinyl chloride. Current results, Ann. M* Acad. ScL. 246 (1975) 195--219. 17 Mantel, N. and W. Haensseh Stausticri aspects of the analysis of data from retrospective studies id disease. J. Natl. Cancer. Inst,. 22 (1959) 719--749. 18 Vars~.eUer. H.J.. W.K. Laibach. R Muller and P, Getiigk. Unusual tplenomegtifc liver a* e'1* di-nced by peritoneoscopy and guided liver biopsy among polyvinyl chlonde production workers. A*NY Acad, Sci.. 246 (1975) 95--134. 19 McMicbae). A.J,. 5.G. Haynes and H.a, Tyroles. Observations on the evaluation of occupation^ rronality data. J. Oceup. Med,. 17 (1975) 129--131* 20 V.JUr. a.. A.S. Teintcin. M. Chuang, l.J. Selikoftf and R. Warshaw. Changes in pulmonary* function c workers exposed to vinyl chionde and polyvinyl chloride, Ann. NY Acad. ScL, 246 (1975) 42--62 21 Nicholson. W.J,, c. Hammond. H. Seidman and 1J. Selikoff. Mortality experience of a eobort of ' Jivl chloride-polyvinyl chloride workers, Ann. NY Acad. $ci.. 246 (1975) 225--230. 22 Putt. F,A.. w.p. Yant and C.P. Waite. Acute re^esn of guinea pin to vapors of some new cm** merciri organic compound*. Public Health Reports* 45 (1930) 1963--1971. 23 Purchase. I.F.H., C.R. Richardson and O, Anderson. Chromosomal and dominant lethal effects *f \1nvl chloride^ Lancet. 11 (1975) 410--411. ;* **- nnu*. V- A. John!** uLm.i.c activation. Am '.f: ,yuu. 5- E.W. Jon*, k ... Millbank Quarterly, .m-nliaw. 1R- *- : .polym.ri. J. Ocfu ,,1,-d Slat*, Envtionm III. vinyl chJorM*.< -..ri, G., C.E. Lane . rhlurid* di**. Ai p L., A. Bicottl an . . Cjnctt Rtv 31 US , ,. tier. R.J.. W, Stf .4m ixpostd to vuty f: ' ;"T\tiy N-innu*. U.t A. Johansson, C. R titleI and C.A. Wachtmemer. Tbe mutagenicity of vinyl chloride after t. > Lbobc activation. Ambio. 3 <1974) 194--197. Mijpiro. S.. EAV Jones and P M, De'nten, A life table of pregnancy termination* and correlate* of fetal tn*s. Mdlbank Quarterly. 40 (1962) 7-^5. r^oershaw, |.R. and W.R, Gaffey. Mortality stuoy of workers in the manufacture of vinyl chloride ,,r ; us polymers. J Occup. Med. 16 (1974) 509*^518. ,u-d Stales En\ironmental Protection Ateno. Samplinc and analysis of select toxic substances, T ?k m. vmvl chloric*. Contract No, 68--01-2646, Jan 20. 1976. .man. C . C.E Lajifc. S. Juhc. G. Stem and L\ Bacbner, Clinical manifestations and course of > \ I chloride disease. Ann. NY Acad Sci., 246 (1975) 6--17. \ P L.. A. Bifioit. and A. Caputo. Oncogenic response of rat skin, lun|i and bones to vinyl chlor.j, , Cancer Rrs. 31 (1971) 516--519. Wjvnoiler. R.J.. w. Sinnger, J.K. Wagoner. J. Jones, H. Falk and C. Carter. Neoplastic risk among w <'rk*ro exposed to ^ myl chlond*. Ann. NY Acad Sci., 271 <1976) 39--48. toxicology and applied pharmacology 39,497-313 (1977) The Effects of Maternally Inhaled Vinyl Chloride on Embryonal and Fetal Development in Mice, Rats, and Rabbits1'1 J, A. John, F. A. Smith, B. K. J. Leong,1 and B. A. Schwetz Toxicology Research Laboratory, Health and Environmental Research, Dow Chemical US^i. Midland, Michigan 4o8640 ReceivedJune 17,1976; accepted September 20,1976 The Effects or Maternally Inhaled Vinyl Chloride on Embryonal and Fetal Development in Mice, Rats, and Rabbits. John, J. A., Smith, F. A., Leong, B. K. J,, and Schwetz, B. A. (1977). Toxicol. Appl, Pharmacol. 39, 497-313. These studies evaluated the effects of inhaled vinyl chloride on mouse, rat, and rabbit embryonal and fetal development. Croups of preg nant CF-1 mice, Sprague-Dawley rats and New Zealand white rabbits were exposed to 300 ppm of vinyl chloride 7 hr daily during the period of major organogenesis. Subsequently, other groups of mice were similarly exposed to 30 ppm of vinyl chloride and rats and rabbits were exposed to 2300 ppm of 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 13% 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 cmbryotoxicUy. 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 Mastromatteo et a!. (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 et al. (1961). Repeated exposure for 6 months to 200 ppm resulted in histologic changes in the centriiobular area of the livers of rabbits, but not in rats, guinea pigs, or dogs. In a study reported by Viola el 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. 1 This work was reported, in part, at the 14th Annual Meeting ofthe Society ofToxfcology, March 10, 1973, Williamsburg, Virginia. 1 Present address: International Research and Development Corporation, Mattawan, Michigan. CopyriaM 01977 br Aeadami* Proa, tag, All rtfhi* of reproduction if! any form Wnd, Printed in Great Britain 497 isptw OMI-OORX irs uuvi-wm* r-' ucc 061059 498 JOHN ET AL. these rats were reported to include severe chronic hepatitis, interstitial pneumonia, as well as tumors of the skin, lungs, and hones. Maltoni and Lcfcminc (1974; reported the oncogenic effects of repealed exposure to inhaled vinyl chloride in rats and mice. Angiosarcomas, /ymbal gland carcinomas, and nephroblastomas developed in rats exposed to concentrations of vinyl chloride ranging from 50-10,000 ppm, 4 hr/day, 5 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 el at. (1975) reported neoplasms in mice and the tentative diagnosis of tumors in hamsters and rats exposed to 50,200, or 2500 ppm of vinyl chloride. The carcinogenic potential ofinhaled 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 teratologic studyivere no( made. Reports on the embryotoxic potential of vinyl chloride in laboratory animals have not been found in the literature. Thus, the purpose of (he studies described in this report was to assess the potential of inhaled vinyl chloride to have a deleterious effect on embryonal ahd fetal development in mice, rats, and rabbits. Since previous studies in this laboratory suggested that the primary metabolic pathway for vinyl chloride is blocked by ethanol (Hefner et at., 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 ofl 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.). t , , diluting gaseous desired conccntr were used for th> spectrophotomci Experimental, bred rats, and J.` daily on Days t additional group of rats and rabt Mira Rats Rabbit Mioe, r METHODS Animals and test material. Female CF-1 mice* weighing 25 to 30 g, Sprague-Dawley rats' weighing approximately 250 g, and New Zealand white rab.bits6 weighing 3.5 to 4.5 kg were used in this study. The day on which a vaginal plug was observed or the day * Mk inhalad were gi gestatio Some o ofgesta on which sperm were seen in a vaginal smear wasconsidered to be Day 0 ofpregnancy for mice and rats, respectively. The day of mating was considered to be Day 0 for rabbits. Table I, some of Between daily exposures, animals were housed in wire-bottom cages in a room con ethanol in their d trolled for temperature, humidity, and light cycle. Commercial laboratory animal food7 and water were available. Food consumption was measured at 3-day intervals gestation. Maternal and for mice and rats and at 2-day intervals for rabbits.* nancy and maten Exposure of bred animals was conducted in stainless steel chambers of 3.7 m1 volume mice and on Day under dynamic airflow conditions. The atmosphere of vinyl chloride was generated by recorded on Day sacrificed by cart * Mice were obtained from Carworth, Portage, Michigan. ' Rati were obtained from Spartan Research Animals, Inc., Haslett, Michigan. Pregnant rabbits * Rabbits were obtained horn Langshaws Rabbitry, Augusta, Michigan. i teriorized througi 7 Ralston Purina Co., St. Louis, Missouri. I of live, dead, and * Ethanol consumption was measured during a separata study in which animals were given only 13% ethanol in the drinking water; these results will be reported separately (unpublished data. The rump length), ant Dow Chemical Company!- , * Vinyl chloride w ucc 061060 oni.i. as tried the id mice. . in mis hr/day, I death, bscrved milarly, 'tumors xposing re more yotoxic erature. rntial of opment rtaboiic \sidered osed to toxic or rx posed >sure to n mice, able 11 Dawley > 3.5 to the day icy for abbits. m conanimal itervals volume ued by w only ila. The inhaled vinyl chloride emhryotoxicity 499 f diluting gaseous vinyl chloride with filtered room air at a rate calculated to give the t desired concentration. Samples of inhibited vinyl chloride monomer (chlorocthylene)9 I 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 ofvinyl chloride. Additional groups of rats and rabbits were exposed to 2500 ppm of vinyl chloride. As summarized in TABLE I U Teratologic Studies with Vinyl Chloride* Vinyl chloride (ppm) Ethanol (%} Mice 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 testation. 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. Table I, 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 fetaI 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 t teriorized through Sntidline incision in the abdominal wall and the number and position I ) of live, dead, and resorbed fetuses were noted. After being weighed, measured (crown- rump length), and sexed (mice and rats), the fetuses were examined for external ano- * Vinyl chloride wm obtained from Matheson Gas Products, Joliet, Illinois. mm ucc 061061 | j j I \ ' i ! 500 JOHN ET AL. mdlics. One-third of each litter was immediately examined Tor 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. The Fisher exact probability test (Siegel, 1956) was used to evaluate the incidence of resorptions among litters. 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 Torrie, 1960). The incidence of fetal ano malies was analyzed by the Wilcoxon test as modified by Haseman and Hoel (1974). Controls. The group of animals which was exposed only to vinyl chloride served as the control 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. RESULTS Maternal toxicity. Among mice exposed to 500 ppm of vinyl chloride by inhalation, there was a decrease in maternal weight gain and food consumption during gestation and in the absolute liver weight at the time of cesarean section compared to control i values (Table 2). These effects were not observed among mice exposed to 50 ppm of vinyl chloride. Mice exposed to a combination of 500 ppm of vinyl chloride by in halation and 15 % ethanol in tbeir 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 1 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 1 decrease in maternal weight gain among rats exposed to 500 ppm is most likely due to the lower body weight ofthe 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 different from that of rats exposed to filtered room air. Maternal food consumption was, however, lower than among control 1 rats in this group. The relative liver weight on Day 21 of gestation was significantly 1 increased among rats exposed to 2500 ppm ofvinyl chloride and 15 % ethanol. Maternal I 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 in , maternal weight gain was observed among;rabbits during exposure to 2500 ppm of vinyl chloride in combination with 15% ethanol (Days 6-18 of gestation), but total \ ucc 061062 VS c -o ** "O 83 o "* * S* < -- O--Q t-> ?23.-o *x '-7 a S: s * 7o o'oCJo*c.:o:Ja- ^< 2w 1 TM 5 2 - LOO S'J=^."n.o0:n2lSS3 S3S 2iO'-J*sc,Sfo,s,c'iS-j> iGi2-fwt- 3=| sn "5~i5~ ^--32 S' P n 2. 2 2 " o ?ryS5"o5 5? 8 3 5' ? 5 y-s^ff55 3;| 3 H c. < w *=" ? 5 o. O n -- '. V o r, C- S "St G c ? ? 3 r. TABLE 2 Maternal Weight Gain, Liver Weights, and Food Consumption of Mice Exposed to Vinyl Chloride by Inhalation* Low concentration* 0 50 00 50 15 High concentration* 0 500 500 0 0 15 Number of dams Body weight on gestation Day 6` Weight gain during gestation Days 6-18c Liver weight on gestation Day 18* Absolute Relative7, _ -- Food consumption during gestation Days 6-15' 2) 302 164 2.75 0.26 59.5 8.7 61 20 31 2 17 6 2.76 0.40 57.8 4.5 6 1 16 31 2 . 11 7* 2.37 0.52* 56.6 7.3 42* 26 29 2 20 3 19 29 3 17 4* 2.75 0.31 55.5 5.5 61 2.49 0.29* 54.4 4.2 o 5 1* 7 30 3 10 V 1.78+ 0.36" 45.8 5.1* 3 1* * Mice were exposed to vinyl chloride or filtered room air by inhalation 7 hr daily on Days 6-15 ofgestation. Some ofthe mice were given ethanol in their drinking water (15%, v/v) on Days 6-15 of gestation. * Top row, vinyl chloride in drinking water (ppm); bottom row, percentage ethanol in drinking water. * Grams, mean SD. * Significantly different from vinyl chloride alone by an analysis of variance, p < 0.05. * Significantly different from control by an analysis of variance, p < 0.05. 1 Milligrams of liver per gram of body weight. o o> o ooC O4x) * MUttCMIUi 8K JOHN E T AL. TABLE 3 Maternal Weight Gain, Liver Weights, and Foe Consumption of Rats Exposed to Vinyl Chloride by Inhalation* -______ Low concentration* 0 500 00 High concentration* 0 2500 0 ev 0 2500 15 Number of dams Body weight on gestation Day 6* Weight gain during gestation Days 6-21' Liver weight on gestation Day 2IC Absolute Relative' Food consumption during gestation Days 6-15' 28 258 26 148 11 14.81 1.79 36.5 4.1 21 2 31 277 20* 125 19* I3.00 1.14 37.1 2.6 22 2 19 288 27 127 15 14.27 1.38 ,34.4 3.3 22 2 16 274 19 138 23 15.55 1.23* 37.8 2.6* 21 2* 16 272 14 120 15' 16.52 1.50 42.1 2.4* 13 2* ------- - - * Rats were exposed to vinyl chloride or filtered room air by inhalation T hr daily on Days 6-1J of gestation. Some of the rats were given ethanol in their drinking water (13%, v/v)on Days 6-15 of gestation. * Top row, vinyl chloride in air (ppm); bottom row, percentage ethanol in drinking water. * Grams, mean SD. * Significantly different from control by an analysis of variance,/) < 0.05. * Significantly different from vinyl chloride alone by an analysis of variance, p < 0.05. 1 Milligrams of liver per gram of body weight. IN H A LE D V IN Y L CHLORIDE EMBRYOTOXICTTY * TABLE 4 Maternal Weight Gain, Liver Weights, and Food Consumption of Rabbits Exposed to Vinyl Chloride by Inhalation* Low concentration* 0 500 00 High concentration* 0 2500 2500 0 0 15 Number of dams Body weight on gestation Day 6* Weight gain during gestation Days 6-29* Liver weight on gestation Day 29* Absolute --^Relative* Food consumption during gestation 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 14 23.2 2.9 7629* 11 4.08 0.21 0.06 0.27 102 16 24.7 2.7 91 36 5 4.39 0.30* 0.01 0.13 122 25 e?7.7 5.9 89 26 16 4.02 0.47 -0.14 0.42 1I630 30.0 6.3 i5 y * RaWti wire to vinyl 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 6-18 of gestation. * Top row, vinyl chloride in air (ppm); bottom row, percentage ethanol in drinking water. * Kilograms, mean SD. * Significantly different from control by an analysis of variance, p < 0.0S, * Grains ofliver per kilogram of body weight. t Significantly different from vinyl chloride alone by an analysis of variance, p < 0.05. cc> Co1 ji 504 JOHN ET AL. weight gain was not different from that among rabbits exposed to 2500 ppm of vinyl chloride alone (Table 4). Food consumption was significantly decreased among the rabbits exposed to 2500 ppm plus ethanol. Observations made at the time 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 of vinyl chloride alone and in combination with ethanol (Table 5). 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 ofvinyl 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 compared to mice receiving 500 ppm of vinyl 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 significantly 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 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 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. Fetal 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 fetal body weight was also observed among rats exposed to 500 ppm of vinyl chloride alone, but not among those exposed to 2500 ppm. Significant decreases in the number of corpora lutea per dam were 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 either 500 or 2500 ppm ofvinyl 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 ofcorpora lutea which is established prior to Day 6 ofgestation, 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. mm ucc 061066 n Ky SE.no. O P" 8- P <g g E *> 2 - B 2rae. o=|<; v-o3* 5 "-3r>, fC2o-2tT-: "in -, "%2"ST&c*BS'?0--. IC~- w I---/. S? 12'*'-S5"nO3s. g=w< cn'-*m^e*.5"^-2?noo3SS^3"s7332i-3<,p3a$SS.SS=1r5~5.i^<3~22--!_8^r32t,r52nOS8tt-.i''"2lo3L3O0Jof3t<2'^3*5m' &2m3orQe-j_^33=f_2noiE..-fC--<3t>gI"tL--w_OWwXo5X2rf--to' 3n-S-,*T <- d g 2. *<. o b ^> 5_' **t 5 3=. s3. ri -3 s"3-3~-^n'^: re S 3 " o WO** g. 3 3- nS5^35'33? iar 23: w* =r +* 2o *2tn 0sr ^5<o3o,r. o 5aO C** o?3 S' 3 S 8 2. n 3 3* v=>*" O--> a. -3 *1 3o3 *o-1 w< TABLE 5 Observations Made at the Time of Cesarean Section of Mice Exposed to Vinyl Chloride by Inhalation* Low concentration 0 50 50 0 0 15 High concentration 0 500 500 0 0 15 INHALED V IN Y L CHLORIDE EMBRYOTOXICITY Number of litters Implantation sites/dam1 Live fetuses/litter1 Implantations resorbed (%) Litters with resorptions (%) Litters totally resorbed Resorptions/litters with resorptions Sex ratio, M:F Fetal body weight (g)/ Fetal crown-rump length (mm/ Maternal deaths/treated dams (%) Percentage pregnancy* 21 12 2 10 4 13(40/261) 67(14/21) 1 2.9(40/14) 30; 50 1.00 0.11 23.0 1.9 0(0/37) 37(21/37) 20 12 + 4 1) 4 8 (18/238) 55(11/20) 0 1.6(18/11) 50:50 1.02 0.10 24.2 0.8* 0(0/27) 74 (20/27) 16 11 4 10 4 11 (19/172) 69(11/16) 2 1.7(19/11) 48:52 0.84 0.14* 22.4 1.5* 0(0/28) 57(16/28) 26 14 2 12 2 7(26/351) 58(15/26) 0 1.7 (26/15) 54:46 1.07 0.06 23.7 1.2 0(0/30) 88 (28/32) 19 13 2 11 2* 13 (33/248)* 79(15/19) 0 2.2(33/15) 52:48 0.99 + 0.11* 23.6 1.0 17 (5/29/ 72 (21/29) 7 10 6* 8 + 6* 19(13/69) 86 (6/7) 2 2.2(13/6) 64:36 0.78 + 0.15* 21.2 1.5* 13(4/30) 31 (9/29)* * Mice were exposed to vinyl chloride or filtered room air by inhalation 7 hr daily on Days 6-13 of gestation. Some of the mice were given ethanol in their drinking water ft 5%, v/v)on Day$6r15 of gestation. Low and high concentrations; top row, vinyl chloride in air (ppm); bottom row, percentage ethanol in drinking water. * Mean SD. * Significantly different from vinyl chloride alone by an analysis of variance, p < O.OS. * Significantly different from control by an analysis of variance, p < 0.03. * Significantly different from control by the Fisher exact probability test, p < 0.03 / Mean of fitters SD. 4 Based oh the presence of fetuses and/or resorption sites observed by gross examination at the lime of Cesarean section. * Significantly different from vinyl chloride alone by the Fisher exact probability test, p < 0.03. 2 i 1 I V 1 3 NHOT TABLE 6 Observations Man at the Time of Cesarean Section of Rats Exposed to Vinyl Chloride by Inhalation' $ Low concentration 0 500 00 High concent!ation 0 2500 2500 0 0 15 Number of litters Corpora iutea/dam* Implantation sites/dam* Pregnancy wastage*1' Live fetuses/litter* Implantations resorbed (%) Litters with resorptions (%) Litters totally resorbed Resorptions/litiers with resorptions Sex ratio, M:F Fetal body weight (g/ Fetal crown-rump length (mm/ Maternal deaths/treated dams (%) Percentage pregnancy* 28 15 3 12 2 32 12 2 1 (4/342) 14(4/28) 0 1.0 (4/4) J2-.48 5.67 0.29 42.6 1.2 0(0/29) 96(28/29) 31 13 2* 13 2 0.4 1* 12 2 3(11/398) 29 (9/31) 0 1.2(11/9) 50:50 5.44 0.38' 43.6 tO.S' 0(0/33) 94(31/33) 19 14 2 12 2 11 12 2 4(9/238) 32(6/19) 0 L5 (9/6) 49:51 5.59 0.27 43.6 1.5 0(0/20) 95 (19/20) 16 15 2' c?4 2 21 13 2 3(6/220) 25(4/16) 0 1.5 (6/4) 53:47 5.62 0.29 43.3 1.1 r- 6(1/17) J 100(17/17) 16 14 2* 12 2 22 12 2 4(7/195) 25(4/16) 0 1.8 (7/4) St .49 5.34 0.32* 42.4 0.9* 0(0/17) 94(16/17) 'Rats were exposed to vinyl chloride or filtered room air by inhalation 7 hr daily on Days 6-13 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. * Mean SD. * 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. * The number of corpora lutca minus the number of implants. * Mean of litters SD. ' Based on the presence of fetuses and/or resorption sites observed by gross examination at the time of Cesarean section. TABLE 7 Observations Made at the Time of Cesarean Section of Rabbi,* Exposed to v,w ucc 061068 f Significantly different hom control by jnalyM* of variance, p < 0-05. ' Significantly different Tram vinyl chloride alone by an analysis of variance, p < 0-05' The number of corpora lutca minus the number of implants. n^H on'ttK presence of fetuses and/or resorption sites observed by gross examination at the time of Cesarean section. TABLE 7 Observations Made at the Time of Cesarean Section of Rabbiis Exposed to Vinyl Chloride by Inhalation' Low concentration 0 500 00 High concentration 0 2500 2500 0 0 15 INHALED V IN Y L CHLORIDE l-MBRYOTOXICITY Number of litters Corpora lu tea/dam* Implantation sites/dam* Pregnancy wastage1*' Live fetuses/litter4 Implantations resorbed <%) Litters with resorptions (%) Litters totally resorbed Resorptions/litters with resorptions Sex ratio, M:F Fetal body weight (g)r Fetal crown-rump length (mm/ Maternal deaths/treated dams (%) Percentage pregnancy* 18 91 9 1 0.4 1 81 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 1* 8 1* 11 72* 9(14/150) 32(6/19) 1 2.3(14/6) 50:50 34.13 4.17 92.6 5.0 0(0/20) 95(19/20) 11 10 2 82 2 1 6 3 22 (19/88) M(7/ll) 2 2.7 (19/7) 61:39 36.46 4.82 92.6 4.7 0(0/11) 100(11/11) 5 10 7 84 2 3 6 4 24 (10/42) 80(4/5) 1 2.5 (10/4) '50:50 33.77 4.48 87.1 5.2 14(1/7) 86(6/7) 16 10 2 92 1l 44 53 (79/149)* 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 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 6-18 of gestation. Low and high concentrations: top row, vinyl chloride in air (ppm); bottom row, percentage ethanol in drinking water. * Mean SD. c Significantly different from control by an analysis of variance, p < 0.05. ' The number of corpora lutca minus the number of implants. * Significantly different from vinyl chloride alone by the Fisher exact probability test, p < 0.05, 1 Mean of titters SD. * Based on the presence of fetuses and/or resorption sites observed by gross examination at the lime of cesarean section. 3 it 508 JOHN ET 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 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 of vinyl chloride in combination TABLE 8 Incidence of Anomalies among Utters of Mice Exposed to Vinyl Chloride by Inhalation* I (No. 5; of vinyl signifies ItiCIDtN --------------- Low concentration High concentration i 30 50 0 500 500 0 0 13 0 0 15 Gross ar i Cross anomalies Soft tissue normlies 221 (20) 74(20) [Nunher examined, fetuses (titter*)) 220(20) 153 (14) 325 (26) 215(19) 75(20) 50(14) 107 (26) 73(19) 56(3) 19(5) Soft tissi Skeletal Skeletal anomalies 221(201 220(20) 133 (14) 325(26) 215(19) 56(5) Bones Bones of the skull* 147 (20> MS ()9) 103 (14) 217(26) 142 (19) 37 (5) Gross ar Gross anomalies Exencephaly Anopthalmia Cleft palate 0 0 1(10) (Fetuaca affected (%) (litters affected, %)] 00 H) KIO) 00 00 1 (MU 2(21) 0 1(5) 2(20) 2(20) 4(40) Omph Soft tissc Micror Soft tissue anomalies Small thymus Skeletal anomalies Stemebrae Unfused 0 3(20) 0 4(7) 3(25) 13(57)* 0 2(19) 00 9(42)* 34 (80)* Dilatei bilat Small I Skeletal i Delayed ossification No. 5 stemebre missing Ribs Extra Spurs 7 (JO) 0 4(30) 4(3J) 4(35) 0 3(30) 5(40) 44(100)* 3(21) 0.6(7) 2(21) MI2) 0 3(31) 4(31) 6(42)* KIO) 3(32) 3(21) 43 000Y 7(40)* 14(60)* 14 (80)* Slernel Unfi Ribs Spur i Vertebrae Forked atlas Missing cervical centra Delayed ossification of 0.4 (J) 0 1(10) 0 4(36)* 0 0 0 0 1 (10) 4(20) 38(60)* Vertebi Miss Skull cervical arches 0 0 1 (14) 0 0 5 (40)f Dela Skull Unfii ! t Delayed ossification Unfused occipital 7 (35) 0 1(37) 0.7(5) 40 (100)* 24(50r 13(54) 1 (12) 30 (5*)* 5(21) 70 (100)* 11 (20) * Rats * Mice were exposed to vinyl chlonde or filtered nom air by inhaUioo 7 hr daily on day* 6-15 of gestation. Some of the mice were give* ethanol In their drmk*ngwatcr(l9%v/v)on day* 613 ofgestation. Low and high concentrations; top row, vinyl chloride in air (ppm); bottom row, percental cttanol in drinking water. * Calculations of the incidence of anomalies ofbooasefthe skutl are based on approximately two-thirds of the tout popu lation of fetuses. The remainder of the fetuses are decapitated during the soft tissue examination. Among litters of mice which were exposed to 50 ppm of vinyl chloride, one litteroeoiiBted ofonly one fetus, which was decapitated^during the soft tissue examination. f Significantly different from vinyl chloride alone by the modified Wilcoxpn test, p < 0,05. 4 Significantly different from control by the modifled Wtlcoxon test, p < 0.05. 0.037, modifled Wjlcoxon test. gestation, gestation. 1 ethanol in * Amon; available r * CalcuJ. thirds of it tissue exar with 15 % ethanol exhibited anopthalmia. The incidence ofcleft palate in this group was * Signifk ' Signifk slightly higher than that observed among mice receiving vinyl chloride alone. No soft tissue anomalies occurred at an incidence significantly greater than control Sin mice. Two fetuses from one litter among rilice exposed to 50 ppm of vinyl chloride plus ethanol exhibited a small thymus. No skeletal anomalies were observed at an ( nation Wi ficantly gr exposed t( incidence significantly greater than in control among mice exposed to 50 ppm of vinyl ossificatio t chloride. Significant increases in the incidence of delayed ossification of stemebrae examinatj mmm mmm ucc 061070 INHALED VINYL CHLORIDE EMBRYOTOXICITY 509 cc, rats, and , 9. and 10, mtnation of rs (Table 8). combination Chloride by (No. 5) and bones of the skull were observed among litters of mice exposed to 500 ppm of vinyl chloride. The incidence of unfused centers of ossification of sternebrae was also significantly higher in this group. Among mice exposed to vinyl chloride in combi- TABLE 9 Incidence of Anomalies amono Litters of Rats Exposed to Vinyl Chloride by Inhalation* Low concentration High concentration rnon 500 IS 56 (5) 1<5) 56 (5) 37(5) 2 (20) 2(20) 4(40) 0 ' 34 (80)' ' 43<ioo>* 7 140)' 14 (60)' 14(80)' 4(20) 38 (60)' 5 (40)* 1 70(100)* 11 (20) t/on Some of th* uruons; top row, of \he iot*l popu> ;er of mic* which <v>s the ioft ns group was >ne. than control inyl chloride served at an opm of vinyl >f sternebrae 0 50$ 0 2500 2500 0 00 0 15 Gross anomalies Soft tissue anomalies Skeletal anomalies* Bones of the skull* Gross anomalies Omphalocele Soft tissue anomalies Micropthalmia Dilated ureter (unilateral or bilateral) Small kidney Skeletal anomalies Sternebrae Unfuscd Ribs Spurs Vertebrae Missing cervical centra Skull Delayed ossification Unfused [Number examined, fetuses (litters)] 339(28) 387(31) 229(19) 214(16) 188(16) 1)3(28) 129(31) 76(19) 73(16) 63 (16) 337 (28) 387 (31) 229(19) 214(16) 188(16) 225(28) 259(31) 153(19} 141 (16) 125(16) [fetuses affected (%) (litters affected, %)] 0 1(3) 0.4(5) 0 0.5(6) 0 0 0 0 2(6) 2(7) 0 2(6) 0 5(10) 0 27(50/ 0 5(19/ 2(6) 0 1(4) 0.3 (4) 16(61) 0 K6) 3(32) 0.5 (6/ 1(12) 9(52/ 14(68) 12(69) 35 (69/ 2(16) 7(53) 4(50) . 21 (81/ 12(61) 0 18(58) 53(90) 6(31/ 3(12/ 3(25) 2 02) * Rais 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 incidence of anomalies of bones of the skull are based on approximately twothirds of the total population of fetuses. The remainder of the fetuses are decapitated during the soft t tissue examination. j ' Significantly different from control by the modified Wilcoxon test, p < 0.05. j ' 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 I exposed to 50 ppm of vinyl chloride plus ethanol, increases in the incidence of delayed ossification of bones of the skull and sternebrae (Nos, 4-6) were observed upon skeletal . examination. The incidences of unfused occipital, unfused sternebrae (Nos. 5 and 6), a'id forked alias were also significantly increased in the 50-ppm plus ethanol group. Among litters of mice exposed to 500 ppm plus ethanol, increases in the incidence of delayed ossification of bones of the skull, sternebrae (Nos. 2-6), and arches of the cervical vertebrae were observed. Significant increases in the incidence of lumbar spurs, missing centra of the cervical vertebrae, unfused sternebrae, and missing fifth sternebrae were observed in this group as well. The incidence of extra ribs was slightly, but not significantly, increased compared to the controls. In rats, no gross anomalies occurred at an incidence significantly greater than among control animals (Table 9). Among litters of rats exposed to 2500 ppm, the incidence of TABLE 10 Incidence of Anomalies amoe Litters of Rabbits Exposed to Vinyl Chloride by Inhalation* Iittcrsi the dn In n control exposet also ob ethanol heart. A of the i ossificat among I with 15' Low 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 pelvis Dilated cerebral ventricle Enlarged right atrium of heart Skeletal anomalies Sternebrae Delayed ossification No. 5 [Number examined, fetuses (litters)] 152(18) 136(18) 69(9) 32(4) 70(9) 50(18) 47(18) 24(9) 10(4) 25(9) 152(18) 136(18) 69(9) 32(4) 70(9) [Fetuses affected (%) (litters affected, %)] 0 0 0 0 100 0 0 0 0 8(11) 0 0 0 10(25) 0 0 0 0 0 8(11) 28(77) 38(94)* 20(44) 16(75) 24(67) * Rabbits were exposed to vinyl 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 6-18 of gestation. * Top row, vinyl chloride in air (ppm); bottom row, percentage ethanol in drinking water. r Significantly different from control 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, only minor skeletal variations were observed at an incidence higher than that ofcontrols. The incidence of lumbar spurs was increased among litters exposed to 500 ppm. No increases in the occurrence ofskeletal 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 There vinyl ch toxicity v rabbits a l and rabt Among r ppm and one mate sumption evidenced occurrenc Exposu species stL higher (13 sorptions; ' the incidci 10% (193/ low incider i exposed ra creases in f not in rab observed ir ureter amc tissue anon of the thre tions; no ir than in the Ingestion haled vinyl < consumptioi percentage r i INIIAI.F-D V1NV1. CHLORIDE EMPRYOTOXICITY 511 h;mol group, incidence of relies of Ihe e of lumbar missing fifth was slightly, than among incidence of Chloride by litters of rats exposed to 2500 ppm of 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, although a cleft palate was observed in one fetus among litters of rabbits exposed to 2500 ppm of vinyl 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 ofdelayed ossification of the fifth sternebra was significantly higher than that of control litters. Delayed ossification of sternebrac 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. - at ion* *)] 70(9) 25 (9) 70(9) . '/.)] 1 (U) 8(11) 0 8(11) 24 (67) on Days 6-18 on Days 6-18 ncidence of exposed to tiers of rats \n incidence mong litters rc observed iification of lebrae were ir spurs and ised among DISCUSSION The results of these studies indcate 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 arc summarized in Table 11 - 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 vinyl 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 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 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 R. ^ -1 ucc 061073 \ TABLE H Summary--Vinyl Chloride Teratologic Studies 50 0. 15 Mice* 500 0 IS 0 500 15 0 Rats* 2500 0 IS Rabbits* 0 500 2500 15 0 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 Ahsolute Relative Implantation sites/dam Percentage resorptions Litters totally resorbed Litter size Fetal body weight Fetal crown-rump length External anomalies Visceral anomalies Skeletal anomalies 6-15 No No 20 16 -- Dec -- Dec -- -- -- -- 0/20 -- -- Inc Dec -- --- - -- 2/16 --R Dec Dec 6-15 Yes Yes 19 7 Dec Dec Dec Dec Dec __ -- Inc' 0/19 Dec Dec -- Dec Dec Dec -- 2/7 Dec Dec Dec 6-15 6-15 No No 21 -- Dec 31 Dec -- -- -- -- -- 0/21 -- Dec Dec -- -- -- -- 0/31 -- Dec Inc 6-15 Yes No 16 -- Dec 16 Dec Dec 6-15 Noo 19 -- Dec Inc Inc -- -- 0/16 -- -- -- -- Inc -- -- 0/16 -- Dec Dec -- Inc -- -- 0/19 -- Dec -- 6-1* No 19 -- Dec -- -- Dec -- 1/19 Dec -- -- 6-18 ----6-18 Yes Yes No 5 16 M ------ -- Dec Dec ------ ------ ------ -- Inc -- 1/5 7,16 2 14 ------ ------ -- -- `--- -- Inc -- Inc Inc -- -- -- Inc -- -- -- -- - Top row, vinyl chloride in air (ppm); bottom row, percentage ethanol in drinking water. * --, No change; Dec, decrease; Inc, increase as compared to control values. * This apparent increase was due to a lower than normal incidence of resorptions among the control group; see Discussion for details. * Dilated ureter. 2 q r- ucc 061074 i a 5 8 a3a t1 -=> t & C30 ^q b> 6* 5 "3 'o voC3JJ 5o57 T=t-3f a is -CO ?35 =S 5ii 3* c .2 ^c Q. icj na 2 3- s itj as* _to. y- r^t u w T.. ?= cci cy -o 5 O h&-- <J *7OIi* raL*os H O"2JS INIIAU D VINYL CtlLORlW. l-MBRYUTOXtClTY 513 I i0 vinyl chloride in combination with 15% ethanol in (hedrinking water. A statistically | significant increase in the percentage ofresorptions was observed among rabbits exposed to vinyl chloride plus ethanol. Fetal body measurements were lower among litters of mice and rats which received ethanol and vinyl chloride compared to vinyl chloride alone. The combination of ethanol and vinyl chloride did not cause a teratogenic response in the three species tested, although higher incidences of some skeletal vari- 1 ations were observed among Inters 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 in these animals seemed to enhance maternal toxicity to an extent greater than embryotoxicity. I 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. Exposure 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. acknowledgments The authors are grateful to Mr. K. D, Nitschke, Ms. H. D. Ioset, and Ms. M. F. Balmer for their assistance in all aspects of this study and to T. R. TerkeNon 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 alizarin red-S. Stain. Technol. I, 123-124. Haseman, }. K., and Hoel, D. G, (1974), Tables of Gehan's generalized Wilcoxon test with fixed point sensoring. J Star. Comp. Simul. 3,117-135, Hefner, R. E., Jr., Watanabe, P, G,, and Gehrino, P. J. (1975). Preliminary studies of the fate of inhaled vinyl chloride monomer in rats. Ann. N. Y. Acad. Sci. 246, 135-148. Keplinger, M. L.. Goode, J. W., Gordon, D. E., andCalamdra, J. C. (1975). Interim results of exposure of rats, hamsters and mice to vinyl chloride. Ann. N. Y. Acad. Sci. 246,219-224. Maltoni, C. (1975). The value of predictive experimental bioassays in occupational and environmental carcinogenesis. An example: vinyl chloride. Ambio 4,18-23. Maltoni, C., and Lefemine.G. (1974). Carcinogenicity bioassays ofvinyl chloride. I. Research plan and early results. Environ. Res. 7, 387-405. Mastromatteo, E., Fisher, A. M., Christie, H., and Danziqer, D. (I960). Acute inhalation toxicity of vinyl chloride to laboratory animals. Amer. Ind. Hyg. Assoc. J. 21, 394-397. Siegel, $. (1956). Non-Parametr/c Statistics foe the Behavioral Sciences. McGraw-Hill, New York. Steel, R. G. D., and Torrie, H. H. (i960). Principles andProcedures ofStatistics. McGrawHill, New York. Torkelson, T. R,, Oyen, F., and Rowe, V. K. (1961). The toxicity of vinyl chloride as j determined by repeated exposure of laboratory animals. Amer. Ind. Hyg. Assoc. J. 22,354\ 361 Viola, P. L,, Broom, A., and Caputo, A. (1971). Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res. 31, 516-519. t d&,vr 12 13; 6 micrc Kci/L. at day plastics industry have been cooducted la . frjtogenic actic 14 produced 65^ of clefts. laboratory animals. The chemicals# routes of .:.c ic idee} , d edui IN-VITRO. Fusion of the isolated 14 day administration, species, and dose levels Art #^ects observed ' foetal palatal shelves vac inhibited by & es follows: vinyl chloride - inhalation - "1; ng of zinc ic. level of retiny1 palmitate ir. the culture tu.ee, rats# rabbits - 50, (nice only), rffn attributed to medium of the ease order as that fouod to 2500 (not mice) ppm: vlnylldene chloride .* ,*rr.c zinc depict: prevent fusion m tne 14 day foetus "in utero". innalation - rat*, rabbits - zq {rats only) 0, 160 ppm, drinking water - rats - 200 pja; 171-184). Whs 77: ion can be ast: Correlation tf the morphology of experimental styrene - inhalation * rats, rabbits - 300 ,'vestigated. M- clefts, tne -iartita-ive parameters and the bw'O ppa, gavage - rats - 160, 300 mg/kg/dei. . * the ten experir types of cleft presenting ir. tar. will be The current TXV's for these cb^icals are .cr.trol diet for : demonstrated and thus give a eruaei** 1, 20 and 10Q ppm, respectively. Each ;T*cn*ncy. The da guartixetive casic model cf the human mal Chemical was admlnlstersd throughout major ;:r:p$ also were r formation. organogenesis. Teratogenic effects were .at their diet c Trj.E investigation was supported by the not observed in any species with any of ;ror the 11th to 1 Medical Research Council of Great Britain tnese monomers. :: qive a dose of and the Scientific and Research Coo&ittee it). On 13th day of the Newcastle University Hospitals# :rcvp each from th :rs, was given no KALTXR, H., Children's Hospital Research .: l ed water ) , 8 0# Foundation, Cincinnati, Ohio. Failure of -.-cut. The dams i JENSH, R.P., J. LUDLOW, 1. WEDCEHS# W.B. testosterone to modify the development of > hours post-injec VOGEL* # T, RJtOER*, and R.L, BRENT. Departments spontaneous eyelid abnormality in mice. .tveis an the concf of Anatomy, Pharmacology# and Radiology, An attempt was made to modify the irrun or later at TDomas Jefferson University# Philadelphia, pronounced sex difference in the frequency * : id enc e of axtarr : Pennsylvania. Studies concerning the effects of orotracte5~prenatal exposure to a c: spontaneous open eyelid In A/JKt mice by ncrmonal means. Primiparous females were >*.tlettl anomalies, .-dicated that, 1 ' non-thermal level of 2450 KHz miccowai^ anesthetized with nembutal (0.05 mg/g, ip) * ect ive in reduc . radiation in the Pregnant rat. oz the 15th day of gestation (about 2 days `-cer.ieity, and 2. Twelve pregnant wistai rats were exposed to before the lids usually close), an incision ;:.-.ceptu and mater 2450 KHz microwave radiation in an anechoic v&s made through the skin of the nape, and i.-.tred upon feedtr charter 8 hours daily throughout pregnancy a:out 1/3 of a 75-mg pellet of testosterone *:ectmg etu. (mean * 115 hours). Since previous studies bac (Creton, Scherlng) was inserted indicated that power intensities to 20 nW/ar , s-ocutaneously and the wound closed with inclusive# were non-thermal, i.e., did not cause an increase in body temperature through out the test period as measured by a rectal thermocouple probe, this dosage level was used. Concurrent control animals were placed in the chamber for similar tune periods. All animals *,rgical staples. The same procedure was fallowed in controls except no testosterone vis inserted. The females were killed 4 days liter, i.e., 1 dsy after the lids usually close and 2 days before expected parturition, itc the offspring were removed, examined, DOROTHY WEI. C*C i 1. kSU*. Department University, Tai^ --Ttts of zinc and le* _^.rolevulinic acid c were killed on the 22nd day of gestation, actj sexed by inspection of the gonads. their uteri exteriorised, and fetal positions Female offspring were externally masculinised, Chick teratogtnesi and resorption sites recorded. All fetuses tat anogenital distance in them being -biiory effect of It were dissected using Wilson's cross sectional indistinguishable to the eye from chat in d-eminolevulinic aci, technique. Data were analyzed and compared males. The resorption rate was similar In 1 :iyity have bees repo- with histone baseline control groups. No the experimental and control groups (17.0 Further studies * significant differences among croups were observed for the following parameters: initial ata 19-42). The hormone had no effect on eyelid development. The overall frequency oi ifst the effect of z1 Tor this vork maternal weight; term maternal weight; maternal open eyelid was 15.42 in the experimental ^ctedby heart pure. weight gam; embryonic and fetal resorption group and 14.02 in the control; and v* 21.1* "t*' chick aabryo. A rates; abnormality rate; and term fetal and it males and 8.92 in females in the former; of Weiasberg et i placental weights. Using these paraaeters as indices, the present results indicate that and 20.82 in males and 7.72 in females in the latter. Perhaps earlier treatment *tcivjty w.i txpr*. ' S>1 "u Pr m* of bloc exposure of pregnant rats to a non-thermal but threshold level of 2450 KHz microwave radiation 6 hours daily throughout gestation does not or administration of a larger dose would be successful. th,t Iinc ; - ibmd the ccivit>- c r. ... D0 inhihinor adversely affect the offspring. (Supported by nih Grant #ESO-il21) liu* t0 l**11 s Th* o*ia*i sol i ' 0.04 ng of ltt# ,ct , and B .G , SHAH*, Bureau cf Qn 4th d#y of ir > CWnucal Safety, Health and Welfare , ,,tJi! c*c.te vms inje * John*, J. A., 7 J. Murrav*, 7, A. 5mith*, ano !i. A. Scfive:2t Toxicology Research LsDoracor>, I>c. Cnerucs! L.S.A., Midland, Michigan TcTe^ologic fevaiuacion of vinyl Cdr.ada, Ottawa. Ontario. Failure of ~ : r.c acetate to reduce teratooer.icn? Ji s~hy1enethlourea in pregnant rats. Ltr.ylenebisd i t h iocar ham a t e f uftt ic idea* t afi of tine .cot.t fl b.tktn th tvo a ` ; = ' diapPelredi hoUfl vss used wi chloride, vir.'lioene chloride, and styrene in la&cratory animal &. t.-eir degradation and a probable i Jlnc could r.duct t cl`,ifc,``jwtiue t-ethylenethiourea (ETl 5 riatemal zinc deficiency during F**'' ' ofono.ohLfAD O#nnl(yj wnoho[ : Teratologic studies cr s series of monomeric substituted v;*\ 1-chemicals widely used in the nancy, have all produced- strikinclF ^ similar anomalies in rats. Mechanic* 'ort.d by , r.seurch r',c. Council), 48A * I TRANSLATION; Mlrkova, E., A. Mikhailova, and M. Nosko: EMBRYOTOXIC AND TERATOGENNIC ACTION OF VINYL CHLORIDE. (Emgriotoksichno i teratogenno deistvie na vinilkhlorid.) Khigiena a Zdraveopazvane3 Vol. 23, No. 5, pp, 440-443, 1977, Institute of Hygiene and Occupational Diseases, Sofia. E. Mirhova, .4 Mihailova, .If. A'ostn -- Embryotoxie and Teratogenic Action of Vinylchloride Summary. The authors examined the embri .toxic ami teratogenic action of vinylchlotide on 40 pregnant white rats of the strain W.star. 1 lie experiment' Acre carried out under Ihe condition* of daily inhalatory pm-nnimi dum- the whole gv-tauun atjnean daily con centration of vinxIchloride (0,15 me n1). There was a manifeMed emhrxoto.xic anil tcratojenic cife. t of \ inylchlciride-elevated total embrvonal mortality, lowered weight of the fotU'CS. induction of external and internal inomilies in the development of the fetus. The authors propose a correction of temporary acting threshold value of concentration in Bulgaria (10 me m*i on the basis of the obtained itwlt*. It Is known that a number of chemical agents used in industry have an un favorable effect on the development of the fetus and progeny [G. Goncharuk, 1968; A. Dyban et al., 1965; L. Ivanova-Chemishanska, 1969; E. Mirkova, 1975, etc.]. One of the most important tasks before public health experts and toxi cologists is therefore to determine the causes of the later effects of poison ing with harmful industrial chemicals, and in particular the necessary evalua tion of the risk of an smbryotoxic and tetratogenic action. Unfortunately, studies on these specific forms of the biological action of industrial chemi cals in cases of exposure to low concentrations are still very inadequate. Studies at these exposure levels make it possible to determine the threshold of the embryotoxie effect, and are the basis for substantiating preventive mea sures for the protection of the prenatal period of human development [I. Sanotskii, 1972], The subject of our experimental study was testing the embryotoxie and teratogenic activity of vinyl chloride during Inhalation exposure in a concen tration of 6.15 mg/m3 -- lower than the accepted provisional Bulgarian standard in force (10 mg/m3). ijcn 061077 MATERIAL AND METHODS The experiments were carried out on 40 pregnant white Wistar rats under conditions o dally Inhalation exposure throughout gestation at an average dally vinyl chloride concentration of 6.15 mg/m3. The animals were sacrificed by decapitation on the 21st day of pregnancy. During the autopsy, the number of Implantation sites in the uterus were counted, the resorptions and autolyses, the number of corpera lutea of pregnancy [corpora lutea vera? -- Tr. Ed. ] in the ovaries, and the number of live and dead fetuses were recorded, and the weight and cranlocaudal dimensions of these latter were determined. The fetuses were macroscopically examined for the presence of external malformations, after which the routine teratologic method of J. Wilson [1965] as modified by A. Dyban et al. [1970] and A. Dawson [1926] was used to look for developmental anomalies In the Internal organs and defects in skeletal ossification. The lethal effect of the vinyl chloride was calculated from the method of A. Malashchenko and I. Egorov [1967]. Using a biological test, a first generation was bred from mothers treated while pregnant and control fathers. Its postnatal development was monitored until the onset of sexual maturity; in evaluating the health of the progeny both the generally accepted criteria of physical development -- weight gain in the newborn and postnatal mortality -- and sensitive functional methods of Investigation were used. The status of the detoxifying function of the liver was studied based on the length of hexabarbltal sleep, while the ex cretory function was examined from the bile cholic acid level (Relnhold method, [1970]), and the activity of the bile enzymes LA? and GGT? (Fermognost tests). The results were statistically treated using the Student-Fisher t criterion at a significance level of p $ 0.05. 2 UCC 061078 RESULTS AND DISCUSSION The prenatal effect of vinyl chloride on the fetus is characterized by a marked embryotoxic action. The level of the overall embryonal mortality in the experimental group is double the control value. A breakdown of this Indicates that the increase is due to an elevated postimplantation lethality. The lethal action of vinyl chloride is directed toward the stages of embryogenesls which occur Immediately after blastocyst implantation. This is indicated by the more than eight-fold Increase in the Incidence of early postimplantation mortality Cembryonal resorption), while the level of late postimplantation lethality is unchanged under the action of the test chemical. When pregnant animals are ex posed to vinyl chloride at the earliest stages of anbryogenesis, there is a tendency toward an increase in preimplantation mortality, but the changes re corded are statistically insignificant. Vinyl chloride's embryotoxic action is also manifested in a reduction in fetal weight. In all probability, the suppression of overall development is due to a disturbance in homeostasis in the mother's body, and particularly to the Impaired condition of the liver, resulting in a deficit of the plastic [nutrient? -- Tr. Ed. ] and energy resources necessary for the vital activity of the developing embryo [A. Mikhailova, E. Mlrkova, unpublished data]. The negative effect of vinyl chloride on the intrauterine development of the fetus Is also manifested by a sharp -- eight-fold -- increase in general anomalies (hematomas) in comparison with the control. These vary in severity over a wide range, and are probably due to primary toxic damage to the fetal vessels. Under the studied exposure conditions, vinyl chloride also had a terato genic effect. External developmental anomalies, malformations of the internal 3 UCC 061079 1 organs, and defects in skeletal ossification are induced. The defects in fetal development which arise do not cover a broad spectrum, and involve only morpho genesis and the brain tissue, the vascular walls, and sternal ossification. The disturbance in morphogenetic processes in the brain leads to the formation of internal hydrocephalus, combined with intracerebral hematomas in 54.4% of the fetuses examined, and to the isolated development of hematomas in 14.3%. Ex ternal brain malformations (encephaloceles) and anomalies in sternal ossifica tion (presence of additional ossification centers) are characterized by a low incidence -- 2.53% and 2.8% of the fetuses, respectively. In the study of the postnatal development of the generation exposed in utero to vinyl chloride, no deviation in overall physical development and sur vival rate were found. The data from the functional studies conducted indicate a disturbance in the adaptive capacity of the progeny, however. Impairment of the liver's detoxifying function is observed in the progeny at the age of one month, as evidenced by prolonged hexabarbltal sleep. The changes are persis tent and are recorded in the second month of the postnatal period as well, af ter the onset of sexual maturity; the degree of their expression is the same in both sexes. As a result of the prenatal exposure to vinyl chloride, the excretory function of the liver is impaired as well. The reduction in bile cholic acid levels which was recorded is an early sign of developing toxic hepatopathy [A. Mikhailova, 1975]. The data obtained on the reduced rate of bile secretion, the reduction in the overall quantity of bile, and the decrease in the activity of the bile enzymes -- LAP and GGTP -- are also evidence of disturbances in the hepatobiliary system of the progeny as a consequence of intrauterine exposure to vinyl chloride. 4 ucc 061080 CONCLUSIONS 1. When rats are exposed to It during pregnancy, vinyl chloride has a marked embryotoxic and teratogenic effect at a concentration of 6,15 mg/m3. The overall embryonal mortality Is Increased, general physical development of the fetus Is suppressed, and external and Internal brain malformations and de fects In sternal ossification are induced. 2. The postnatal development of the first generation of rats exposed in utero to vinyl chloride is disturbed. The changes recorded In the detoxifying and excretory functions of the liver are persistent. 3. The existence of a marked embryotoxic and teratogenic of vinyl chloride at a concentration of 6.15 mg/m3 -- lover than the provisional Bulgarian health standard In force (10 mg/m3) -- as veil as the data on the impaired adaptive capacities of the progeny during the postnatal period, are the basis for re viewing this provisional MAC and Indicate the need for lovering it. 5 UCC 061081 REFERENCES 1. Goncharuk, G.: In. the. book: Gig. Primeneniya Pest, i Klinika Otravl. (Public Health Aspects of Pesticide Use, and the Clinical Picture of Poisonings.) Kiev, 1968, pp. 7-8. 2. Dyban, A., I. Akimova, and N. Svetlov: Dokl. AN SSSR, No. 163, p. 1,514, 1965. 3. Dyban, A., V. Laranov, and I. Akimova: [Arkh. ] Anat. Histol. Embriol.t No. 10, p. 89, 197?. 4. Ivanova-Chemishanska, L.: Dissertation, Sofia, 1966. 5. Malashehenko, A. and I. Egorov: Genetika, No. 3, p. 59, 1967. 6. Mirkova, E.: Dissertation, Sofia, 1975. 7. Mikhailova, A.: Dissertation, Sofia, 1975. 8. Sanotskll, X.: In the book: Vopr. Gig. i Norm, pri Izuahavane Otdel. Poesl. Vozdeistviya Prom. Veshchestv. (Problems in Public Health and Standardization in Studying the Long-Term Sequelae of Exposure to In dustrial Chemicals.) Moscow, 1972, p. 5. 9. Dawson, A. and S. Stain: Technology, Vol. 1, p. 123, 1926. 10. Relnhold and Wilson: Cited by N. Gabrielyan (dissertation), Moscow, 1970. 11. Wilson, J.: Embriologigal Consider, in Teratology. N. Y., 1965, p. 251. 6 UCC 061082 134 abstracts: fourteenth annual melting )I Once daily for 7 days. Since pretrcalmenl with 1254 at this dose caused loss of body weight and striking enlargement and pallor of the liver, lower doses were included. Controls were given H;0 by mouth. On the eighth day, after a 16-hr fast, animals were either sacrificed for determi nation of microsomal cytochromes b,, P-448, P-450, NADPH-P-450 reductase and NADHcytochrome-c reductase, oxidative-iV-demcthylasc, and glucose-6-phosphatasc, or exposed to . 5 % VCM for 6 hr and sacrificed 24 hr later. Liver injury as indicated by ccntrolobular vacuoliza tion, focal midzonal necrosis, and increased serum transaminase activity was not apparent in HjO-, SNL-, 3-MC-, or PCN-pretreated rats, while slight, moderate, and severe injury were consistently found in HCB-, PB-, and 1254-pretreated animals, respectively. In animals pre treated with 150 jtmol 1254/kg, a dose which nearly tripled P-450, VCM produced the most severe injury of the combinations tested. Although liver injury was only found in animals pre treated with an agent which induced P-450, induction of P-450 alone did not correlate with injury. PCN which induced P-450did notpotentiate VCM injury. Othercellular components in addition to P-450 may be important in the potentiation of VCM toxicity. (Supported by NIH Grants ES-00002, OH-00315, AM-16183, and HL-06370.) ! I f i 29. Results ofa Vinyl Chloride-Teratology Study in Mice, Rats, and Rabbits, B. A. SCHWKTZ, B. K. J. Leono, F. A. Smith, M. Balmer and P. J. Gehring, Toxicology Research Laboratory, Health and Environmental Research, The Dow Chemical Company, Midland, Michigan. The potential of inhaled vinyl chloride monomer (VCM) to have a deleterious effect on the developing embryo and fetus of laboratory animals has been evaluated. In an initial experi ment, CF-1 mice, Sprague-Dawley rats, and New Zealand white rabbits were exposed to 500 ppm VCM 7 hr daily during organogenesis. Some of the VCM-exposed mice were also treated with 15% ethanol (EtOH) in the drinking water as their only source of liquid to determine if concomitant ingestion of ethanol would alter the toxicity and/or teratogenicity of VCM. No signs of toxicity were observed in the adult rats or rabbits during exposure or at the time of necropsy and caesareaj|<^igp| Arnwifjthe rats, there was a significant decrease in fetal body weight but the incidence of externatissue and skeletal malformations was not different than among control litter;. There was no effect on fetal body measurements or the incidence of external, soft tissue or slUkUl alforpiG$?ns in rabbits. Mice were more susceptible to VCM > capable of from 0.3 ti\ aerosol ant sampler f which the c of respiratc for brooch for particle tion of drui Its usefulnc through th< body orhet tion from t 31. Bronch B. S. Br> Lederle I The pote deleterious The clearer adult cynor with techiv restrained i completion a 3 x 3 in. s after inhala occurred di clearance c rapidly that aerosols on than either of the other two species. There was a slight decrease in the weight gain among mice and maternal deaths wemtaer<ed.(fijieath$/30 exposed mice). The percentage of pregnancy among mice was slightly lower fnaifStitjtigiontrols. There was a slight increase in the incidence of resorptions and fetal body weights were slightly lower than control values but the incidence of external, soft tissue and skeletal malformations was not different than among controls. Concomitant treatment ofmice with EtOH in the drinking water accentuated some of the toxic effects associated with exposure to VCM alone. The maternal weight gain and the pregnancy rate was further decreased, but the incidence of maternal deaths was not increased by con comitant EtOH ingestion. The incidence of cleft palate and certain skeletal anomalies was significantly increased among mice receiving EtOH plus VCM. Thus, based on the results of this initial study, exposure of bred mice, rats, and rabbits to 500 ppm VCM during organo genesis did not result in a teratogenic response. Mice were slightly more susceptible to the toxic effect of VCM than either rats or rabbits. Simultaneous exposure ofmice to VCM by inhalation and EtOH in the drinking water resulted in toxic effects greater than that observed among mice exposed to either one alone. These results are being used to design additional studies to assess the toxicity of VCM, > 32. In Vitr Instiliatii Industria The effec Two group 1.0 mg/rat vehicle con' ability wert Cells from 5a with He number of were used PAM viabi peared to b numbers p> 30. A Laboratory Aerosol Systemfor Conducting Inhalation Toxicity Studies. B. S. Brar, C. E. Traitor, C. R. Boshart and J. F. Noble, Toxicology Research Section, Lcderle Labora tories Division of American Cyanamid Company, Pearl River, New York. An aerosol system has been developed for the safety evaluation of drugs to be wd in the respiratory tract. Design features of the equipment include: (1) a spinning rfe generator . 33. Studies 4-ipomcar Environm Tennessee 4-Ipomeai toxicity in se Toxicology. 11 (1978) 45--54 Elsevier/North-Holland Scientific Publishers Ltd, EFFECTS OF VINYL CHLORIDE EXPOSURE ALONE AND IN COMBINATION WITH TRYPAN BLUE - APPLIED SYSTEMATICALLY DURING ALL THIRDS OF PREGNANCY ON THE FETUSES OF CFY RATS GY. UNGVARY, ARANKA HUDAK, ERZSEBCT TATRAI, M. L6RINC2 and G. FOLLY' Departments of Experimental Pathology and Chemistry, State Institute of Occupational Health. Budapest. H-1450 Budapest P.O.B. 22 and Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, H-1450 Budapest P.O.B, 67 (Hungary) (Received December 29th, 1977) 1 Revision received May 22nd, 1978) (Accepted May 23rd, 1978) SUMMARY Vinyl chloride (VC) has been shown to be present in the fetal and maternal blood as well as in the amruotic fluid after the exposition of pregnant CFY rats to VC at an atmospheric concentration of 5500, 18 000 or 33 000 mg m3 i '-2000. 7000 or 12 000 ppm) for 2.5 h on the 18th day of preg nancy, indicating the permeability of the placenta to the agent. Teratologica! investigation of the offspring of pregnant rats exposed continuously to VC at an atmospheric concentration of 4000 mg/m3 air 11500 ppm) during the first, second or last third of pregnancy has shown that VC has no teratological effect in the rat and has no embryotoxic effects either, when applied during the second or last third of pregnancy in the above concentration. Exposition to VC during the first third of pregnancy resulted tn an increased fetal mortality and in the manifestation of embryotoxic effects. Fetal losses and induction of central nervous system malforma tion due to trypan blue administration were not potentiated by a combined exposure of pregnant rats to VC and the dye. INTRODUCTION VC is ranking 23rd from among the 50 most widely used industrial chemi- Suppor.ec. i_n part, by the Scientific Research Council, Ministry of Health, Hungary. m.'i-0401-os-i/mu. Abbreviation VC, vinyl chlonde 45 cals. Carcinogenicity of the compound has been reported in rats, mice and hamsters 11--3]- In man the occurrence of hemangiosarcoma -- a rare malignant neoplasm -- of the liver has been brought into possible causa] relationship with VC exposure [4--8]. VC or its metabolite, chloroethylene oxide, have been shown to induce mutagenic effects [9--14], Chromosome aberrations have also been described in plant workers after VC exposure of various lengths [15--17], However there are only a few data on the terato genicity of VC. The results of the epidemiological studies are controversial. Infante [18] reported an increase in the incidence of congenital malforma tions of the centra] nervous system among people living near chemical plants working with VC. Further epidemiological study [19] did not confirm this finding. Infante et al. [20] found higher mortality in families where the father was subject to occupational exposure to VC. Paddle [21], however, questioned methodological aspects of this approach. The only study in experimental teratology with VC was conducted by John et al. [22]. They found no teratogenic effect after inhalation of VC at an atmospheric con centration of 50, 500 or 2500 ppm for 7 h daily, during organogenesis in S-D rats, CF-1 mice or N-Z rabbits. The large scale production of VC and the wide usage of the polymer PVC, the great number of people exposed, the reported hazardous effects, muta genicity and carcinogenicity of the compound as well as controversial data of epidemiologic studies of teratogenicity call for a detailed study of experi mental teratology of the compound. The present work was aimed to answer the following questions: (1) Does VC, inhaled by the pregnant animal, cross the placenta and result thus in a direct intrauterine exposure of the fetus? (2) Does VC in itself possess teratogenic or embryotoxic effects? (3) When given in combination with a known teratogenic agent does VC potentiate the teratogenic effect of the former compound? materials and methods Female CFY rats of 240--280 g body wt. were mated in a harem system. The day of finding sperm in the vaginal smear was considered as the first day of gestation. The animals were kept on a standard rat pellet [LATI*. Gbddlld] and tap water ad lib. Body weights were recorded once a week. Groups of 3 rats were exposed to VC for 2.5 h on the 18th day of gesta tion at 5500,18 000, 33 000 mg/m3 (~2000,7000, 12 000 ppm) atmospheric concentrations. At the end of the exposure the animals were sacrificed and maternal and fetal blood and amniotic fluid samples were collected for VC determination performed by the method of Lirincz [23]. Allocation of other pregnant rats to experimental groups can be seen in Table I. Groups IA, IC, HA and IIIA inhaled air in an inhalation chamber for 24 h/day on the days of pregnancy 1--9, 8--14 and 14--21, respectively. Groups IB, ID, IIB and IIIB were exposed to VC in an atmospheric concen- * Institute of Laboratory Animals, Godbllfi. 46 _'"V to*, v l AllU. t SUMM AltJ/J- M DA llA (>1- KXl'KItlMI N I A f, f! II O UI'S Ol I1 lfl\<. N A N T CI V It A in n nii-n 1 2 1 h/<Ut llavs of liri'l* ll'HICV Nn of lit (in s M ,i 1" rn.i | U'l'H'hl i;.nnr* (%} N if fi Insrs Lit r 111 ail lie* xrosu* IO V1NVL CIILOTtlDK. Tit YFAN OUJKOrt DOTH ----------- --- - ------------- --- -- hi Ml lltss r-) Mi .m lull I M/r Mean fetal iv Hr hi (ck Mean plari'nt.il iv eight 00 Wright 11 .mil (1' fMmrs m 11 vi-r w 1 /ImmIi wl ralU> m lavcr w 1 i n-ilui ml hrulv nl * Mlio (%) to A Aii inhalation I'll vs. sal s r.r fl V(' mli.iMl inn* |*li v s. mI s.r.r C An inhalation Ti v ii,m Muc s.t .8 I) VC iiih.ilalmn* 1 i v|i in Miir s.< K fill A An' toll.il4if i<ali 1-0 7--8 1-9 7-8 1 -fl 7--b i-n 7 fl B -14 0 VC inhalation* fl-M <IU} A Air inhalal ion 14--21 0 VC inhalation* 1 4--21 {IV} llntiraU'd i'iutiiil 11 54 I'll 1 171 2.IB 10 55 IB i 22.1 2 0!1 13 5 5.58 < l.ll 2.80 18 51,12 * 108 2 51 11 49,38 157 1 85 2R 51.70 374 1.12 18 52.72 t 212 2 81 22 40.8,1 * 24 1 1,07 28 52.4 5 ' .115 1 23 'l In |M'r eenl of s(a riiiifS liorty weight. ^ In per mil of Intut Implantation silcs. r i'rr mil of (ivlng fetuses weighing less than 1,3 it, ri Maternal weight {total weight of ret use* + placentas) c 0.5 ml/|f)0 f body wt,/ctay * 4000 ihc/iu* ( t 500 ppm) **0 5 ml/lOO g botlv w( /<fav of IOh fw/v) solution, * /* . 0,05; ** f < 0 01; (Meat) t r ' 0,05; ft: V < 0.01; (Mann Whitney U test); R.F M. 1 3 3 .1 1 1 -- , 2 12 12 34 5 IB 12 14 10 1 .7 G.5t 2l.ltt IS.7t+ 11 15 1 0.84 11.58 * 0.38 10.15 * 0.G4 1100 0,08 3,81 < 0.02 3.74 * 0.03 3.71 ' 0.03 3.75 . 0.03 1.18 4,54 1 l .20 * 0.01 13.38 * 0,37 3.00 0.04 3.88 f 0.02 5.8 11.78 1 3*85 0,04 0.03 5,4 11.18 i 3.84 0.74 0.03 1 .17 11.25 1 1,8 3 0.54 0.02 0.53 0.006 0.58 i O.OOG 0.57 O.OOB 0.54 A 0.000 0.55 ` 0.007 0.52 * 0.004 0,52 i 0.007 0.57 i 0.007 0,51 < 0.005 2.3 87 8.3 10 G 0.3 3.21 11.8 12.3 2.0 3.71 i 4.41 0.00 0 13 4.25 i 4 .02 * * * 0,00 0.00 1.18 * * 4.70 ;* * 0.08 0.00 - 4.81 ' * * 5.31 * ** 0 14 0,17 4,05 * 0.08 4 26 i* 0.06 4.B7 i 0.08 5.03 i * 0.07 3.75 * 0 08 3 60 i 0.05 4.29 0.11 4.12 0.07 3,80 1 0.08 4,50 * 0.09 061086 t*t -4 I'm-?- , <U*4 -<*' im MaHNlMMlt trarion of 4000 mg/m3 (--1600 ppm) for the same length during the same periods of gestation as tbeii respective controls. The volumes of the inhala tion chambers were 0.13 m3, the vertical flow rate of the air 2 m3/h at a regulated temperature of 24--25C and 50--55% relative humidity. VC concentration in the inhalation chamber was determined by means of a type 5840 A Hewlett Packard digital gaschromatograph [24]. The rats in groups 1C and ID were given subcutaneously 2 injections of 50 mg/kg body wt. trypan blue (1% solution) on the 7th and 8th day of gestation. Group IV was kept in the animal quarters during the whole period of gestation. On the 21st day of gestation the animals were anesthetised with ether. Abdominal wall and uterine horns were cut open and the number, position of fetuses living, dead or resorbed were noted. Fetuses and placentae were excised, weighed and macroscopic investigation was carried out. Half of the fetuses of each mother were put into Bouin's fixative and dissected after fixation under the stereomicroscope [25]. Organs with macroscopic abnorm alities were embedded and hematoxylin-eosin stained sections were studied further. Histological investigation of representative other organs was also carried out. In order to investigate the skeletal system the other half of the fetuses were fixed in alcohol and stained with ali2arin-red-S [26], The mothers were dissected and their livers were processed in routine histology. .Arithmetic means and standard errors were calculated. Student's t-test was used for statistical comparison. The litter was regarded as the experi mental teratological unit [27]; affected over total fetus ratios were calculated. Mann-U'hitney U-test was used for the statistical comparison of the ratios obtained. RESULTS Considerably high VC concentrations were found in the blood of pregnant rats as well as their fetuses, when the mothers were exposed to VC at an atmospheric concentration of 5500,18 000 or 33 000 mg/m3 (~2000, 7000, 12 000 ppm) for 2.5 h on the 18th day of pregnancy. The presence of VC in the amniotic fluid was also detectable (Table II). Maternal loss was not encountered in the experiments. No difference in the weight gain of pregnant rats expressed as percentage of the starting body weight was found with the exception of group IllB exposed to VC during the third week of pregnancy. The weight gain in this group was lower, than in the other groups (Table I). The maternal liver weight and liver weight/body weight ratio increased in response to trypan blue as well as to VC applied in the first or second week of pregnancy (P < 0.01 and P < 0.05, respectively) while no difference was seen in these parameters after VC exposure during the third week of pregnancy (Table I). No pathological change was observed in the liver of VC treated mothers at the light microscopic level. There was a marked periportal histiocytic reaction in the liver of trypan blue injected animals. The number of resorbed fetuses as well as the fetal loss taken as percent- 48 TABLE n VINYL C FLUID O EXPOSUP Inhalation (mg/ml) 0 5500 (-2000] 18000 (-7000 j 33000 (-12000j , S.E.M. age of tl exposed tendency group ex different third we fetuses a Combine effective increasec A slig. the grou with try placenta, was seen Althougl or VC ir different of novel The ; shown t attribute uiscuss Conti; le' weight c 128], O, TABLE n VINYL CHLORIDE LEVELS IN' MATERNAL AND FETAL BLOOD AND AMNIOTIC FLUID OF CFY RATS ON THE 18TH DAY OF PREGNANCY AFTER 2.5 H OF EXPOSURE Inhalation chamber (mg/ms) Maternal blood (ug/ml) Fetal blood (Mg/ml) Amniotic fluid (ag/ml) 0 5500 (-2000 ppm) 18000(-7000 ppm) 33000 (-12000 ppm) 0:0 19.02 : 1.70 32.40 2.12 48.43 : 1.95 010 12.80 2.92 22.67 i 2.75 30.52 1 3.77 01 0 4.27 0.42 4.93 0.18 13.50 1 2.99 s, S.E.M. age of the total number of implants was significantly increased in the group exposed to VC during the first 9 days of pregnancy (P 2: 0.05); there was a tendency of increased resorption and fetal loss, though not significant in the group exposed to VC during the second week of pregnancy (P< 0.1) and no difference in the parameters was seen after an exposure to VC during the third week of pregnancy. There was an increase in the number of resorbed fetuses as well as fetal loss in the group injected with trypan blue (P< 0.01). Combined trypan blue administration and VC exposure was not more effective than the dye injection alone. The number of dead fetuses was increased by the trypan blue treatment (Table I). A slight tendency of increase in the ratio of retarded fetuses was seen in the groups exposed to VC during the first 9 days of gestation or injected with trypan blue, but no significant change in the mean weight of fetuses or placentae and in the ratio of fetuses with weight retardation (less than 3.3 g) was seen no matter during which time of gestation the VC exposure occurred. Although the ratio of retarded fetuses was higher in the groups inhaling air, or VC in the inhalation chambers during the third week of gestation, this difference was not significant and probably may be due to the stressor effect of novel environment. The findings of the dissection and skeletal investigation of fetuses are shown on Table III. None of the malformations or anomalies could be attributed to VC. DISCUSSION Continuous exposure of rats to VC results in a permanent elevation of its ulood level [2SJ. A permanent increase in blood level and the low molecular eight of VC facilitate a rapid extravascular distribution of the chemical [28]. One can assume, that during a continuous exposure of pregnant rats 49 ucc 061088 ~IE ! OoI TABLE HI FINDINGS OF DISSECTION AND SKELETAL INVESTIGATION OF FETUSES Treatment Inhalation 24 h/day of pregnancy Air VCb Air 1-9 1-9 1-9 VCb 1-9 No. of litters examined No. oT live fetuses S,c. injection days of pregnancy Phys. sal* 7-8 Phys. sal Trypan hc 7-8 7-8 13 19 13 171 223 131 Trypan 1>C 7-8 18 198 Cxlcrnat malformations Exoncophaly Umbilical hernia No of fetuses dissected -- 83 -2 ---- 108 63 " 1 95 Internal malformations Inlrmal hydrocephalus Anophthalmia Microphl halmia Polycystic lungs Thymus with processus Pyelecl asia DiLil.it i< ni ir urjimry Uliidilci -- ~ 4 2 --1 12 1-- 2 69 4 ft 1 2 2 ~ 9 2 fi Air VCb 8-14 8-14 ---- ---- 14 28 157 374 ---- ---- 75 185 1-- ---- ---- ---- 69 6 20 18 Air 14-21 vcb 14-21 -- 18 22 212 244 ---- - 102 117 1-- ---- ---- 31 99 6-- 7-- - - 28 315 -- 166 -- -- -- 3 - 680190 000 v. d r. Mif'lclnl Ht.llllalion sjBns Foorlv .vwflrd sfrritrhinc Biparl. verfehra centra Shfirl.Tr^c ,.f > m.t 82 isn 1 to 127 143 07 ucc n 061090 Nil. f 11 .ill / 'Mrl si ,u 111 'll frl ||*;r *1 Skeletal relaxialion signs Poorly ossified sterncbrae Bipnrl. vertebra contra Shortness of 13 th rib. M^ 2 8 -- I 1 ( 7 6 -- Skeletal anomalies Fused sterncbrae Supernumerary ribs. -- 2 Skeletal malformations Missing orhita Multiplex -- -- * 0.5 ml/100 g body wt./day. h 4000 mg/ms (1500 ppm). c 0.5 ml/100 r body wt./day of 1% (w/v) solution. 1 !) -- 2 m.i 4 G 1KO 110 73 9 31 11 14- 22 12 l 4 m 7 12 - 2 111 1 2 1 I to VC the fetuses like the mothers are also permanently exposed. Our detection of VC in the amniotic fluid and in the blood of fetuses of pregnant rats exposed on the 18th day of gestation (Table II) justify this assumption. In order to study the possible teratogenic and embryotoxic effects of the compound, rats were exposed to VC at 4000 mg/m5 (1500 ppm) atmos pheric concentration continuously during the first, second or third week, of pregnancy. In spite of the exposure at this very high level of concentra tion no obvious alteration in the behaviour of experimental animals was observed; their food and water consumption and weight gain did not differ from that of the controls and activation of self protective mechanisms [29] was not seen either. Although our study of the great number of fetuses gave essentially negative results, some tendencies found -- not reaching the level of signifi cance -- might deserve further attention. There was an increase in the number of resorbed fetuses in the groups subjected to VC during the first and second week of pregnancy. Though the increase in the number of implantations and higher birth rate might explain this difference in the group exposed during the second week; no increase in the number of implants was seen in the rats exposed during the first week. Thus it is most probable that the toxic effects of VC might explain the increased fetal loss close to the level of significance (P a 0.05). This is all the more probable, because toxic agents independent of their chemical nature have been shown to result in similar embryotoxic effects, when applied during the first third of pregnancy [30]. Among the offspring of mothers exposed to VC during the first week of pregnancy one case of microphthalmia and an other case of anophthalmia occurred. In spite of the fact that this was not consistent with a significant increase in the incidence of congenital malformations, these cases deserve further attention for the following reasons. None of these malformations was observed in the group exposed to air in the chamber, or in the untreated controls. Both malformations are related to the central nervous system, and an increased incidence of congenital malformations of the central nervous system have been brought into causal relationship with VC exposure by Infante [18]. On the basis of our results VC exposure in itself has no teratogenic effect in CFY rats, but an embryotoxic effect of VC exposure during the early stages of pregnancy at high atmospheric concentrations should be taken into consideration. A similar lack of teratogenic effect of VC has been reported by John et al. [22]. Their conclusion is based on studies of the effect of VC applied during arbitrarily chosen short periods of organogenesis. One could emphasize here that it is a minimal requirement of experimental studies aimed to reveal the teratogenic effect of any particular chemical that the pregnant mothers are exposed to the chemical in such a way as to provide continuous exposure of the fetuses during the whole period of organogenesis. The fulfilment of this 52 req uirem up by in chemical whole pe We are the resui human a concentn VC [31] age requi. Infants of the cei is due to context v to induce a teratog embryo tc Negative to VC di trypan b (exencep! fetal loss* blue treat ACKNOWL The te< Szomoldn REFERENt P.L. Vi C. MaJt C. Malt JX. Cr. L.B.T1 L. Mai J. Clan P.M. S 447. H. Zirr 51. 9 H. Bart 10 C. Mai Jacquin : i E. Hub* Lop; Cercigm G.TurcJ ucc 061091 requirement is particularly important with VC and other toxic agents taken up by inhalation and exhaled rapidly. In our view the teratogenic effect of a chemical cannot be excluded in studies using shorter exposure times than the whole period of organogenesis. We are not aware of any data on the effect of VC in early pregnancy. Even if the results of our animal experiments cannot be applied directly to the human and considering that an exposure to VC at 250 ppm atmospheric concentration would result in the saturation of the metabolizing capacity of VC [31], the hazards of an occupational exposure of women in the fertile age requires thorough consideration. Infante-[IS] claims that the high incidence of congenital malformations of the central nervous system in the neighbourhood of PVC producing plants is due to VC. Edmonds et al. [19] were not able to confirm this view. In this context we studied the possibility that even if VC has no teratogenic effect to induce malformations of the central nervous system it might potentiate a teratogenic effect of other agents. Trypan blue has been reported to be embryotoxic and bring about malformations of the nervous system [32--34]. Negative results have been obtained in this respect; a concomittant exposure io VC did not affect either the teratogenic or the embryotoxic effects of trypan blue, as no higher incidence of congenital neural malformations i exencephaly. anophthalmia, microphthalmia, aplasia of the orbit) or higher fetal losses were encountered in the group with combined VC and trypan olue treatment, ACKNOWLEDGEMENT The technical assistance of Mr Gy. Krasznai, Miss A. Csonka, Mrs Gy. SzomolSnyi, Mrs J. Nyilas is gratefully acknowledged. REFERENCES ] P.L. Viola, A. Bigotti and A. Caput, Cancer Res., 31 (1971) 516. 2 C. Malloni and G. Lefemine, Environ. Res., 7 (1974) 387. 3 C Maltoru and G. Lefemine, Ann, N.Y, Acad. Sci., 246 (1975) 185. l J.L. Creech and M.N. Johnsson, J. Occup. Med., 16 (1974) 150. 5 L.B. Thomas and H. Popper, Ann. N.Y. Acad. Sci., 246 (1975) 268. 6 L. Makk, F. Delmore, J.L. Creech, Jr., L.L. Ogden, II., E.H. Fadell, C.L. Songster, J. Clanton, M.N. Johnason and W.M. Cristopherson, Cancer, 37 (1976) 149. 7 P.M. Smith, D.MJ. Williams and D.M.D. Evans, Bull. N.Y. Acad. Med., 52 (1976) 447. t- H, Zimmermann and H. Eck, Virchows Arch. A. Pathol. Anat. Histol., 368 (1975) 51. 0 H, Baruch, C. Malaveille and R. Montesano, Int. J. Cancer, 15 (1975) 429. iu C Malaveille, H. Bartsch, A. Barbin, H.M. Camus, R. Montesano, A. Croisy and P. Jacquinan, Biochem. Biophys. Res. Commun., 63 (1975) 363. E. Huberman, H. Bartsch and L. Sachs, Int. J. Cancer, 16 (1975) 639. ` - N Loprieno, R. Barale, S. Baroncelli, C. Bauer, G, Bronzetti, A. Cammellini, G. Cercignani, C. Corsi, G Gervasi, C. Leporini, R. Nieri, A.M. Rossi, G. Stretti and G Turchi. Mutat. Res.. 40 (1976) 85. 53 13 U. Rannug, R, GOthe and C.A, Wachtmeister, Chem.-Biol. Interact., 12 (1976) 251, 14 A.J. Cairo, J.B. Guttenplan and P. Milvy, Mutat. Res., 38 (1976) 81. 15 V. Ducatman, K. Hirachorn and LJ. Selikoff, Mutat. Res., 31 (1975) 163. 16 F. Funes-Cravioto, B. Lambert, J. Lilisten, L, Ehrenberg, A.T. Natarajan and S. Osterman-Golkar, Lancet,) (1975) 459. 17 J. Szentesi, E. Hornyak, Gy. Ungviry, A. Czeizel, 2. Bognir and M. Timir, Mutat, Res. 37 (1976)313. 18 P.F. Infante, Lancet, ii (1975) 1098. 19 L.D, Edmonds, H. Falk and J.E. Nissim, Lancet, ii (1975) 1098, 20 P.F. Infante, J.K. Wagoner, AJ. McMichael and R.J. Waxweiler, Lancet, i (1976) 734. 21 G.M. Paddle, Lancet, i (1978) 1079. 22 J.A. John, F.A. Smith, BJC.J. Leong and BJt. Schwetz, Toxicol. Appl. Pharmacol, 39 (1977) 497. 23 M. LOrincz, Munkavddelem, 24 (1978) 47. 24 NIOSH Manual of Analytical Methods, Method No.P and CAM-127 (1974). 25 J.G. Wilson and F. Warkany, Methods for administering agents and detecting malformations in experimental animals, in Teratology; Principles and Techniques, The University of Chicago Press, Chicago, 1965. 26 R E. Staples and V.L. Schnell, Stain. TechnoL, 39 (1964) 62. 27 J.K. Haseman and M.D. Hogan, Teratology, 12 (1975) 165. 28 J.R. Withey, J. Toxicol. Environ. Health, 1 (1976) 381. 29 E.S. Reynolds, M.T. Molsen, S, Szabd and RJ. Jaeger, Res. Commun. Chem. Pathol. Pharmacol, 12 (1975) 685, 30 J. Elis, Proc. Eur. Soc. ToxicoL, 16 (1975) 133. 31 H.M. Bolt, R.J. Laib, H. Kappus and A. Bucbter, Toxicology, 7 (1977) 179. 32 L. Denker, Teratology, 15 (1977) 179. 33 J, Gillmann, C. Gilbert, I, Spence and T. Gillmann, S, Aft. J. Sci., 13 (1948) 47. 34 J. Warkany, J.G. Wilson and J.F. Geiger, J, Comp. Neurol., 109 (1958) 35. j f i f j j I | J ; } Toxicology D Elsevier/.' EMBRYO METHYL ARANKAI Department H 3450 But (Received 5 (Revision rt (Accepted 1 SUMMARY CFY ra 1500 mg/ h/day fro toluene f< (266 pprr mice wen 2-i h/day inhaling p None o tions did anomalies Benzene ; ment. The gr orcgnanct nveiene. t '-NTRODL 1 The te j and tera ( -'fupatjt i m respect Most o TRANSLATION; Ungvdry, Gy., A. Huddk, E. Tdtrai, H. Lttrincz, and G. Folly: STUDY OF THE TERATOGENIC AND EMBRYOTOXIC EFFECT OF VINYL CHLORIDE IN CFY RATS*. (A vinilklorld teratogen fis embriotoxikus hatdsdnak vizsgdlata CFY patkdnyokon.) Eg&szBSgtudomany, Vol. 21, pp, 363-369, 1977. National Institute of Occupa tional and Industrial Hygiene and Hungarian Academy of Sciences Research In stitute of Experimental Medicine, Budapest. Gy. Ungviry, Arank* Hud 4k, Em4bt T4tri, M. Ldrinot, G. Folly: Study *( tha Teratogenic and Emkryotoxie Effect of Vinyl Chloride in CFY RaU CFY rata in the 8--14 deya ofgestation were kept in inhalation chamber* which wen oontino* oualy ventilated vertically with air containing 4000 mg/m' (co 1900 ppm) vinyl chloride. Two control group# of the eame geetation time were uaed. The firet group waa kept in a chamber ventilated with dean air and the aeoond one under normal animal-bou*e condition*. The were lacrificed on the 21et day ofgeetation andtheoffapring* wereaubjeotedto teratologic*!*tudiea. Group# of 3 CFY rata on the 18th day of geetation were aubjeoted to inhalation with vinyl chloride of 5.500, 18.000 and 33.000 mg/m* concentration reapectively for two and a half hour and the vinyl chloride concentration waa determined in the Mood of the mother* end offspring# and in the amniotio fluid*. The vinyl chloride failed to have teratogenic effect. Applied in the middle third period of geetation the above mentioned concentration did not induce embryotoxic effect. The vinyl chloride gete through the placenta ofthe rat foetna anditiademonatrabto in the foetal blood and amniotio fluid. Vinyl chloride (VC) is in 23rd place on the list of the SO moat widely used industrial chemicals. The yearly vinyl chloride production is ca. 8 bil lion kg (Teynolds et al. [20]) -- 2.3 billion kg in the U.S.A, (Boden [2]), and several million kg in Hungary. VC has a carcinogenic effect in rats, mice, and hamsters (Viola et al. [26], Maltoni and Lefemine [15, 16]), The first human case of hepatic angiosarcoma for which a causal relationship with exposure to VC could be demonstrated was autopsled in 1973 and published in 1974 [Creech and Johnsson [3]). In the WHO International Agency for Research on Cancer In ternational Technical Report, 14 reported cases of hepatic angiosarcoma were mentioned in June 1974, and already 43 reported cases were mentioned in January 1975 [27, 29]. The occurrence of hepatic angiosarcoma due to exposure to VC *Based on research conducted on the subject "Toxicological Research on In dustrial Chemicals. Investigation of the Teratogenic and Mutagenic Effects of Vinyl Chloride", accepted for the ministry level research project No. Eii M 4: "Public Health Protection...". UCC 061094 has been corroborated by several authors (Thomas and Popper [25], Makk et al. [13], Smith et al. [22], Zimmermann and Eck [31], etc.). After the confirmation of the carcinogenic effect, light was soon shed on the mutagenic effect of VC as well (more exactly, of chloroethylene oxide, a metabolite of VC) (Bartsch et al. [1], Malaveille et al. [14], Huberman et al. [9], Loprleno et al. [11], Rannug et al. [19], Barro et al. [7], etc.). Chromo some aberrations In persons exposed to VC for variable lengths of time have also been reported (Ducatman et al. [4], Funes-Cravloto [6], Szentesl et al. 124]). Data on the teratogenic effect of VC are scanty, and the results of the epidemiological surveys are Inconclusive. An Increased Incidence of malforma tions of the central nervous system due to VC in the vicinity of PVC polymeriz ing plants was reported by Infante (cited by Edmonds et al. [5]). Edmonds et al. [5], rechecking the relationship with VC, were unable to confirm these data. Infante et al. [10], who examined the wives of workers handling VC, observed Increased fetal loss; this report was not considered accurate by Paddle [18] from a methodological point of view. Experimental studies were reported only by Schwetz et al. [21]: short-term exposure to VC at a concentration of 500 ppm for 7 hours/day during organogenesis failed to elicit a teratogenic effect in S-D rats, CF-1 mice, and N-Z rabbits. Exact and detailed experimental teratological studies are definitely jus tified by the production of large amounts of VC, its wide use In the form of PVC, by the large number of persons exposed to VC, as well as by the demon strated carcinogenic and mutagenic effects of the substance, and by the contro versial epidemiological results. In our study, we wished to examine the teratogenic or embryotoxlc effect 2 UCC 061095 of VC In rats. He. also fished to determine if the VC inhaled by the mother is able to pass Into the fetus through the placenta, thereby causing direct fetal exposure. Fenale CF7 rats weighing 240-280 g were mated In a harem system. He con sidered the day on the morning of which sperm were found In the vaginal smears taken daily from the animals was considered day 1 of the pregnancy. The ani mals were kept on standard LATI feed and tap water ad libitum throughout the experiment. The animals were weighed weekly. From day 8 to 14 of the pregnancy, 28 animals were kept in two chambers with a volume of 0.13 m3 each, through which air containing 4,000 mg/m3 VC was circulated In a vertical direction at a rate of 2 m3/hour. The temperature was 24-25*C and the humidity was 50-55Z in the chambers. The VC concentration In the air of the chambers was measured by means of a Hewlett-Packard 5840 A digi tal gas chromatograph. Fourteen control animals were kept in a similar chamber, through which only clean air was circulated, between days 8 and 14 of the pregnancy. Twentyeight animals were used as untreated controls. On day 21 of pregnancy the animals were killed by ether anesthesia. After opening of the abdominal wall, the uterine horns were opened, and the position and number of the live, dead, and resorbed fetuses were recorded. The fetuses and placentae were removed one by one, examined, and weighed. Half of the fetuses from each mother were fixed in Bouin's fluid and then autopaled under a steromicroscope (Wilson [29]). The other half of the fetuses were stained with alizarin S after fixing with alcohol (Staples and Schnell [23]) for the examination of the skeletal system. The mothers were autopsled and the livers removed and weighed. 3 ucc 061096 Groups of 3 animals were, exposed to VC concentrations of 5,500, 18,000, and 33,000 mg/m^, respectively, for 2.5 hours on day 18 of pregnancy. We de termined the VC levels in the maternal and fetal blood and In the amnlotic fluid to clarify the transplacental passage of this substance Into the fetuses. The method described by LOrincz [12] was used to determine the VC. The data obtained were subjected to statistical analysis: we calculated the arithmetic means and standard error and enployed the "two-tailed" [unsure of this -- Tr. Ed.] t test. We considered the litter (pregnant female) as a teratologlcal unit (Haseman and Hogan [8]); we determined the Incidence of all affected fetuses per litter: The statistical decisions were made by means f the Mann-Whltney U teat performed with these incidences. RESULTS There was no maternal mortality. The gain In weight during pregnancy, ex pressed In Z of the Initial body weight, showed no differences among the three groups (Table 1). The number of resorbed fetuses, as well as the fetal loss, expressed in X of the total number of Implants, showed no statistically significant differences among the three groups, even though the number of resorbed fetuses was higher In the group which had Inhaled VC; the number of Implanted fetuses was also higher in this group (Tables 1 and 2) . There were oniy two dead fetuses in the experiments: one in the group exposed to air, and another in the untreated con trol group. The average fetal and placental weights, as well as the ratio of the underweight fetuses (below 3.3 g) showed no significant differences among the three groups (Table 1). The liver of the mothers inhaling VC was enlarged in terms of both the 4 UCC 061097 TABLE 1. DISTRIBUTION OF THE MOTHERS AND FETUSES IN THE GROUPS AND SOKE CHARACTERISTIC PARA METERS. KEY: (a) treatment; (b) number of mothers; (c) gain in weight1, Z; (d) number of fetuses; (e) fetal loss2, X; (f) average number of fetuses/mother; (g) average fetal weight, g; (h) average placental weight, g; CD underweight fetuses3, Z; (j) Inhalation of air between days 8 to 14 of pregnancy; CD inhalation of vinyl chloride between days 8 to 14 of pregnancy; Cl) live; and Cm) resorbed, dead. Kel&i (a) -- Icvagft fathoUott , . * Urbwafg--14. Cj) napjin vinllklorid inhaUott *Urhe4gS--14. . . napjin CD Any&k (b) U TVaUtily- ^ ^liwgMtok gyorapodiW1 Cc)% 414 Cl) rusorbeilt 68.46 14* 318 11 maggoti Tmioff' /Cek) % 8.37 14 40,30 187 1.88 6 3,18 U 81,70* 874 1.1* 17 4,64 4Uag mtgut- 11.25* 0,64 11.80 0,81 13,38* 0.37 Alkig ougxat.- 8.85 * 0,03 8,85* 0,04 3,08 0,02 at log placenla- OJy g Ch) 0,61 * 0,005 MulyivtiiriiuU pmgzalokd u CD_'" 2,90 0,65 0,007 0,30 0,62 0,(104 3.21 Expressed in Z of the initial body weight. 2Number of resorbed and dead fetuses, expressed in Z of the total number of implantations Clive + dead + resorbed fetuses). 3Number of fetuses weighing less than 3.3 g, expressed in Z of all live fetuses. 860190 oon TABLE 2.. INCIDENCE Op FETAL RESORPTION. KEY; (a) number of Implanted fetuses per litter; (b) number of resorbed fetuses per litter; (c) untreated controls; Cd) Inhalation of air; and (e) exposure to vinyl chloride. absolute liver weight and the liver weight expressed In Z of the body weight. The latter value was also calculated for the adjusted body weight of the mothers (difference between the maternal body weight and the total weight of the fe tuses and placenta). The difference proved to be significant In this case as well (Table 3). Microscopically visible developmental malformation was seen in only 1 out of the 847 embryos examined, in the group inhaling VC. The vertebral column of this underweight (3.0 g) fetus was excessively curved. Staining with alizarin S revealed the absence of thoracic vertebrae V, VI, and VII, and Irregular, malformed vertebrae above and below the gap. Left ribs Nos. 5, 6, and 7 and right ribs Nos. 5 and 6 were also missing, and ribs 7 and 8 showed a blade shaped fusion. Autopsy of the fetuses revealed the following Internal anomalies: thymus 6 ucc 061099 TABLE 3. EFFECT OF yiNYL CHLORIDE ON THE BODY WEIGHT AND LIVER WEIGHT OF THE ANIMALS. KEY: (a) treatment; (b) number of mothers; (c) body weight at time of sacrifice, g; (d) liver weight, g: (e). liver weight/body weight, Z; (f) liver weight/ /adjusted* body weight, Z; (g) Inhalation of air between days 8 to 14 of pregnancy; and (h) Inhalation of vinyl chloride be tween days 8 to 14 of pregnancy. KM (a) -- bregS inh14nirt teriMa*4g (g) 8--14. nipjtn TfaOklorid inhtlioM 4 tarhcMtg 8--14.n*pj4n '`n' Anyik ftr OlM iMttdr Xc) 14 *w,0 8.20 (d) * 15,51 0,31 M4j/tert* Air Ce) % 3,89 0,08 Uij/reduk4h*twta01j Cf)% 4,50 0,09 14 380,0 1M3 A05 A87 8,88 0,47 0,08 0,08 28 395,0 18.85 * 4.25 5,03** 3.78 0,31 0,08 0,07 ^Maternal weight -- (weight of fetuses + placentas). *p <0.05. **p <0.01. with appendage, thymic hypoplasia, hydrocephalus internus, wide renal pelvis, dilated ureter, and dilated urinary bladder. All these anomalies were seen in the group Inhaling VC at the same frequency as In the control group. The fetal skeletal system was examined by alizarin S staining. We found the following anomalies. Signs of . skeletal retardation: broad c.ranial fusions, large fontanels; two or more of the 6 ossification centers of the sternum are missing, or the majority of the ossification centers are hypoplastic; the cores of the ventral vertebral bodies are dumbbell-shaped or reduplicated, and rib No. 13 Is of reduced length. Skeletal anomales: fused. Irregular ossifi cation centers In the sternum; supernumerary ribs or rib pairs. While the frequency of the signs of retardation was numerically higher in the group in haling air than In the untreated controls and in the group inhaling VC, there 7 ucc 061100 was no statistically significant difference either in the frequency of these signs or in the frequency of the anomalies (Table 4). TABLE 4. INCIDENCE OF SIGNS OF SKELETAL RE TARDATION. KEY: (a) number of affected/ex amined fetuses per litter; (b) number of lit ters; (c) untreated controls; (d) inhalation of air; and (e) inhalation of vinyl chloride. CuUdonUnt (a) irintatt/Tizagilt migtatok wAm* 0/S 0/8 0/7 0/6 ; 0/5 0/i 0/S 0/1 1/8 1/6 S/5 m 3/8 4/7 (b) culidok mini* kmeletlen lajpujoll levegd vinilJclorid 42 11 4 71 21 1 1 2 2 1 1 1 4 8 8 5 1 l Multiple skeletal developmental malformation was found in a single fetus from the VC group, which was already mentioned. A considerable VC concentration was found in the blood of the mothers ex posed to VC concentrations of 5,500, 18,000, or 33,000 mg/m3 for 2.5 hours on day 18 of pregnancy, as well as in the blood of their fetuses. VC was also, detectable in the amnlotic fluid (Table 5). DISCUSSION To Investigate the effect of VC on organogenesis, we exposed the rats t ca. 1,500 ppm (4,000 mg/m3) VC in the air continuously between days 8 and 14 of pregnancy. This concentration is 80 times higher than the MAC effective in 8 ucc 061101 * TABLE 5. VINYL CHLORIDE CONCENTRATIONS. KEY: (a) chamber, mg/m3; (b) maternal blood, yg/ml; Cc) fetal blood, pg/ml; and Cd) amniotic fluid, pg/ml. Kunrm Anymi v4r mg/m1 (a; (b) Ml/** Magzkti v4c (c) /</* [d) /* 0 5 500 00 19,0* 1,70 00 13,80 3,83 00 4J7 0,43 18 000 33,80 3,13 33.07 3,78 4,83 0,18 33 000 48,i8 1,08 30,83 3,77 13,40 3,98 Hungary (MAC value in Hungary: 50 mg/m3 ca. 20 ppm), i.e., it la a high con centration, Yet this high VC concentration failed to cause any noticeable change in the animals' behavior: their feed and water consumption was the same as that of the controls, and there was, in principle, no reason to expect the so-called "self-protective mechanlsm"-induclng effect of VC, either (Reynolds et al. 120]). We should mention, however, that the liver weight of the animals exposed to VC was Increased, and the cause is currently unknown. Continuous exposure generates a steady VC level In the blood of rats (Withey [30]). This steady blood level and the rapid interstitial migration of the lowHSplecularweight VC (Withey [30]) suggest that not only the mothers, but also the fe tuses are exposed to VC steadily. This is also supported by the results re ported in this paper; VC was detectable In the blood of the mothers exposed on day 18 of pregnancy, and also In the fetal blood, as well as In the amniotlc fluid. The hemoendothelial placenta of the rat represents no barrier to VC. These examinations, carried out on a large number of fetuses, have basically yielded a negative result. Yet two findings which, though not sig nificant statistically, show a numerical difference, deserve to be analyzed briefly: 9 UCC 061102 k 1. The. number of cesorbed fetuses was Increased la the group- inhaling VC,* the number of Implantations as well as the number of the live fetuses per mother were also higher in this group. The Increased resorption may possibly be at tributed to the higher number of Implantations. 2. The frequency of the signs of skeletal retardation was numerically higher in the group inhaling air than in the group exposed to VC; this value was higher in both these groups than in the untreated control group. This phenomenon can be explained by the stressful effect of the unusual environment In the inhalation chambers. The stress effect was fended off partially by the VC, having a narcotic effect. Consequently, it can be said in the light of these results that VC as such is not teratogenic in C7T rats, nor does it have an embryotoxlc effect in the concentration applied in this species. Schwetz et al. [21] also arrived at a similar conclusion. However, they drew their conclusion on the basis of ex aminations following exposures during short, arbitrarily chosen periods of organogenesis. We wish to emphasize the importance of exposure of the animals to the test substance at all relevant moments of organogenesis in experimental teratology. 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