Document MMe49RBaxXoxX1ygQwJYe7eLy

> )X i,nvir<uwwn(al Health /Vrvprf lives Vnl 21. m>, 71 7M, l<)?7 Dominant Lethal Studies with the Halogenated Olefins Vinyl Chloride and Vinylidene Dichloride in Male CD-I Mice by Diana Anderson,* M. C. E. Hodge,* and I. F. H. Purchase* The mutagenic iictmtv of tin.*I chloride (VO mxl vimlidene dichloride i\ DC) a! three exposure le'cls ssns assessed in fertile male CD-I mice with the dominant lethal test. Kadi compound was assessed in u separate studs. Male mice were exposed hy inhalation to VC at 3000. 10.000, and 30,000 ppm and to VDC at 10, 30, and 50 ppm for 6 hr/day Tor 5 days. By comparison with control males exposed to air, no mutagenic effects on any maturation stage of spermatogenesis in treated males were detected. T here was no significant increase in the number of postimplantational early fetal deaths as shown hy (he number of female* with one or more early deaths or the number or early deaths/prcgnuncy or the number of early deaths total implonts'prcgnuncy. There was no evidence of prc-implantational egg losses as indicated hy the total implants pregnant female. There was also no reduction in fertility. (The reduction in fertility at 50 ppm VDC was unpmtenb The tack of effect was not due to the insensitivity of the system used, since both the VC and VDC study a mutagenic effect was clearly demonstrated in male mice dosed iP with the positive control compounds cvclophosphamide (CTX) and/or elliyImcthanc sulfonate (KMS). During dosing these animals were housed under similar exposure conditions to those animals exposed to the test substances but with a flow of 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 polytvinyl chloride) ha*? been found to cause tumors in rats (/) and man (2). It has also been shown to produce chromosome breaks in exposed workers (3-6) and causes mutations in Salmonella typhinumum (7, 8).. Another chlorinated monomer, VDC is also known to cause mutation in Salmonella typhimnrium (7,8). We. therefore, carried out dominant lethal studies to determine if there were any mutagenic effects of Mnipcri.il C liL-niik.il Industries I.id.. Ccntml To\icuU>vy Luhuridory. Aldcrley l';irk nr. Macclesfield. Cheshire SKIO 4TJ. F.npland. this type in mice after V'C and VDC exposure at three levels. At the same time negative control ani mals 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 1 td., Worslev. Walkden. I.ancs,. U.k. VDC was obtained from 1(1 l td.. Moral Division. Runcorn. Chcshiie, U.K. CMS was obtained fiom koch-Light 1-ld., Colnbrook. Ducks. U K., and CTX (hndoxar.a) from Ward Hlenkinxop Ltd.. London. U.K. December 1977 71 t; GC 00020Q Animals CD-I mice tC'hailes River, Manston, Kent) were used throughout the experiment. Unclosed females were 8-10 weeks old when mated ansi male mice immediately after dosing were 10-12 weeks old. Males were caged individually and females in pairs. They received food and water ad libitum. 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 be 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 fluid-, feed/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 dean air. The volume of air required as calculated from Eq. (1). Air flow (l./min) = Dining dosing the mice weie housed individually iu chambers made of stainless steel mul glass with an internal capacity of lliiee litres. Most of the negative conlml animals and till ani mals dosed with Cl X or EMS were housed under identical conditions during the dosing period but with a (low of air through the chambers. Those dosed with (TX were injected II* on day 5 of ex posure and those dosed with EMS were given tin 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 animtils which w'ere 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 studv. Of the 17 animals, three in the CTX treatment group also did not mate in the fertility study. Eilty animtils 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. Syringe size (ml/cm) x density (g/ml) x 24 x lO" Mating Injection rate (min/cm) x ppm x mol. wt. Fertility Testing. Male mice were caged with (i) virgin female mice, I male and 2 female mice in Group VC Study Group 1 Group 2 Group 3 Group * Group 5 Group ft VDC Study Group 1 Group 2 ` Croup 3 Group 4 Gioup * Table 1, Treatment groups. No. survivors/ no. animtils treated (mii'C) Treatment 20/30 1 ft/20 19/20 9/20 15/15 25/25 30/30 2000 IK/20 ft/20 2/20 17/17 Air (ncuulive control) 3000 ppm VC, ft hi dav. 3 daw 10.000 ppm VC, ft lit'day, 5 days 30.000 ppm VC. ft hr/dav, 3 dais 200 mg (1 X mi water kg hod} weight, once lw IP injection on d;i\ 5. 200 mg IIMS in Wiilcr'kg both weight orulls, once'rtiiv. ^ do\s 13 aii. 13 mum.il vnnililunis utce.itive umtioll 10 ppm VIX , ft tn 'd.i\. 5 this * Ml ppm VI)< ft hr d;i\. * thus 50 ppm VIX . ft hi day, 2 dass 75 pprn VOC . ft hrdnv for 1 day, then 50 ppm VIX . ft hr d.i\, for I dav 200 mp C I ,\ in water kp l-odi weight, nine Iw IP uiicetion on dav s 72 Environmental Health Perspectives ,I t >i t r ** 4 Ct'? c;ich cuyc. After 5 duys the females were trnns- feried to otherc;tgcs. Tlie female mice were killed 15 days after Iasi introducing them to the males and examined for pregnancies. The 106 males which survived dosing in the VC study and 115 males in the VDC stud^ and were successful in fertilising at least 1 female in their cage were selected for con tinuation in the expeiiment. ICxiicrinicnltil Mutiny. Two virgin female mice 8-- 10 weeks old were put into each of the 106 and 113 cages in which the males were individually housed. After 5 days the females were removed and rehoused in paiis, A week after the initial introduc tion the males were caged with another two virgin females anil again left for 5 days. This process was repeated until the treated male mice had been mated at weekl) inters uls for 8 weeks with virgin females. The males were then killed and not examined fill (her. No attempts w ere made to establish whether or when mating had occurred. Instead it was assumed that most matings leading to fertiliza tion would occur 2 or 3 days after introducing female mice to the cages containing males. female mice weie killed 13 days after the asslimed dale of leitili/aiion, i.e., 15 or 16 days after caging females with males. Assessment Uteri of killed mice were examined for live im plantations, early deaths, and late deaths Statistics 1 he data have been statistically analyzed as re ported previously (10), Results Mating weeks after treatment are represented in Tables 2-13 by numbers 1-8 and the mating week before treatment is repiesented by week 0. As sessment of females which became pregnant during the fertility test yielded the data for week 0 bill only data from those animals that survived treatment hare been included in weeks 0-8. Tables 2-7 relate to the VC study and Tables 8-13 relate to the VDC siudv. Table 2. Number and percentage of male mice which survived treatment successful!) niatinu at each week. Week 0 1 2 3 4 5 b 7 X "/> " 0.001 Group I, air (negative control) No. of rff mice 20 100 20 KM) 20 100 20 100 20 loo pi 95.0 20 100 20 too 20 KM) Gioiip 2. V( . 4 .OIK) ppm fb hr < 51 No. of Cf mice IK 100 IK 100 IK 100 IK 100 IK KM) IK KM) IK 100 IK 100 IK KM) Ciloup l, VC , 10.1)00 ppm (ft hr 5) No of rA mice 19 KM) IK 100 19 Kill |9 KM) 19 100 19 100 19 KM) 19 100 19 100 Ciloup 4. VC , 40.000 ppm (6 hi X 5) No, if n mice 9 100 7 77.K 9 100 9 KM) 9 too 7 77.K 9 100 9 1(H) X XX. 9 Gump V CI\. 200 mgkg IP No of r/( mice 15 ion 14 93.3 14 . 93.3 14 93 4 It 1(H) It 100 14 Kill 14 93 3 13 92 9 Group 0, F:\IS. 200 mgkg (oral) x 5 No, of r< mice 25 100 y 12.2 23 92.0 23 92.0 25 too 25 100 24 10(1 25 too 25 100 Table 3. Number of mated females beenming pregnant. Week Group 1. air (negative control) Group 2. VC . 4.IMM) ppm (ft hr x 5) No. No. No. No. piegnanl maled pregnant maled 0 .VS 1 35 3ft 3 44 4 }H S 4S ft 40 7 ,47 X 47 "l> o ooi. > "ooi 40 .40 40 4.4 40 .4ft 40 .40 40 .44 40 .44 40 .44 40 4 1 40 32 3ft 3ft 3ft 4ft 3ft 3ft 3ft 3ft 3ft Group 3. VC. 10.000 ,<pm (ft III S) No. No. pregnant mated 2K IK 32 3b 33 4K 44 tx u >K 3b IK 4ft 4K 47 3K 31 47 C Jump 4, VC 30.000 ppm ift in Si No. No. piegnanl mated It IK 11 IK It IX Ift IK 17 IK 12 IK IK IK IK IK 14 IH Group t. C r\. 21MI ing.'kg n> No. No pi tenant mated 23 30 2b 30 25 30 2 b 30 30 30 2K u> 2ft 2K 71 30 23 2S CiiiUip b. IMS. 2IM) mg kg t oral 1 N S No No piegnanl maled 37 50 4" 50 3V' so 40 Ml lb SO 17 Ml P 4X 41 50 4S 50 December 1977 73 GGC 000210 Week 0 1 2 3 4 5 6 7 8 "p < 0.01. p < 0.05. Week 0 1 2 3 4 5 6 7 8 "p < 0.01. Week 0 1 2 3 4 5 6 7 K */> < 0.01. Week 0 1 2 3 4 5 6 7 8 "p < 0.05. "p < 0.01. 74 Group 1, air (negative control) 1 1.0 12.9 12.8 12,5 12.4 13.7 11 7 11 8 12.7 Table 4 Mean Infill implants per pregnant female. Group 2. VC 3.01)0 ppm (6 hr x 5) 12.6 13.6 12.7 12.5 12.1 12.7 11.9 11 6 12.1 Gmifp V VC. 10,000 ppm <0 In x 3) 12 0 13.4 1 3,0 12 4 13.7 14.1 12.5 11.8 12.6 (lump 4, VC . W,000 ppm <b hr - M 13.0 16.1 13.7 II 2 10,0"' 12 8 12 6 12 0 12.1 < il'Mip ( 1 X. 200 rug'kg IE (1.3 8 8' (0.9 10.9 12 6 12 3 12.5 11 3 12 5 (tump f> 1 MS, 200 nig'kg foi ^ > * 3 II 8 4,0" X 4' 12 3 12 7 12,7 12.1 12,4 12,5 Table 5. .Number of pregnant females with one or more early deaths (Kl)t. Group 1, air (negative control) Group 2. VC. 3.0(H) ppm (6 hr x 5) Group 3. VC, 10,000 ppm (6 hr x 5i Group 4. VC. 30.000 ppm (6 hr x 5) Group 5. CTX. 200 nrg/kg IP Gump 0, EMS, 200 me kg (orall x 5 0 SIF.D f ^ 1 ED 0 s 1 ED 0 s 1 ED 0 18 17 21 9 16 22 10 5 22 15 20 16 17 10 21 6 5 1 19 17 18 18 13 20 6 9 2 18 l(i 16 14 19 15 9 6 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 3" 21 25" 1 23" 5 18 15 16 19 16 25 1.4 24 12 22 10 22 ^ 1 ED 26 3 26" 25 27 22 21 19 23 Table 6. Mean number of early deaths per pregnancy Group 1. air (negative control) 0.77 0.86 0.83 0.91 0.89 1.06 0,97 0.76 1.03 Group 2, VC. 3.(MX) ppm (6 hr x 5) 0.33 0.91 1.00 0.74 0.79 0.71 0.91 0.87 0.88 Group 3. VC. 10,000 ppm (6 hr x t) 0.54 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,(6 4.27" 4.84" 1 34 1.13 l.ll 0.65 , n.x: 0.70 Table 7. Karly death* us a percentage of tola! implants per pregnant feinalc. Group 1. air (negative control) 6.5 6.6 6.4 7.3 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 5.9 6.5 55 7.6 7.3 7.2 Group 3, VC. 10.000 ppm (6 hr x 5) 4.4 7.5 9.6 7,0 4.5 7.3 6.0 5.7 8.1 Gmup 4. VC. 30.000 ppm (6 hr x 3) 31 2.7 6.9 7.8 6.5 7.9 III 7.4 5.9 Group 5. CTX. 200 mg/kg IP 1 4" 49 I" 44 A" 14.1 V f> 9.1 5,2 7,0 5.7 Group 6. IMS. 200 mg/kg (mu!) x 5 0.59 3 50" 2.58" 1.45 0.83 0.72 0 85 0 63 0.73 Gmup f. IMS. 2(H) mg/kg (omh * 3 '1 77 78\\ 11.6 h7 57 7.0 31 Knviruumeutal Health Perspectives > * . ;i I t i i t ; V V J 1 1 t ] j 4 ' *r -V if }? i 1 (- (. Tahir K. Niimlwr and percentage of male mice which surii\cd (realmcnl successfully: mating al each week. Week 0 1 2 1 4 S 6 7 8 Ciump 1 (pooled negative control) No, of mice % 50 100 50 9 50 96 50 94 49 98 47 94 49 8ft 49 92 4ft 92 < MOIIp 2. VIX , 10 ppm (6 hr x 5) No. of mice 20 20 20 20 20 20 20 20 19 % too 95 100 100 100 100 100 90 95 ( rump 1, Vl)( . til ppm (6 hr < 51 No. of mice 7c 18 100 IK too 18 94 18 89 18 89 17 82 17 100 17 100 17 88 f lump 4. Vl)( , 50 ppm (6 In v S) No of mice ' 8" lh 7" 7' V 7' 6' T % 38 75 86 43 43 57 43 50 57 Group 5, (IX. 200 mg/kg IT No. of mice 17* 17" 16 17 15 17 17 I6" 19 7c 76 65 94 94 100 [00 88 75 86 "p < 0 1)01. V < O.m. 'p < 0.05. ------------- Week 0 I * 4 s 6 7 8 Table 9. Number of maled females becoming pregnant. Group 1 (pooled negative control) No. No. pregnant mated 73 100 82 100 75 100 82 100 85 98 80 94 75 98 77 98 73 96 Group 2. VDC, 10 ppm (6 hr x 5) No No. pregnant mated 30 40 32 40 36 40 34 40 36 40 34 40 33 40 33 40 28 39 Group 3, VDC. 30 ppm (6 hr x 5) No No. pregnant mated 25 38 33 36 32 36 26 36 27 36 26 34 32 34 28 34 26 34 Group 4. VDC. 50 ppm (6 hr x 5) No. No. pregnant mated 5* 16 7" 16 7" 14 5" 14 6* 14 6* 14 5* 14 7 13 7 14 p< 0.01. *p < 0.001. f p < 0.05. Group 5. CTX. 200 mg/kg IP No, No. pregnant mated 17* 34 20* 34 25 33 28 34 26 32 29 34 25 34 19r }\ 28 39 Week 0 1 2 3 4 S 6 7 8 V < o oi. V < o.ooi 7> < 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) 11.0" 13.1 11.7 12.1 12.1 11.7 12.4 13 0 13 6 Group >. VDC. 30 ppm (6 hr x 5) 12.0 12.8 12.1 13.2 13.0 12.x 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 11.5 9 2H 9.5* 11.0" 12.1 12.0 1 t.r 11.7' 11.6 !In*! 1 if % & $ b 1 f E |V f 75 (:- Table 11. Number of prcuniint females v*ith one or more curb deaths Week 0 1 2 l 4 5 6 7 X "p < o.oot. V < o.oi. Gump 1 (pooled negative control) 0 1 ed 40 33 41 41 39 3ft 45 37 40 45 41 39 40 35 43 34 37 3ft Gmnp 2, vdc. 10 ppm (ft hr x 5) o ^ i i:d 14 Ift IX 14 IX IX IX 16 17 19 15 19 14 19 15 IS 13 15 Group 3, Vl)( , 30 ppm (6 hr 'x 5) 0 3 1 ID 10 15 19 14 17 15 15 II 11 tft 11 15 15 17 15 13 13 13 Gioup 4 VIH . 50 ppm hr 5) 0 ^ 1 KD 14 ft 1 34 41 :4 33 23 52 34 Group 5. CTX. 200 mg1 kg IP 0 3 I ED 9X O' 20 ?" Tl 7" 21 12 14 12 17 12 13 10 9 15 5 Table 12. Mean number of early deaths per pregnancy. Week 0 1 2 3 4 5 6 7 X -p < 0.001, Group 1 (pooled negative control) 0,59 0.72 O.fW 0.7ft 0.79 0.7X 0.XX O.fiS 0.X5 Group 2, VDC. 10 ppm (ft hr x 5) O.XO 0.72 0.75 0.6X (I.X3 0 ftX 0,91 0.X5 0.75 Group 3. VDC. 30 ppm (ft hr x 5) 0.X4 O.fil 0,5ft 0.4fi 1,00 0.73 0 94 0.57 0.X5 Group 4. VDC. 50 ppm (ft hi x 5) 2.00 0,29 0.71 O.XO 1.0(1 0.67 1.00 0.57 0.71 Tahle 13. F.arly deaths as a percentage of total implants per pregnant female. Group 5, CTX. 200 mg/kg IP 1.0ft 4 00" 4 04" 2.32" 0 X5 O.Xfi n.ftj 0.74 0.30 W'eek 0 1 2 3 4 5 ft 7 X "p < 0,001. Group 1 (pooled negaiive control) 4.X 5.7 5.5 ft.4 ft.9 ft.ft 7.0 4.9 7.0 Group 2. VDC. 10 ppm (6 hr x 5) 10 1 5.3 6.9 6.3 7.1 ft.l 7.ft ft.fi 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 Group 4. VDC. 50 ppm (6 hr x si 17 3 2.2 5.7 S} 10.3 5.6 7.5 4.3 5.X Group 5. CTX. 2(H) mg/kg IP 9.1 45.6" 41 4" 2I.X" 6.9 6.9 5.6 ft.l 2.4 Fertility Successful Mat ini' Frequency. The numbers of mules successfully milling ill cuch week are shown in Tables 2 and 8. Numbers remained high during the experimcnis. No statistically significant differences in the mating frequency were found be tween VC tieminent groups and the control at any week by using a chi-squmed test. There was. how ever. a significant difference between the HMStreated group and the negative control group in week I. In the VDC study, the milting frequency was 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-S 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. Pregnancy Frequency. The numbers of females in each group which became pregnant at each week of mating are show n in I ablcs 3 and 9. In the VC study, statistical differences between treated groups and the negative control group weie found only in the FMS-lieuicd group at weeks I and 76 (environmental Health Perspectives i I t t t * I 1 i * * 2 by using a chi-square test. In the V!)( study there weie significant ililTetenees in the highest VDC exposure group at weeks 0-6 aiul the positive control group in weeks 0. I. anil 7. Again this was probably due to infertility of the males. These tesults indicate that VC' at the three ex posure levels and VDC' at least at exposures of III and 30 ppm did not cause a reduction in fertility. Any reduced fertility at ?0 ppm VDC was un proven. Total Implantations The mean total number of implants per pregnant female in each gtoup is shown in Tables 4 and Id. The mean values weie 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 /-test. In the VC study statistically significant differences were evident in week I in the CTX-trealed gionpand weeks I and 2 in the EMS-treated group. A significant difference (p < 0.05) in week 4 was also found between I he group exposed to the highest dose of VC (Group 4) and the negative contiol group. In the VDC study the CTX positive contiol 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 from the negative control group in the prc-experimental nontreatment week. Thus there was no indication of a preimplantation loss of eggs in either study except in week 4 after 30,000 ppm of VC. Early Deaths with IMS tieaiment. In the VDC stmlv only the (IX positive contiol group was significantly difleient limn the neuali'c contiol eioup m weeks I, 2. and 3 ( I able 12). Farly Deaths as a Percentage oj Total Im plants )ter Pregnant Female. Again, it was necessary to stabilize the variance prior to analysis. In the VC study CTX and EMS heatmeut gtoups were significantly different from the negative con trol groups in weeks I and 2. whereas VC-treated groups were not l iable 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 (Table 13). Tims with all these different methods of analy sis of the data of early deaths no statistically significant difleiences from the negative control gioups were seen in the VC' or VDC treated groups. Late Deaths In each study late deaths were randomly distiibuted throughout all the groups and did not appear to be Irentmcnt-ielated. Conjoined Placentae Conjoined placentae were seen in this strain. They were the result of very close implantation sites and were not monozygotic twins (/2). In the experiments they were classified as double implan tations. Since they occurred with equal frequency in all groups they did not appear to he correlated with treatment. The data for early deaths have been presented in various ways. A'umber of Pregnant Females with One or More Farit/ Deaths. In the VC study. CTX and EMS 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 OTX-trcaled group and in week 2 for the EMStieatcd gioup. 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 11). The Mean Snmher of Farly Deaths per Pregnancy. A large number of low or zeio values were encoimteted. so it was necessary to stabilize the variance prior to analysis. There were no statis tically significant increases in early deaths aflct VC treatment ( Table ft). However. dilTcienees wete evident in weeks I and 2 with CTX treatment and Discussion The best indication of mutagenic activity of a substance in (lie dominant lethal test is an increase in the number of post-implanlational foetal early deaths (/.f). from the data for eatly deaths there was no evidence in either study of a mutagenic ef fect with VC oi VIK' at the administered exposure levels, I his did not appear to be a resull of lack of sensitivity of the animals used, since there was a marked response to ("IX and EMS. High doses of ("I X anil E'MS weie used in out studies to obtain a highly significant positive result. However, the dominant lethal study m our hands (/-/) is at least as sensitive as that reported elsewhere We have shown ethyl methane sulfonate on pievions occasions to give a positive result with a single IP dose of 130 mg/kg body weight, which is computable to that repotted previously (/.H lor the same mouse strain. December 1977 77 For reasons described earlier (10, II), different evaluation methods were used for the early deaths' data and much of the data in the studies were sub jected to various methods of statistical analysis. Pre-implantation egg losses, while representing some of the mutagenic effect, are not as important as postimplantational 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 in females mated with treated males and those mated with control males, as suggested by Epstein (16), rather than counting corpora lutea. There was no preimplantational egg loss by comparison of VC- or VDC-treated groups with the negative control ` group, except in the highest VC exposure group in week 4 (p < 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 due 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 increase in early deaths at this time. Late deaths also are not considered as important as early deaths in the assessment of the mutagenic potential of a test substance (13). Late deaths were excluded from analysis to increase test sensitivity. Gropp and Kolbus (17) have shown, however, that trisomies in mouse foetuses cause death and elimi nation before term. It might be argued that sampling for late deaths at a later stage of pregnancy might lead to different results for 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. However, 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 size to perform the experiment. A fertility study was undertaken prior to the ex periment also for ieasons described earlier (10, 11). From 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. In this study where exceptionally high doses of VC were used, which would never be encounteied by workers, no mutational effect in the germ cells was observed. A possible explanation is that the active metabolites did not reach the germ cells. Similarly the lower but more toxic levels of VDC did not produce germ cells mutations. It can be concluded, therefore, that VC and VDC do not cause dominant lethal mutations in male CD-I mice at 30(40. 10,000. and 30,000 ppm and 10, 30, and 50 ppm. respectively. The authors would like to thank the Inhalation Section fur exposing the males to VC and VDC, Mrs. s. Painter foi her technical assistance, and Mr. T. Weight and Mr. S, H. Ellis for their statistical evaluation of the data. All personnel mentioned are at this laboratory except Mr. 5. H. Ellis, who is at IC1 Pharmaceuticals Division, Alderley Park. REFERENCES 1. Maltom, C., ct al. Vinyl chloride carcinogenesis--current results and perspectives. Med. Lasaro 65: 421 (1974). 2. Creech- J. I... and Johnson. M. N. Angiosaicoma of the livei in the manufacture of polyvinyl chloride. J. Occtip. Med. 16: 150 <19741. 3. Ducatmnti, \.. Hirschom. E.. and Selikolf. I. J Vinyl Chloride cxpoMiic and human chromosome abcualions. Mutalion Res. 31: 163 (I975l, 4. Euncs-Cravioto. F . et al. Chromosome aberi.uions in uoikers exposed to vinyl chloude Lancet 1:459 (19751. 5. Purchase. I. L. II.. Richaidson. C. R.. ami Anderson. D. Chromosomal elfcets in peripheral lymphocytes. Pioe. Roy. Soc. Med. (London) 69: 290 (1976). 6. S/cnlcsi. 1 . et al. High late of chromosomal alienation in PVC workeis Mutation Res. 37: 313 (1976). 7. Hnrlsch. IL. Malaveille. C . and MonlCsano. R. Human, rat and mouse liver-mediated mutagenicity of vinyl chlotide in S, strains. Ini. J. Cancer 15' 429 (1975). H. Bartsch. H..etal. Tissue-mediated mutagenicity of vinylidene chloride and 2-chlotohutadiene in Sulnioiicllii ivplimiurmni. Nature 255: 641 (I975t. 9. Gage, J, C. Toxicity of epicliloihydrin vapoui. Urn. J. Ind. Med. 16- 11 11959). It). Andcisoit D,. McCnegor. D, U . Purchase. I. I , H, Dom inant lethal studies with paraquat and diqual in male CD-I mice. Mutation Res. 40: 449t|976l. 11. Anderson. D.. Hodge. M. C. L.. Purchase. I. I- H. Vinyl chloride: dominant lethal studies in male CD-I mice. Muta tion Res. 40: 359(1976) 12. Uutcnian. A. J. Dichorial. one-egg twins in the mouse Naluic 1X7: 349 (19601 13. Bateman. A. J., and Epstein. S. S. In: Chemical Mutagens. Puneiples and Method' for I lieu Detection. A Hollandei, Ed . Plenum Press. Nc" X ork-l.ondon 1971. pp 54I-56X. 14. Anderson. I),. et al. Dominant lethal lest results with known mutagens in two lahoialones. Mutation Res. 43: 231 (1977). 15. Ray. V .........id llyneck. M. I.. Some puntaiy considera tions in the iiilcipiclntion of the dominant lethal a".n . Inviron Health Petspcct. 6 24(1973). 16. I pslom. S S. I lie use ol the dominant lethal lest to detect geneiic activity of one iioniucnlal chemicals I nvitott. Health I'etspecl 6, 2' (|974>, 17. Giopp, A . and kollms. I I xeuccphalv in the sv lulionte of liisomy no. 12 ol the loeial mouse Nalme 249; I4'(|9?4p, 78 Environmental Health Perspectives GGC 000215 194 Annals New York Academy of Sciences Popper about the reversibility of the phenomenon, particularly if caught early. If workers are exposed to moderate to low levels for relatively short periods of time, has there been any evidence that there is any irreversible change, or does the progression that you were documenting take time? Dr. Popper: The only real experience we had was a case which Dr. Berk presented. In this case, after discontinuation of the exposure, fibrosis persisted, but other features were absent. The characteristic disturbing activation of the hepatoevtes was no longer seen. I am not surprised. From experience with hepatic fibrosis in circumstances other than vinyl chloride, it is known that fibrous tissue may not disappear once present. I suspect, but do not know, that the person may be left with innocent scars in the liver which possibly may not interfere with his life expectancy. One patient, however, is really no proof. Part H. Carcinogenesis associated with Vinyl Chloride A. Experimrittut Studies CARCINOGENICITY BIOASSAYS OF VINYL CHLORIDE: CURRENT RESULTS Cesarc Maltoni and Giuseppe Lcfemine Imtuio di Oncologta and Centro Tumor! Bologna, Italy Introduction Vinyl chloride (VC) has been produced in many countries for more than 30 years. It is the constituent of polyvinyl chloride (PVC). which nowadays is the plastic industrial material most used and diffused in the world. Moreover, it is used as a copolymer in Saran and other plasties, as a chemical intermediate, as a solvent, and as a propeilant: in the past, lor a short period, it was also used as an anesthetic. VC is at present produced at a rate of near 12 million tons per year. The following population groups may be exposed to VC: (I) workers engaged in the proauction of PVC ana oi VC and in the use of VC for other industrial purposes; (2) workers manufacturing PVC, (3) residents in areas neighboring on factories producing PVC and VC: and to a much lesser extent (4) people using products containing VC, as some propellent sprays: (5) people having contact with resins made with VC and with materials containing these resins: and (6) populations consuming food contained in plastics made with VC. Bioasxays on the long term effects cl VC wfcre not available until 1970. This situation reflects the limited consideration which has been, up to the present, given to experimental bioassavs m predicting the oncogenic effects on man of environmental and occupational agents. In part this is due to industrial and economical interests, in part to deficiencies in techmcal-icientific facilities, to the dilettantism of many experimental workers, and to the madequaev ot many experiments performed in the past. What is now happening with vinyl chloride and the *torv of vinyl chloride carcinogenicity should bring about greater consideration of experimental bioas says and finally induce a new turn in the field oi occupational and environmental carcinogenesis. In other words, experimental prediction on the pathogenetic potential of occupational and environmental agents before they are produced and released on a large scale into the human environment should preclude the need for latei epidemiological evidence. History In 1970 Professor Viola presented, at the X International Cancer Congress (Houston!. the results ot a pilot experiment, which then were extensively pub lished m 1971.' Professor Viola reported to have observed in 26 rats exposed bv inhalatory route to 30,000 ppm of VC 4 b daily, 5 days weekly, for 10-12 months, surviving 10 months or more, 17 skin tumors, 7 pi nary tumors, mainly carcinomas, and 5 osteochondromas. GGC 0 0 0 2 19t AlltlJld 1UIN rtt.JoLl.iJ l/l JtMl In 1967 our Institute started a large investigation, among workers of several Italian industrial plants, to determine the effects of the exposure to different chemicals on respiratory epithelia. The pathological changes were studied by sputum cytology. In 1970 this research was extended also to workers in VC-PVC industries. A significant increase of cellular changes in bronchial epithelium, such as marked squamous metaplasia and squamous dysplasia, were found among the workers more heavily exposed to VC. Immediately after the Houston Congress, we contacted Protessor Viola who kindly put some slides of his experimental tumors at our disposal. According to our interpretation, the malignant tumors observed by Professor Viola in the skin were carcinomas arising from the Zymbal gland fa sebaceous gland of the ex terior acoustic duct), which is responsive to a large number of carcinogens. The malignancies of the lung, in our opinion, were metastases from Zyrnbal gland tumors. At that time {end of 1970) we planned a project of correlated investigations to further clarify the type and degree of the carcinogenic risk from VC. The project was encouraged and supported by Montedison (Italy), which later was joined bv other major European companies, that is, IC1 (U.K.), Solvay (Belgium), and Rhonc-Progil (France). The Head of the Health Services of Montedison supplied us with all the technical data dealing with occupational exposure: this made possible the repro duction in our experiments of the most important parameters of occupational conditions. Planning, Materials, and Methods This project included a series of experiments, which were started in sequence. Thev were designed to study the etfccts of VC. administered through dilTcrcnl routes (inhalation, ingestion, and peritoneal and subcutaneous injection I, indiffer ent concentrations, for varying periods of time, bv continuous or intermittent treatment, on animals of different species irats. mice, hamsters!, strains (Sprugue- Daw lev and Wistar rats!, sex and age (adults, new corns, embryos). The planning of the program and the preparation ot suitable experimental conditions, particularly the building of an apparatus for inhalatory exposure, took us until May 1971, In July 1971 we started experimenting. The chambers of exposure are built basically ol stainless steel and glass. They have been desiened to deliver concentrations varying from 30.000 to I ppm of VC. and to treat simultaneously nearly 1,500 rodents. The VC concentration in the chambers.is controlled bv gas chromatogranhy. The VC i> supplied by Montedison. Each stock is analysed in ihc Montedison Research Unit. The impurities and their maximal levels in the VC employed have been as follows: H,0 !(X) ppm Acetic aldehyde 5 ppm Acetylene 2 ppm Allene 5 ppm Butane 8 ppm 1,7-flutadiene 10 ppm Chloropnene 10 rpm Diacctylene 4 ppm Vinyl acetylene 10 ppm Propme 3 ppm Methyl chloride 100 ppm .ijiiom A: Lcleu Lutk...,;,....... -J The experiments utilized mainly Spraguc-Dawley rats; Wistar rats, Swiss mice, and hamsters have also been used. All the animals, except the hjmsters. arc bred m our Institute for years and. whatever their use, arc all examined at death by com plete autopsy, giving us extensive information concerning their current palhologv The animals have been weaned and classified by sex when 4-5 weeks old, at which time they have been numbered by ear punch and divided into groups by litter distribution. From weaning, the animals have been fed. ad libitum, an adequate commercial diet. During the period of treatment, the animals are housed in groups of 10 in stainless steel wire cages with a solid bottom of the same metal. After the period of experimental treatment, the animals are kept in groups of 5 in makroion cages, with tops made ol stainless steel wire. A shallow layer of white wood shavings serves as bedding. The animals arc kept in a temperature-controlled laboratory at 19-20'C. Fifteen experiments have been started in sequence. Some of them were pro grammed at the beginning; for some others the need appeared on the basis of the first experimental results. The plan of the experiments and the up-to-date situa tion is shown in Table 1. Experiment l studies the effect of the atmospheric exposure to 10.000, 6,000. 2,500, 500, 250, 50 ppm of VC, 4 h daily, 5 days weekly, for 12 months. Two groups of animals were added as control: one untreated, as for all the following experiments, and one treated with vinyl acetate (VA) at 2,500 ppm. under the same conditions. This dose of VA appeared to be the maximum possible dose for a chronic exposure. Experiment 2 was performed to investigate in a larger number of animals the effect of doses bciween 250 to 50, that is 200, 150 and'100, under the same con ditions as in Experiment 1. It was started after it was clear that a dose of 250 ppm was still showing oncogenic effects. Experiment 3 was planned to study the effects of a shorter period of exposure, keeping all the other conditions of Experiment I. Experiment 4 studies the effects ot the same type of exposure as m Experi ment 1 in another species (mice). The treatment lasted 7 months in consideration of the higher mortality and the shorter life span nf the mice of our line as com pared with rats. Experiment 5 was undertaken as a pilot investigation on the possible effects of the atmospheric exposure, during pregnancy, on offspring. Experiment f> was planned to reproduce the same conditions as in Professor Viola's experiments. Experiment 7 was planned to study the effects of (he some type of exposure as in Experiment I in another strain of rats (W'istarl. Experiment S studies the effects ot the same exposure as in Experiment 1. in a third species i hamstersi tor the same period as in Experiment 4. Experiment 9 was planned, before tumors were observed at 50 ppm in experi ment BTl. to assess further whether inhalation ot VC has anv effect at a dose Icvci of 50 ppm. Experiment 10 studies whether a high, but limited and/or intermittent at mospheric exposure has any effect. Experiment 11 was pertermed to evatuaic the effects of VC by ingestion. Experiment i2 studies the third general roinc ot exposure. Expeiunent 13 was started to assess wnethcr \C acts on tissues directly or through metabolites. Experiment 14 has been included to studv the responsiveness of newborns. GGC 0 0 0 2 1 7 Annals New York Academy of Sciences c( Taik.e 1 Flan of rut Hxperimints up to May 5, 1974 VoOo Treatment tip So Route Doses of VC Length hpecn-s Strain ---- -- ---- --- --1 ' BT1 |nha latiort B1 2 BT3 Inhalation Inhalation DT4 * BT5 B16 BT7 Inhalation TranspUcenLit Inhalation Inhalation BIS Inhalation B19 Inhalation - -- -------io.ooo, o ooo, 2.500, 500, 2^0, 50 ppm Umu.iUij wonttols Irc-ncd controls: VA 2 5oo ppm jpo. lio, loo rrm I'nirc-iti'ii controls 10.O00. 6,000. 2,500. 500. 23d, 50 ppm Untreated controls IU,1)1)0, 6.000 2.500, 5(H), 250, 50 ppm Untreated controls 10 0>0, 6.000 ppm 30 000 ppm lOlrH). o.UOO, 2.500, 300. 2'0, 50 ppm Untreated Controls lU.tHH), 6,000. 2,300, <00. 250. 50 ppm Untreated control) 50 ppm Untreated controls 4 h daily, 5 days weekly. Rat 52 wk 4 h daily, 5 days weekly, 32 wk 4 h daily, 5 days weekly, 17 4k U.it Rat 4 h dally, 5 days weekly, Mouse weekly, 30 wk 4 It d *tly, 7 days (from 12th to UUli day of pregnancy) 4 h djily, 5 days weekly, 52 wk 4 It daily, 3 days weekly, 32 wk K.it Rat Rat 4 h daily, 3 days weekly, Hamster 30 wk 4 h daily. 5 days weekly, Rat 52 wk SpragueD.iwley SpraguePawley SpragueDawley Swiss SpragueDawley SpragueDawley Wistar Golden SpragueDawley Animals Age (weeks) No cf Total Per group Length of the Experiments at the Present Date wecks) 13 268 309 377 64-96 135 (Ended) 13 280 263 545 120-185 21 262 288 550 60-190 11 230 260 510 60-150 19 (breeders) 12 days (cm- bryos) 17 110 30 36 146 30 60 11 -- 220 220 30-54 60 30-40 It -- 268 268 32-70 11 200 200 400 100 (c) 300 (0 43 68 43 77 43 43 30 15 btio BTII BTI2 DTI J BTU 0TI3 I Inhalation Ingestion Enduperitoncal injection Subcutaneous injection Inhalation Inhal.itmn 10,000, 6,000 ppm Untreated controls 16 6. 33 2. 50 mg/kg body weight in oltvc oil Controls, olive oil 4 25 mg m I 0 cc oltvc Oil Controls. 1.0 cc olive oil 4 23 mg in t 0 cc olive oil Controls; 1 0 cc, olive oil 10,000, 6,000 ppm 25, |(1, 5 ppnt Untreated controls 4 h daily, j days weekly 5 wk; 4 h daily, 1 day weekly, 25 wk, I h daily, 4 days weekly, 25 wk 5 times weekly, 32 wk Rat Rat 4, 3, 2. umes by 2 months and once Rat 1 injection Rm 4 h dally, 5 day) weekly 3 wk 4 h daily, 3 days weekly, 52 wk Rat Rat SpragueDawley SpragueDawley SpragueDawley Sprague* Dawley SpragueDawley SpragueDawley 11 420 420 840 120 15 i) 160 160 320 80 !} 13 150 150 300 60 15 21 80 70 no 75 . 15 1 day 13 45 44 89 240 240 480 43 46 120 10' Just started Maltoni & Lcfcminc: Carcinogenicity Bioassays GGC 000218 VVOO Annals New York Academy of Sciences Taiii I! 2 l-xpLUiMiNr 13T1: Ulxiji.ts Ainu 135 Weeks (End of tiif. Experiment) Croups and Treatment Ai.itfi tl. -li.itiley K. tsj Or* Total rcftnl So 1 eland *` Ncpiirofiln stomah*1- * * 4 Average (/f9 1 tWll< ) IltUR No. (Wtf Averse % latency time No. {w k; Animals with Tumors A ii(; os.iKtmm I.iw < Average Onrt Intcmy lime (Hk) Subcii- tancoui Other sites annin* lUrtS carets nomas Hepa tomas Brain ncuto- Mastfl. mas Other type and/or site' Tout' 1: VA, 2,500 ppm II; VC, lO.IHiO ppm III: VC, 6,000 ppm IV. VC, 2,500 ppm V: VC. 500 ppm Vi: VC, 250 ppm VII: VC, 50 ppm VIII: No treatment 'ifi 69 72 74 67 67 64 63 49 61 16 26 50 m 7 12 62 54 3 35 54 4 7 79 59 59 58 5 8 59 4 7 65 6 It) 74 4 7 83 6 It) 80 1 2 135 9 15 64 13 22 70 13 22 78 7 12 81 4 7 79 I 2 135 3' 4 3` 3 3` 3 2* 1 2 I* 1 3i 1i 12 I3 4 1 7 1" 3 8' 5 4> 4'* 3" 9' 10- 38 31 32 22 16 10 6 Total 577 964 29 26 4? 14 12 11 7 15 45 155 Maltoni & Lcfeminc: Carcinogenicity Bioassays corr*ecAtelivdenaunmimbearl.s after 2fi weeks, when the first tumor (a Z>mbal gland carcinoma) was observed. The percentages are referred to the * Metastases to lung * Metastases to liver, lung, spleen, and bram. 4 Metastases to lung. * Several cases of breast fibroadenomas, adrenal and pituitary tumors (generally adenomas) have not been considered since their distribution m the different I'.roupt docs not vary. 1 / Several annuals with 2 or more tumors. * One nnuiovurcoma of the lips; | angiosarcoma of the nose; I intr;iabUoniin| angiosarcoma (next to liver). * Two Z>inh;il gland adenomas, 3 mammary carcinomas: I neurilemmoma; I ovarian cysioadenocarcinoma. (nex1tOtnoeliavnegr)i.osarcoma in subcutaneous fibrosing angioma; I ossifying parauricular angiosarcoma; 1 intraabdominal angiosarcoma ' l our Z>mb:il gland adenomas; I salivary gland adenocarcinoma; 2 hepatic and 1 peritoneal angiomas. 4 Two intraabdnminal angiosarcomas (I next to spleen and I nest to ovary); I ossifying angiosarcoma of neck. 1 One Zymbjl gland adenoma; I mammary carcinoma; 2 ependymomas. " One pulmonary angiosarcoma, l angiosarcoma of uterus. " fbie mammary carcinoma; 2 lymphomas; I pulmonary fibrosarcoma. * One intraahdoimnal angiosarcoma (next to spleen); I mtiathoracic ossifying angiosarcoma. 'One /ymhal gland adenoma; I mammary carcinoma; I lymphoma. One mtr.iahdommal dill useJ angiosarcoma. 'Three Zyml.al gland adenomas; 2 mammary carcinomas; I subcutaneous angiopciicytoma; 3 uterine adenocarcinomas fl with sarcomatous component) One invasive acanthoma of /jmbal gland; I subcutaneous fibrosarcoma; 2 peritoneal libroangiomas; 2 uterine adenocarcinomas (l wuh sarcomatous component); I uterine leiomyosarcoma; I ovarian fibrosarcoma; 1 pulmonary rliabdomy osarcoma; I lymphoma. 0C 000219 202 Annals New York Academy of Sciences Experiment 15 was started as soon as wc found 1 liver angiosarcoma, I extrahepatic angiosarcoma, and 1 nephroblastoma in 3 animals of the first ex periment. exposed to 50 ppm of VC for 1 year, and surviving 135 weeks from the beginning of the treatment. The animals are controlled weekly and weighed every 2 weeks during the period of treatment and monthly after the treatment is over. All the detectable cross pathological changes are recorded during the control. All the animals arc kept under observation until spontaneous death. The animals, when moribund, are isolated, in order to avoid cannibalism, A complete autopsy is made on each animal. Histological examinations are performed on Zymbal glands, interscapular brown fat, salivary glands, tongue, lungs, liver, kidneys, spleen, stomach, different segments of the intestine, bladder, brain, bones of the legs and feet, and any other organ with pathological lesions. All the animals exposed to the highest doses (30,000 and 10,000 ppm), with or without tumors, are examined radiologicalty, during treatment and/or at death; moreover, radiological examinations are made on all animals bearing tumors, although exposed to the lower doses. Results The current results of several experiments are shown in the Tables 2-9. Some others are stiii not available. Tumors of different types may be present in the same animals. Zymbal gland carcinomas may be bilateral. Histologically they may repro duce, to some extent, the structure of the normal Zymbal gland (Figure 1), but often they present squamous, glandular, solid, anaplastic, and polymorphous patterns. Several of these different pictures may be observed in the same tumor. Metastascs of these tumors are sometimes found in the lung (Figure 2). Nephroblastomas are often bilateral. Their histological picture reminds us of an embrsonal kidney (Figure 3). They metastasize to the liver, spleen. lung, and brain. Hyperplasia of ncphroblastoma-hke tissue is often observed in the kidney of treated rats, with or without nephroblastoma. Angiosarcomas and angiomas may be found at more than one site in the same animal. In liver they are often polycentric. Hepatic and extrahepatic angio sarcomas are more or less differentiated (Figures 4-9). They are morphologically Table 3 Experiment BT6: Results After 41 Weeks Animal* Sprague* Lra ley Rjls.1 | Animals ith Tumors Group* and Treatment I: VC, 30,000 ppm !I Cor- | Tout reeled tio.* Survivors u> j 1 1 60 60 i 48 1 ZvmS.it iJjDd carcinoma* [ L iAvenre \cph ro- blasiomas ".I - !*:? 1 j ("k) Angio sarcoma* Liver Other she* Other type and/or site 12 1 20 J 35 i1 Animals alive after 24 weeks, when the first tumor (a Zymbal gland carcinoma) was observed. The percentages arc referred to the.cprrected number,. Malioni & Lcfcmine: Carcinogenicity Bioassays 203 Table J Experiment BT2: Results After 41 Weeks Groups ant) Treatment Anim.ils iN|>rneuef)awty kat*i Total Survivors Animals with Tumors Zymbal f land carci nomas Nephro- *__ bmu..a.t.ut - sarcoma* Other type andor utt I: VC. 200 ppm II: VC. 150 ppm III: VC, 100 ppm IV: No treatment Total 120 115 120 KN 120 112 135 171 545 507 i i * One subcutaneous fibroangioma similar in rats. mice, and hamsters. Angiosarcomas frequently metastasize; as far as the liver angiosarcomas are concerned, the lung is the most frequent site of distant metastascs. Blood vessels ectasias, endothelial hyperplasia, associated or not associated with cellular atypia, are often observed in the liver and in other organs and tissues in treated animals, with or without local or distant angiosarcomas or angiomas. Therefore, the effect of VC on blood vessels and endothelia should be considered systematic. Angioblastic proliferation, angiomas, and angiosarcomas in the liver and in other sites may or may not be associated with fihronla'ij. From the histological observations, the sequence of local changes in the genesis of malignant vascular tumors appears to be as follows: endothelial hyperplasia, angiomas, atypias of endothelial cells in hyperplasia (dysplasiai and angiomas, angiosarcomas. Fibroangioblastic proliferation undergoing fibrosis is frequently observed in the spleen of the treated rats and mice. Skin carcinomas in treated rats appear frequently to arise from sebaceous glands, of which they may reproduce in some extent the morphological charac ters {Figure 10). The hepatomas observed in treated rats are usually well differentiated. Metastascs have been observed in the lung. Brain neuroblastomas are very similar to the human medulloblastomas (Fig ure 11). Since they may be quite small and therefore not easily macroscopically detectable, wc are now making serial sections of the brains of the treated rats, to obtain more precise information on their real incidence. The lung adenomas in treated mice arc usually multiccmric; their number is higher in animals treated with heavier doses. Manv of these tumors appear to undergo early malignant transformation. Mammary carcinomas may he solitary or multiple. They are usually well differentiated adenocarcinomas, with monomorphous or polvmorphous arrange ment. The most distinct feature of these tumors is the frequency with which they present areas of squamous metaplasia (Figure 12). Up to the present no acroostcolvtic lesion has been observed on our treated animals. Several of the tumors produced by VC arc quite rare or exceptional. To our knowledge lis-er angiosarcomas, nephroblastomas, and neuroblastomas have GGC 0 0 0 2 2 0 Annals New York Academy of Sciences " 1i (( to O Table 5 Experiment DT3: Results After 66 Weeks* Groups ant] I rt.itini'ni Amnuls (Sprtrue JKtwJry RjlM 2ymb.il ulan d NcpfiroMns- carcinomas tonus Animals with Tumors Angiosarcomas l.ivcr Other sites Other type and/ blastomas or site Total I; VC, (0.000 ppm 11: VC, 6.0(10 ppin III: VC, 2,500 ppm IV: VC, 500 ppm V; VC. 250 ppm VI: VC, 50 ppm VII: No treatment Total 60 29 60 42 60 51 60 54 60 39 60 41 IW 166 550 422 4 (15) 2 (31 I (2) -- (I) - (4) - (2) - (4) 1 7 (21) I 00) - (6) - (6) - (5) - (1) - (2) - (2) - (2) -- (1) - (20) - (5) 3 (10) if (12) 2t (3) (3) 15 (2) (1) 111 5 (21) 8 (35) 4 (17) 1 (M) 1 (4) l (3) 1 16 (73) * In parentheses arc recorded the tumors found in experiment BTI after 66 weeks, t One fihroepttheli.il papilloma of (lie skin, t One squamous carcinoma of the skin, I subcutaneous angioma. One lymphoma. 11 One lymphoma. Maltoni & Lefemme: Laruiiop.iiit-K) uioassay o j: *^2 577* , <n n< -? o ^ C 3 CL 3 3 3~ * --p- T * t naO T o3 y ^ *** n1 *-t3t o o Q. "' ti = C.^ ? 3 O* C" cr> K,, O t/i --- 3 W* a 8^ Sr. v - CX 3 *1 C*. u a 3 -* O> no / o z o- P- 3n tP;- ^3 ^ 5`tJ c_ G C3 S3 H C. r, 2z--o*r o 5rs r. o 3 *_<- 5 _ S*n t* -/ t* n 3 2.; ri 2tj_ 3: 'i5i 1. -g O 5' 3 _C 3 3-1 n3 rs ^-- ,, ex rr _ "c o* o &. --3 r~ V3 'SaP5TI., Tnx=C3 < a 3 e3 o?-is;C- w* IT 3W/* >L ^--t- i ^o ; o^ ?2 o- <u* 3V* 5O Hc ,, < TM O " o cr - o* < C/) V | QCr>- cOn' *I I u ' 33 gt (/4 VI (U 733 *=2 otp/> owU* oo oC 35 3 cj ns -n 2Z? p~e* n.-no'fe._-o in c-l'r* M-- * a T3 *3 a 33 3 IMrfj ** aUI LOJ 3o o3 ** i s? eos 2 , m* oo a. a. I 0a*3*a s* V! V Ik !t ? q' csl S' aB _gg2K urrTt -r-atIgs'NC3*.'i 33y EH i o S- 5| -) n ea-3* c*r rt y a H o 2,5 p* - ro >i 2? oO I C -i :1r* ^arat 3ftj T3 -aS ill 3*3 T3 2 ^ l33ls -"33 7733 33 a 3 -a "Wm ti<s* a v "1 a* =! > -9 sr = 0O-3* ?5 -1-U GGC 000221 206 Annals New York Academy of Sciences F ig u r e l. W e ll iliffc re m ille d Z y m b a l gland c a rc in o m a in ->. H & E , x 190. c- T^ni.L tt I xnmimi n i HI l KI Ml is Amu 41 Wi i KS . Mu. ' Stnmsli mill 1 union 1 f.His .r>'l | u ,ilm< nl n ,1 V V -rv a Pnliivu, ti, M muTUi) i it . 'M,*in ,s La er t,*r 1 (iiilirImI Oilier >r? No As t r.u*c i.ili m > Into- No 'ir Itlcnc) t ,Oil, <>| oltier (uniors t\lT ut Hie .i,n! nr sk in an,); or `Me Tour' a Mil I VC . 10,000 ppm (>0 i) "" .so 1 7 H 27 S4 31 II vi , (Mini* ppm 1(1 60 :o 's 51 -1 : K> 10 -i III W, 2,>'W> ", ", m \ ", 51 IS 21 12 S') 55 |ipm IV \ ( . 500 ", til mi 2') 2') 5" 7 :i 2H 16 27 .14 ppm V V ( . 2.M ppm 30 ' "i 2u 5 l`> 11 33 11 19 31 VI \ l , 50 |,|,m to 1 to 00 27 to 57 1! H 2" VII Nil treat Ml 70 1 Ml 71 67 Ml M W 108 1 ,1 7 3` ment | q IK 8 1-1 A7 > 3 6 10 7 12 :s \\ 32 33 30 3S 1 1' V 28 > 1- 3' l' 27 4 i- 2' 13 4 i1 1- 17 30 4' 15 8 1 Total 2WI 2:><l 51,1 230 Ml 471 102 m 246 89 16 - - , *- _ Alive .minuU .tiler It* works, when the msi itimoj m m.mimarv carcinoma) was observed Corrected number 17 15 8 1 109 -- "L- - r- --,s ,-i` -- -- -- The percentages arc referred to ihe AdononiiN. some cO whioh undergoing malignant translormaimn In tom.los 4 Several anmi.iU wtil) 2 nr more tumors ' Throe Mih.vititneiiiis angiomas, 2 liver lihroangumias. I heart lihmangiom.i. I ossifying mterscapnlar angioma , One squamous carcinoma and I invasive acanthoma of the skin ' One hepatic ,iM|'i<inu One squamous caieinoma and 1 acanthoma o| the skin, 1 squamous carcinoma of the nose, One lvnifihomu 1 One renal angiosjreoma 1 One squamous caremoma and 1 acanthoma oi (he skin I 1 One subcutaneous angioma One acanthoma of the skin One hepatic angioma Two suoetitancoiis angiomas, 2 hepatic hbruangiumns w >o o 0"en*ef^0ilw-5a3' -o33*na js*r -- ft ^ J<2 3Or* -S3^ i*3n -so2n<* 0us ^ O 3 => d z*r 01 n * o- H o <<<<==" prfepp too ;sc S"gST!-j 3 =33 0 1 9at. H S i =b3 3jr te3 wos o3 --2 no3 n95 t2<- o o. 3 3 tn 3 Sir fi) 1/1 <n/ 2 o9v3* S* ' b* T*3 #pvai Oi** es g&. uS 7 208 Annals New York Academy of Sciences r> ! I 2. The range of induced tumors varies lo some client trom species 10 species. When given by inhalation VC produced: in rats. Zvmbal gland carcinomas, neph roblastomas. angiosarcomas, and angiomas or the liver and other sites, skin car cinomas. hepatomas, and bram neuroblastomas; in mice, lung adenomas (fre quently undergoing malignant transformation), mam mars carcinomas, of a peculiar type, angiosarcomas and angiomas ot the liver and ol other sites, skin epitheliai tumors; in hamsters, liver angiosarcomas and, as early evidence seems to suggest, skin trichoepitheliomas and lymphomas. Incidentally, liver angio sarcomas have been observed in ail three animal species. 3. In the BT1 and BT4 experiments, VC shows a carcinogenic cITect at 50 ppm. 4. From the BT1 experiment (the only one completed) a dose-response rela- Maltoni & Lcfcminc: Carcinogenicity Bioassays 2Q\ OOOfV o oCP f *> - S-' VC * ?! r>* Vm i * ' *** f rf ^ 1^ Figure 3. Nephroblastoma in rat. H&E, x 470. tionship clearly emerges, as far as angiosarcomas and nephroblastomas are cor cerned. in the lower dose ranges: from 500 to 50 ppm for angiosarcomas an from 250 to 50 ppm for nephroblastomas. 5. A comparison of the results available at the present moment in rats C' posed for 12 and 4 months (BT1 and BT3 experiments! shows that the neoplax: response, as far as angiosarcomas and nephroblastomas arc concerned, is aBectt by the length ot exposure to VC. 6. The comparison of the results obtained in rats of two ddferent strains, i.s Sprague-Dawley (BT1) and Wistar (BT7). at the present moment, seems to st> gest that the strain factor considerably affects the neoplastic response. 7. The onset of two subcutaneous angiosarcomas m the offspring of breede; exposed durinc pregnancy tor 7 days appears to indicate a transplacental efle. of VC. 210 Armais New York Academy of Sciences Maltoni & Lefcminc: Carcinogenicity Bioassays 211 GC 000224 8 The observation of a few cases of ossifying angiosarcomas suggests a new irientation in the pathogenetic interpretation of acroosteolysis in workers ex posed to VC, The results presented in this report should be considered both preliminary and ncompletc: prekminary since most of the experiments are still in progress, and ome at the initial stages: incomplete because a more profound and systematic Microscopical examination, which is now under way, of further serial histological ections. may disclose new initial, minute, undetected tumors. For the latter rcaon previous reports have been even more incomplete. The release of the prclimilary and incomplete results has been made necessary because of the importance ol the topic, the pressure of the events and the requests. Since there have been more than 100.000 slides up to now. J am sure that tho-c cncaced in patholoeicai screening ol experimental material arc aware of our diflicult task. Our data have been periodically transmitted to the European Companies which have supported our research and are available to all interested parties. Before the end of 1972, a short time after it was known that \ C w as inducmg in rats not onlv Zvmha! gland tumors, hut also nephroblastomas and liver ancro'sarcomas, these results were also mace known to the MCA (U S AT. This intermation promoted clinical observations and cpidemioloeiea! investigations, which in December 1973 led to the first discovery of a liver angiosarcoma, as an occupational disease, in a worker ot a U.S.A. factory producing \ C-PVC. : v, $ V " ` 212 Annals New York Academy of Sciences Maltoni & Lcfcmine: Carcinogenicity Bioassays 21 GGC 0 0 0 2 2 5 Figure 7. Liser angiosarcoma in mouse associated with fibrosis. H&E, x 190. /I..'ll,.'. 1 Ul (i rti-JU^lil} Ui OUCH CCS Makoru it Lclcuimc. s-ji 4,1 J GGC 0 0 0 2 2 6 t~-v. ---VT--; ,* s S ' N -*1 ,** *- ^^1. *j- ; .^r v _ * *; 5=--v ^N i*V 4 s_* *?.I x ;.-*r *'S- V f - A Ut* **a Fjcure S. Liver angiosarcoma in mouse associated with fibrosis. HAE. X 190 Figure 9. Kidney angiosarcoma in mouse. HAE, x 190. I 216 Annals New York Academy of Sciences Maltoni & Lefemine: Carcinogenicity Bioassays 217 GGC 0 0 0 2 2 7 Figure 10. Skin sebaceous, well differentiated, carcinoma in rat. H&E. X 190. Figure 11, Brain neuroblastoma with typical rosettes in rat. H&E, x 470. I 218 Annals New York Academy of Sciences Figure 12. Mammary adenocarcinoma with squamous metaplasia in mouse. H&E. X 190. May I recommend again that the VC carcinogenesis may serve as a convinc ing example for urgently promoting experimental bioassays for all new industrial compounds and for those compounds that arc already widespread but whose effect we still ignore, to avoid occupational cancers. Our Institute is now undertaking projects of carcinogenicity bioassays of other monomers employed in the plastic industry, RETCRENCES 1. Viola, P. L., A. Btcom & A. Caputo. 1971. Oncogenic response of rat skin, lungs and bones to vin\l chloride. Cancer Res. 31: 5 16--5 19. 2. Maltom. C. & G. Leeemine. 1974. Camnoccmciiy to hioassays of vinyl chloride. I. Research plan and early results. Environ. Res. 7: 3B7-405. OOO((0NN0 INTERIM R RATS, H \ O oa M. L. Kepiin. Indus: A The following data are p. Bio-Test Laboratories, Inc. companies b> the Manutact. complete but because of the:and OSHA on April 15, 1974 In this study which was i' hamsters, and mice of both posed to either 0, 2500. 200. week. Mice will be exposed Rats and hamsters will be r months. The rats are COBS Char: hamsters are Golden Synar. posure. water is present c"0 - Moribund and dead ic : photographed. The major or nation. Ontv the tissues from a shows the results to date 'er cal comparisons can be se lected in the same way. Or. Interim Summary or E*-io*urf Group Vo of Mor Tout Mil Control t 50 ppmf 200 ppm t 2500 ppm: 0 4 15 30 : : ; * Prehmmarj findings : Laboratory April. 1974 t Seven male and 2 lens, had tumors in spleen, hser. I Only mice with gresi.,-