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ip- y fv ----- Emirortmental Health Perspectives Voi. 21, pp, 71-78, 1977 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 activity of vinyl chloride (VC) and 'imiidene dichloride (VDC) at three exposure levels was assessed in fertile male CD-I mice with the dominant lethal test. Each compound was assessed its 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 $ 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 une or more early deaths or the number of early deaths/pregnancy or the number of early deaths/tout implants/pregnancy. There was no evidence of pre-implantational egg losses 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 insensitlv ity 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 compound* cyclophosphamide (CTX) and/or ethylmethanc sulfonate (EMS). During dosing these animal* were housed under similar exposure conditions to those animals exposed to the test substances but with flow of air through the exposure chambers. Thus, neither V'C 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) has been found to cause tumors in rats (!) and man (2). It has also been shown to produce chromosome breaks in exposed workers (2-<5) and causes mutations in Salmonella typhimurium (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 "Imperial Chemical Industries Ltd., Central Toxicology Laboratory'. Alderley Park nr. Macclesfield, Cheshire SKIO 4TJ. England. this type in mice after VC 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 uere assayed. Materials and Methods Chemicals VC was obtained from Air Products Ltd.. Worslev, Walkden, Lancs.. U.K. VDC was obtained from 1C1 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 ; V !,' SL 100763 Animals mic_e^CharleN River. Mansion. Kent) used throughout the experiment. Undo^ed females were 8-10 weeks old when mated and male mice immediately after dosing were 10-- I - weeks old. Males were caged individually and females in pairs. They received food and water ad libitum. Dosing of Male Mice Dose lev.els 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 5Q.J30.jind JO. ^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, w here it is vaporized by a calculated volume of dry clean air. The volume of air required as calculated from Eq. (1). Air flow (l./min) = Syringe size (ml/cm) x density (g/ml) x 24 x 106 Injection rate (min/cm) x ppm x mol. wt. in During dosing the mice were housed individually in chambers made of stainless steel and glass with an internal capacity of three litres. Most of the negative control animals and all ani mals dosed with CTX or EMS were housed under identical conditions during the dosing period but with a flow 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 u'ere housed under normal conditions. Before test mating began, groups of mice were treated as shown in Table 1. In the VDC study, in order to obtain a group with sufficient animal numbers some of the anjmjds^ which were infertile in weeltj) were included^ in group 4. Of the 8 survivors, 5 did not mateTrTthe 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 provide 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 Testing, Male mice were caged with virgin female mice, 1 male and 2 female mice in Group VC Study Group I Group 2 Group 3 Group 4 Group 5 Group 6 VDC Study Group t Group 2 Group 3 Group 4 Table 1. Treatment groups. No. survivors/ no. animals treated (mice) Treatment 20/20 18/20 I9'20 9/20 15 15 25 25 50 50 * 20/20 I8,2(L 620 2/20 17,17 Air (negative controll 3000 ppm VC. 6 hr/day. J days 10.000 ppm VC. 6 hr/day. 5 days 30.000 ppm VC. 6 hr/day. 5 days 200 mg CTX in water/kg body weight, once by IP injection on day 5. 200 mg EMS in water/kg body weight orally, once/day. 5 days 15 air, 35 normal conditions (negative control) 10 ppm VDC. 6 hr/day, 5 days 30 ppm VDC, 6 hr'day. ? days 50 ppm VDC, 6 hr,May. 2 days ,, 75 ppm VDC, 6 hr'day for 1 day. then 50 ppm VDC. 6 hr'day. for 1 day" - 200 mg CTX in water/kg body weight, once b\ IP injection on day 5 72 Environmental Health Perspectives "VOO each cage. After 5 days the females were trans ferred to other cages. The female mice were killed 15 days after first introducing them to the males and examined for pregnancies. The 106 males which survived dosing in the VC study and 113 males in the VDC study and were successful in fertilizing at least l female in their cage were selected for con tinuation in the experiment. Experimental Stating. 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 and again left for 5 days. This process was repeated 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 further. 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 3 days after introducing female mice to the cages containing males. Female mice were killed 13 days after the as sumed date of fertilization, 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 The 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 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 1. Number and percentage of male mice which sursived treatment successfully mating at each week. Week 0 l 2 3 4 5 6 7 8 "p <0.001. Group 1, air (negative control) No of r? mice 20 100 20 100 20 100 20 100 20 100 19 95,0 20 100 20 100 20 100 Group 2. vc. 3.000 ppm (6 hr * 5l No. of r? mice 18 100 IX 100 18 100 18 100 18 100 18 100 18 100 18 100 18 100 Group ?, VC. 10 000 ppm hr a 5i No. of mice 19 100 IK 100 19 100 19 100 19 I0O 19 100 19 100 19 100 19 100 Group 4. VC. 30.000 ppm (6 hr x 5i No. of 9? mice 9 100 7 77.8 9 IOO 9 100 9 100 7 77.8 9 100 9 100 8 88.9 Group 5. CTX, 200 mg/kg IP No. of mice 15 100 14 93.3 14 93,3 14 93.3 15 100 15 100 14 100 14 93.3 13 92.9 Group 6. EMS. 200 mg'kg (oral) x 5 No. of 9} mice 25 100 3" 12.2 23 92.0 23 92.0 25 100 25 100 24 100 25 100 25 100 Table 3. Number of mated females becoming pregnant. Group 1, air (negative control) Group 2, VC. 3,000 ppm (6 hr x 5) Week No. No. No. No. pregnant mated pregnant mated 0 35 40 30 36 1 35 40 33 36 2 36 40 36 36 3 34 40 30 36 4 38 40 34 36 5 35 40 34 36 6 36 40 33 36 7 37 40 31 36 8 37 40 32 36 "p < 0 U01. 'p -.0 111. Group 3, VC. 10.000 ppm (6 hr x 5) Nc No. pregnant mated 28 ?.X 32 36 33 IX 34 3N 33 vx 36 kx 36 'N 37 'S 31 37 Group 4, vc. 30.000 ppm (6 hr x 5) No. No. pregnant mated 15 18 II 18 15 18 16 kX 17 18 12 IX 18 18 18 IX 14 18 Group 5, CTX. 200 mg/kg IP No. No. pregnant maled 25 30 26 30 25 30 26 30 30 30 28 30 26 28 22 30 23 2X Group 6. EMS. 200 mg kg (oral) x 5 No. No. pregnant mated 37 50 4" 50 31* 50 40 50 46 50 47 50 47 48 41 50 45 50 December 1977 73 xo^6S Week 0 1 2 3 4 J 6 7 8 "p <001. bp < 0.05. Week 0 1 3 4 5 6 1 % *P < 0.01. 0 1 3 4 5 6 7 8 "p < 0.01. Week 0 1 2 3 4 J 6 7 8 ap < 0.05. V < o.oi. 74 Group 1, air (negative control) 11.9 12.9 12.8 12.5 12,4 13.7 11.7 11.8 12.7 Tdhie 4. Mcjn lolal imptjnH per pregnant female. Group 2. VC 3.000 ppm (6 hr x 5i 12 6 13.6 12.7 12.5 12.1 12.7 11.9 11.6 12.1 Group V \c. 10 000 ppm (6 hr 5> 12.0 13 4 13 0 12.4 13 ' 14 1 12 5 11 8 12 6 Group 4. VC. 30,000 ppm (6 hr x 5i 13 0 16.1 13 7 11 2 10 0" 12 8 12 6 12,0 12.1 Group 5. CTX. 200 mg/kg IP 11.3 8 810 9 10.9 12 6 12 3 12.5 11.5 12 5 Group 6 EMS. 200 mg kg (oral! t 5 11 8 4 0" 8 4" 12 ? 12 7 12.7 12 1 12.4 12,^ Table 5. Number of pregnant females with one or more early deaths (ED). Group 1, air (negative control) Group 2, V C. 3,000 ppm 16 hr < 5i Group 3. VC. 10.000 ppm (6 hr x 51 Group 4. VC. 30.000 ppm (6 hr x 5) Group 5. CTX. 200 mg/kg IP Group 6, EMS. 200 mg kg (oral) x 5 0 s> 1 ED 0 s. | ED o a | ED 0 * 1 ED 0 18 |7 15 20 19 17 18 16 16 22 17 18 17 19 18 19 13 24 21 9 16 22 16 17 10 21 18 18 13 20 16 14 19 15 17 17 20 13 18 16 14 22 II 22 21 15 13 18 21 16 14 18 15 16 10 5 65 69 96 97 39 7 11 8 10 86 >> 1 2 8 14 12 13 10 13 1 ED V 25' 23* 18 16 16 13 12 10 0 21 I 5 15 19 25 24 22 :: * 1 ED 26 3 26' 25 27 22 23 19 23 Group 1. air (negative control) 0.77 0.86 0.83 0.91 0.89 1.06 0.97 0.76 1.03 Table 6. Mean number of early deaths per pregnancy. Group 2. VC. 3,000 ppm (6 hr x 5) 0 33 0.91 1.00 0.73 0.79 0.71 0.91 0.87 0.88 Group 3. VC. 10.000 ppm (6 hr x 5l 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 J, CTX. 200 mg/kg IP 0.16 4.27* 4.84* 1.54 1.13 1.11 0.65 0.82 0.70 Group 6. EMS. 200 mg,kg (oral) x 5 0.59 3.50* 2.58* 1.45 0.85 0.72 0.85 0.63 0.73 Table 7. Early deaths as a percentage of total implants per pregnant female. 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 J-9 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 4.5 7.3 6.0 5.7 81 Group 4, VC, 30.000 ppm (6 hr x 5) 3.1 2.7 6.9 7.8 6.5 7.9 II.1 7.4 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 6. EMS. 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 Tble 8. Number and percentage of male mice which survived treatment successfully: mating at each week. Week 0 1 2 3 4 5 6 7 8 "p < 0.001 "p < 0.01. fP < 0.05. Group | (pooled negative control) No. of mice % 50 IOO 50 98 50 96 50 94 49 98 47 94 49 88 49 92 48 92 Group 2, VDC, 10 ppm (6 hr x 5) No. of mice 9e 20 100 20 95 20 100 20 100 20 100 20 100 20 100 20 90 19 95 Group 1, VDC. 30 ppm (6 hr x 5) No. of mice % 18 100 18 100 18 94 18 89 18 89 17 82 17 100 17 100 17 88 Group 4, VDC, 50 ppm (6 hr x 5) No. of mice ____% 8" 38 8" 75 7* 86 7" 43 7- 43 7* 57 7 43 6r 50 7r 57 Group 5. CTX. 200 mg/kg IP No. of mice ___ % 17* 76 17" 65 16 94 17 94 15 100 17 100 17 88 16e 75 19 86 1 sole v. Number of mated females becoming pregnant. Week Group 1 (pooled negative control) No. No. pregnant mated 82 75 82 85 80 75 77 73 p< 0.01. `p <0.001. ' p < 0.05. 100 100 100 98 94 98 98 96 Group 2. VDC. 10 ppm (6 hr x 5) No. No. pregnant mated 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 36 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 J* 16 7* 16 7* 14 5* 14 6* 14 6* 14 5* 14 7 13 7 14 Group 5, CTX, 200 mgfkg IP No. No. pregnant mated 17* 34 20* 34 25 33 28 34 26 32 29 34 25 34 19* 33 28 39 Week 0 1 2 3 4 5 6 7 S "P < 0.01, *P < 0.001. 'p < 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 3, VDC. 30 ppm (6 hr x 5) 12.0 12.8 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 3, CTX, 200 mg/kg IP 11.5 9.2* 9.5* 11.0" 12.1 12.0 11.I* 11.7" 11.6 75 & Table ll. Number of pregnant females nith one or more early deaths (ED). Week 0 t - 4 6 7 8 "p < 0,001. "p < 0.01 Group 1 (pooled negative control) 0 s l ED 40 33 41 41 39 36 43 37 40 45 41 39 40 35 43 34 37 36 Group 2. VDC. 10 ppm {6 hr x 5) 0 ^ 1 ED 14 16 18 14 18 18 18 16 17 19 15 19 14 19 15 18 13 15 Group 3. VDC. 30 ppm (6 hr x 5) 0 =5 1 ED 10 15 19 14 17 15 15 II II 16 II 15 15 17 15 13 13 13 Group 4, VDC. JO ppm (6 hr x 5) 0 s l ED 14 61 34 41 24 23 52 34 Group 5. CTX. 200 mg/kg IP 0 *IED 98 0" 20 2" 23 7* 21 12 14 12 17 12 13 10 9 15 5 Table tZ. Mean number of early deaths per pregnancy. Week 0 1 T 3 4 5 6 7 8 "p < 0 001. Group 1 (pooled negative control) 0.59 0,72 0.69 0.76 0 79 0.78 0.88 0.65 0.85 Group 2. VDC. 10 ppm (6 hr v 5) 0.80 0 72 0.75 0.68 0.83 0 68 0.91 0.85 0.75 Group 3. VDC. 30 ppm (6 hr x 5) 0.84 0.61 0.56 046 1.00 0.73 0.94 0.57 0.85 Group 4, VDC. 50 ppm (6 hr x 5) 2.00 0.29 0.71 0.80 1.00 0.67 1.00 0.57 0.71 Table 13. Early deaths as a percentage of total implants per pregnant female. Group 5, CTX, 200 mg/lcg IP 1.06 4.00* 4.04* 2.32' 0.85 0.86 0.64 0.74 0.30 Week 0 1 > } 4 5 6 7 8 "p < 0.001. Group 1 (pooled negative control) 4.8 5.7 5.5 6.4 6.9 6.6 7.0 4.9 7.0 Group 2. VDC. 10 ppm (6 hr x 5) 10.1 5.3 6.9 6.3 71 6.1 7.6 66 5.4 Group 3. VDC. 30 ppm (6 hr x 5) 7,3 5.1 4.4 3.6 7.4 6.2 8.3 4.5 7.1 Group 4. VDC, 50 ppm (6 hr x J) 17.3 2.2 5.7 5.3 10.3 5.6 7.5 4.3 5.8 Group 5. CTX. 200 mg/kg IP 9.1 45.67 41.4* 21.8* 6.9 6.9 5,6 6.1 2.4 Fertility Successful Mating Frequency. 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 oejweeh vTTtreafment groups and the control at any wiekJJiLusing_a chi - squaredjest. There was. how ever. a significant difference between the E\1Streated group and the negative control group in week I. In the VDC study, the mating frequency was high in the two groups exposed to the lowest doses of VDC in ail weeks by comparison with rhc negative control group. The mating frequency, nowever, 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 nf the males used. "" Pregnancy Frequency. The numbers of females in each group which became pregnant at each week of mating are 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 EMS-treated group at weeks I and 76 Environmental Health Perspectives 2 by using a chi-square test. In the VDC studyc with EMS treatment. In the VDC study only the there were significant differences in the highest/ CTX positive control group was significantly dif VDC exposure group at weeks 0-6 and the positive- ferent from the negative control group in weeks 1. control group in weeks 0, 1, and 7. Again this was-' 2. and 3 (Table 12). probably due to infertility of the males. / Early Deaths as a Percentage of Total Im These results indicate that V_C at the three ex plants per Pregnant Female. Again, it was posure levels and-VDC at lea^~at exposures oTTO necessary to stabilize the variance prior to analysis. and 30 ppm did not cause a~reduction inTertilitv,' In the VC study CTX and EMS treatment groups Any reduced fertility at 50 ppm VDC was un were significantly different from the negative con proven. trol groups in weeks 1 and 2. whereas VC-treated groups were not (Table 7). The CTX treatment Total Implantations group also showed a significant difference in the week before treatment. In the VDC study only the The mean total number of implants per pregnant CTX treatment group was significantly different female in each group is shown in Tables 4 and 10. from the negative control group in weeks I, 2. and 3 The mean values were adjusted to take account of (Table 13). the unequal number of pregnant females per male Thus with all these different methods of analysis and were compared statistically by using an of the data of early deaths no statistically significant analysis of variance and a /-test. In the VC study differences from the negative control groups were statistically significant differences were evident in seen in the VC or VDC treated groups. week 1 in the CTX-treated group and weeks 1 and 2 in the EMS-treated group. A significant difference Late Deaths (p < 0.05) in week 4 was also found between the group exposed to the highest dose of VC (Group 4) In each study late deaths were randomly distrib and the negative control group. In the VDC study uted throughout all the groups and did not appear to the CTX positive control group was statistically be treatment-related. significantly different from the negative control groups in weeks 1, 2. 3, 6, and 7. Only the VDC' group to be exposed to 10 ppm showed a significant Conjoined Placentae difference from the negative control group in the Conjoined placentae were seen in this strain. pre-experimental nontreatment week. They were the result of very close implantation Thus there was no indication of a preimplantation sites and were not monozygotic twins (12). In the loss of eggs in either study except fn week 4 after experiments they were classified as double implan 30,000 ppm of VC. tations. Since they occurred with equal frequency in all groups they did not appear to be correlated Early Deaths with treatment. The data for early deaths have been presented in various ways, .Vumber of Pregnant Females with One or More Early Deaths. In the VC study, CTX and EMS treatment caused increases in the number of pregnancies with early deaths (Table 5). The effect vs as significant (chi-square) in weeks l and 2 for the CTX-treated group and in week 2 for the EMStreated 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 1, 2, and 3 (Table 11). The Mean Number of Early Deaths per Pregnancy. A large number of low or zero values were encountered, so it was necessary to stabilize 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 1 and 2 with CTX treatment and Discussion The best indication of mutagenic activity of a substance m the dominant lethal test Is an increase in the number of post-implantational foetal early deaths (13). f rom the data for early deaths there was no evidence in either study af a mutagenic ef fect with VC or V>C at the administered exposure levels. TRIs did not appear to be a result of lack 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 (14) is at least as sensitive as that 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 (15) for the same mouse strain. December 1977 77 ^00'1 69 sc> 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), Pre7 `mplantational egg losses have been studietT~&y~~ comparing vaTueTof total implants in females mated with treatecPmales and those matetTwith control males, "as suggested by bp~stein~( 16). rather thancounting 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 reasons 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 encountered 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. U can be concluded, therefore, that VC and VDC do not cause dominant lethal mutations in male CD-I mice at 3000, 10,000, and 30.000 ppm and 10, 30, and 50 ppm. respectively. The authors would like io thank the Inhalation Section for exposing the males to VC and VDC. Mrs. S. Palmer for 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. S. H. Ellis, who is at ICI Pharmaceuticals Division. Alderley Park. REFERENCES 1. Maltoni. C, et al. Vinyl chloride carcinogenesis--current results and perspectives. Med. Lavaro 65: 421 (1974). 2. Creech. J, L.. and Johnson. M- N. Angiosarcoma of the liver in the manufacture of polyvinyl chloride. J. Occup. Med. 16: 150 (1974). 3. Ducatman. A-. Hirschorn. E.. and SelikofT, t. J, Vinyl chloride exposure and human chromosome aberrations. Mu tation Res. 31: 163 (1975). 4. Funes-Cravioto. F., et al. Chromosome aberrations in workers exposed to vinyl chloride. Lancet I: 459(19751. 5. Purchase. I. F. FI.. Richardson. C. R.. and Anderson. D. Chromosomal effects in peripheral lymphocytes. Proe, Roy. Soc. Med. (London) 69: 290 (1976). 6. Seentesi. I.. et al. High rate of chromosomal aberration in PVC woricers. Mutation Res. 37: 313(1976). 7. Bartsch. H,. Malaveille. C . and Montesano. R. Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in J. typliimitriinn strains. Int. J. Cancer 13: 429 (1973). 8. Bartsch. H.. et al. Tissue-mediated mutagenicity of vinylidene chloride and 2-chlorobutadiene in Salnumrlla trphimuriinn. Nature 25J: 641 (1973). 9. Gage. J. C. Toxicity of epichlortiydrm vapour. Brit. J. Ind. Med. 16; 11 (1939). 10. Anderson. D., McGregor. D. B.. Purchase. I. F. H. Dom inant lethal studies with paraquat and diquat in male CD-I mtce. Mutation Res. 40: 349 (1976). 11. Anderson. D., Hodge. M. C. E., Purchase. I. F. H. Vinyl chloride: dominant lethal studies in male CD-I mice. Muta tion Res. 40: >59 (1976). 12. Bateman. A. J. Dichorial. one-egg twins in the mouse. Na ture 187: 339(1960). 13. Bateman. A. J.. and Epstein. S. S. In: Chemical Mutagens. Principles and Methods for Their Detection. A Hollander, Ed.. Plenum Press. New York-London. 1971. pp. $41-366. 14. Anderson, D.,etal. Dominant lethal test results with known mutagens in two laboratories. Mutation Res. 43:231 (1977). 15. Ray. V. A., and Hyneck. M. L. Some primary considera tions in the interpretation of the dominant lethal assay. Em iron. Health Petspect. 6: 23 (1973). 16. Epstein. S. S. The use of the dominant lethal test to detect genetic activity of environmental chemicals. Environ. Health Petspect. 6: 23 (1973). 17. Gropp. A., and Kolbus. U. Exencephaly in the syndrome of trisomy no. 12 of the foetal mouse. Nature 249: 145 (|974>. 78 Environmental Health Perspectives 100T70 SI*