Document JJrGV1x7ong7eQq3QwMyr6bQZ

Environmental Ih ulth /Vrt/xrmc.v V,,l 21, mi. 71-7,1, !<J77 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 or vinyl chloride (VCI and vinylidene dichloride | VI)C) at three exposure levels was assessed in fertile male CD-I mice will* the dominant lethal test. Each compound was assessed in a separate study. Male mice were exposed hy inhalation to VC at 31)00. 10,000, and 30,000 ppm and In VI)C at 10, 30, and 50 ppm for A hr/day for 5 days. By comparison w ith control males exposed to air, no mutagenic effects on any maturation stage nr spermatogenesis in treated males were detected. There was no significant Increase in the number of postimplantational early fetal deaths as shown hy the nunihcr of females with one or more early dealhx or the number of early deaths/pregnancy or the number of early deaths-total implants'prcgnancy. There was no evidence nf prc-implanlational 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 as unproven). The lack of effect was not due to the insensitivity or 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 cyclophosphamide (CTX) and/or elhylmcthane sulfonate (EMS). During dosing these animals were housed under similar exposure conditions to those animals exposed to the test substances hut with a flow nf 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(vinyi chloride) has been found to cause tumors in rats (/) and man (2). It has also been shown to produce chromosome breaks in exposed workers 13-6) and causes mutations in Salmonella typhinwriam (7, #). Another chlorinated monomer, VDC is also known to cause mutation in Salmonella lypliimnrium (7,8). Wc. therefore, carried out dominant lethal studies to determine if there were any mutagenic effects of "Imperial Chemical Industries Lid.. Central Toxicology Laboratory, Aldcrley 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 find given EMS anchor CTX were assayed. Materials and Methods Chemicals VC was obtained from Air Products 1 id.. Worsley. Walkdcn. Lanes.. U.K. VDC was obtained from IC'I Ltd.. Mond Division. Runcorn. Cheshire. U.K. EMS was obtained from Koch-Light Ltd., Colnbrook. Bucks. U.K.. and CTX (hndoxar,.i) from Ward Blonkinsop Ltd.. London. U.K. December 1977 71 OGC 003463 Animals During dosing the mice were housed individually CD-I mice (Charles River. Mansion. Kenl) were used throughout the experiment. Undosed females were 8-|0 weeks old when mated and male mice immediately after dosing were 10-12 weeks old. Males were caged individually and females in pairs. They received food and water cut libitum. 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 F.MS were housed under identical conditions during the dosing period but with a flow of air through the .chambers. Those dosed with CTX were injected IE on day 5 of ex Dosing of Male Mice posure and those dosed with EMS were given an oral dose by gavage for 5 consecutive days. Both of Dose levels for VC and VDC were selected on these substances were prepared as aqueous solu the basis of preliminary toxicity studies. A dose of tions immediately before use. Some of the animals 30.000 ppm of VC was found to be in the toxic in the negative control group controlling the VDC range and this was chosen as the highest exposure study were housed under normal conditions. Before level since it was desirable that the maximum toler test mating began, groups of mice were treated as ated dose or higher should be used. Other levels of shown in Table I. 10.000 and 3000 ppm were also used. The required In the VDC study, in order to obtain a group w ith concentrations of VC were generated by mixing sufficient animal numbers some of the animals known volumes of VC and comp ;ed air using which were infertile in week 0 were included in rotameters as indicators. VDC was much more group 4. Of the 8 survivors. 5 did not mate in the toxic to the mice. Exposure levels of 30. 30. and 10 fertility study and one had a higher than average ppm were selected. The required concentrations of number of early deaths. However some of these VDC were generated by a controlled fluid-, males mated in the main study. One male surviving feed/atomizer technique (9). The method involves 50 ppm for 2 days died in week 2 of the study. Of continuously passing a known volume of the com the 17 animals, three in the CTX treatment group pound through a concentric jet atomizer, where it is also did not mate in the fertility study. Fifty animals vaporized by a calculated volume of dry clean air. were included in the negative control group to pro The volume of air required as calculated from Eq. vide a better data base against which to compare the (I). treated groups. There were no statistical differ ences between males exposed to air and housed Air flow (l./min) = under normal conditions. Syringe size (ml/cm) x density (g/ml) x 24 x 10* Mating Injection rate (min/cm) x ppm X mol. wt. Fertility Testing. Male mice were caged with (l) virgin female mice, 1 male and 2 female mice in Croup VC Study Croup 1 Croup 2 Croup 3 ' Croup 4 Croup 3 Croup ft VDC Study Ciroup 1 Croup 2 Group 3 Group 4 IjlOtlp * Table 1. Treatment groups. No. survivors/ no. animals treated (mice) Treatment 20/20 IK/20 19/20 9/20 15/15 25/25 50/50 2020 IK/ 20 ft/2<> 2/20 17/17 Air (negative control) 3000 ppm VC, ft In`day. 5 dtivs 10.000 ppm VC. ft lirMay, 5 days 30.000 ppm V'C'. 6 hr/day. 5 davs 200 mg ('7 X in water, kg bmlj weight, once by IP injection on dav 5, 200 mg RMS in waler-kg bod> weight orallv . ooce'das . 5 da\s 15 aii. 35 noimal condition^ (ncuaioo cotittoll 10 ppm \ DC, ft In'day. 5 (lays 30 ppm Vl)(', ft In.'day. 5 days 50 ppm VDC. ft In day. 2 days 75 ppm VDC . f hr day for I day, then 50 ppm V|X`. f* hr dav. for 1 dav 200 mg C f X in water ke body weight, once In IP injection on day 5 72 Environmental Health Perspectives ' V- -I I 0 i 0 I t t * I { l i .1f ** .1II :t ,t :tI each cape. After 5 days the females were trans ferred to other capes. 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 1 female in their cage were selected for con tinuation in the experiment. llxpcrimcntul Matin". 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 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 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 w'ere 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 w'hich became pregnant during the fertility test yielded the data for week 0 but only data front those animals that survived ti m. nent 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 niice which tunived treatment successfully mating at each week. Week 0 1 \ 4 5 ft 7 X 7' < 0.001. Group 1. air (negative control) No. of Of mice 20 too 20 too 20 100 211 too 20 LOO 10 93.0 20 too 20 too 20 too Gioup 2, VC. 3.0(H) ppm tfi hr v ?) No. of C mice IX too IX 1(H) IX 1(H) IX 100 IX too IX KM) IX 100 IX 100 IX 100 Clump t. VC . I0.IKH) ppm (ft hr .< 5) No. of c; mice 19 100 IX 100 19 100 |9 |(M) 19 100 19 KM) 19 l(M) 19 l(M) 19 IOO Group 4, VC. 30.000 ppm (ft hr * Si No. of *}r mice 9 100 7 77.X 9 KM) 9 KM) 9 100 7 77.X 9 IIK) 9 KM) X XX.9 Group 5. CTX 200 nig/kg IP No, of ct mice IS 100 14 93.3 14 . 93.3 14 93.3 IS KM) IS 100 14 KM) 14 93.3 13 92.9 Group ft. tiMS. 200 tng'kg (oral) x 5 No. of C? mice 25 100 3" 12.2 23 92.0 23 92.0 25 KM) 25 KM) 24 KM) 25. 100 25 100 Table 3. Number of mated females becoming pregnant. Group 1. air (negative control) Group 2. VC. 3.0(H) ppm (ft hr * 5) Week No. No. No, No. ptegnant mated pregnant muted 1) 35 40 .30 3ft 1 35 40 13 3ft 2 3ft 40 3ft 3ft 3 34 40 30 3ft 4 3 40 34 3ft 5 35 40 34 3ft 6 3ft 40 33 3ft 7 .17 40 31 3ft X 37 40 32 36 P n.iHtt. 7> < (1.01. Group 3. VC . 10,0110 ppm Ih 111 M Noi. No. pregnant mated 2X 3X 32 3ft 33 3N 34 IS 3) 3X 3ft 3X 3ft 3K 37 3X 31 37 Ciroiip 4, VC . 30.000 ppm ift In < Si Nri. Ni>. picgnnnt mated IS IX 11 IX IS IX Ift IX 17 IX 12 IX IX IS IX IX 14 IX Group 5. C IX. 2lX) mg-'kg II 1 No. No. pregnant mated 25 30 2ft 30 25 30 2ft M) to 30 2N to 2ft 2N 55 30 23 2K Clump ft. IMS. 2<M) mg ke total) x S Nit. No, pregnant netted 3' SO 4- SO 31- Ml 40 SO -Ilf Ml 17 Ml 47 4X 41 50 45 50 December 1977 Mm 11 Eft GGC 003465 32 Week 0 1 2 3 4 5 6 7 8 *p < 0.01. V < 0.05. Week 0 1 2 3 4 5 6 7 8 V < 0.01. Week 0 1 2 3 4 5 6 7 8 "p <0.01. Week 0 | 2 3 4 5 6 7 8 -p < 0,05. V < o.oi. 74 Group 1, air (negative control) 11.0 12.9 12.8 12.5 12.4 13.7 11.7 11.8 12.7 Table 4. Mean Inlal implants per precnnnf female. Group 2. VC 3.0(81 ppm (ft hr x 5) 12.ft J3.6 12.7 12.5 12.1 12.7 11.9 ll.ft 12.1 Ginilp V VC. 10.000 ppm (ft hr x 5) 12.0 13.4 13.0 12.4 13.7 14.1 12.5 11.8 I2.fi ( 1!(Hip -1, VC . 30.(88) ppm (ft hr * 5) 13.0 Ifi.l 13.7 11.2 10.0'' 12.8 12 ft 12.0 12.1 ( lump 5. C 1 X. 200 mg'kg It' 11.3 8.8' w.'> 10.9 12.ft 12 5 12.5 11.5 12.5 ( MUlip ft. IMS. 2(81 mc'kg lot,alt a 5 11.8 4.0" 8,4" 12.5 12.7 12.7 12.1 12.4 12.5 Tattle 5. Number of pregnant females with one or more early deaths (Kl)i. Group 1, air (negative control) Group 2, VC. 3.0(8) ppm (6 hr x 5) Group 3. VC. 10,(88) ppm (6 hr x si Group 4. VT, 30.0(8) ppm (6 hr x 51 Gioup 5. C IX. 200 mg/kg IP Gump ft. E.MS. 218) mg kg (oral) >' 5 0 * 1 ED 0 > 1 ED 0 7* 1 ED 0 * 1 ED 0 18 17 21 9 Ift 22 10 15 20 16 17 10 21 6 5 22 5i 19 17 18 18 13 2(1 6 9 2 18 16 IA 14 19 15 9 6 8 16 22 17 17 20 13 9 7 14 17 18 18 1ft 14 22 17 19 II 22 21 15 3 9 12 7 II 13 18 19 13 18 21 16 8 10 10 13 24 14 18 15 16 8 6 13 * l ED I) 3" 21 ;_V' 1 2y 5 18 15 16 19 Ift 25 13 24 12 22 10 22 * 1 ED 26 3 2ft" 2$ 27 2> 23 19 23 Table 6. Mean number of early deaths per pregnane}. 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,(88) 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. 10.0(8) ppm (6 hr x J| 0.54 1.00 1.15 0.88 O.fil 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 0.16 4.27" 4.84" I.M 1.13 l.ll 0.65 * 0.82 0.70 Table 7. Early deaths as a perccnlsiK? implants per prtpiaiit fWiutlc. 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.(88) ppm (6 hr x 5) 4.4 7.5 y.fi 7.0 4.5 7.3 6.0 5.7 8.1 Group 4. vc. 30.000 ppm (6 lu x 5) 3.1 2.7 6.9 7.8 6.5 7.9 III 7.4 5.9 Group 5, CTX. 200 mg'kg IP 1.4" 49.1'44.6'* 14.1 9.0 9.1 5.2 7.0 5.7 Group ft. IMS. 2(8) mg/kg (oral) > 5 0.59 3.50" 2.58" 1.45 0.85 0.72 0.85 0.63 0.7) Clump ft. IMS. 2(8) mg/kg total) * 5 5,1 77 7g" i|.5* H t. h7 57 7.0 5.1 59 Environmental Health Perspectives V V Table 8. Number and percentage wf male mice which survived treatment siiccesstudv: mating at each week. Week 0 1 2 3 4 5 6 7 X *p < 0.001. V < o.oi. 'p < 0.05. (roup 1 (pooled negative control) No. of mice <7r 50 100 50 98 50 96 50 94 49 98 47 94 49 88 49 92 48 92 (ii'nltp 2. VIM . 10 ppm (6 hr x 5) No. of mice % 20 100 20 95 20 100 20 too 20 100 20 100 20 100 20 90 19 95 < iinup 5. VIM . 30 ppm (ft hr x 51 No. of mice % 18 100 IX 100 18 94 18 89 18 89 17 82 17 100 17 100 17 88 fimup 4. VIM . 50 ppm (ft hr x 5) No. or mice % 8' 38 8" 75 7* 86 7" 43 V 43 V 57 V 43 6' 50 T 57 Group 5. OX. 2(8) mg/kg ir No. of mice c/r. 17* 76 17* 65 16 94 17 94 15 100 17 100 17 88 16' 75 19 86 Table 9, Number of mated females becoming pregnant. Group 1 (pooled negative control) Week 0 1 2 3 4 % 6 7 X No. No. pregnant mated 73 100 82 100 75 100 82 too 85 98 80 94 75 98 77 98 73 % p< 0.01. p < 0.001. ' p < 0.05. 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 3tf 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 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 W 33 28 39 W'eck 0 1 3 4 s ft 7 8 -p < 0.01. hp < O.tMtl. rp < 005. 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 II.X 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 HO 13.4 12.7 10.7 12.6 12.7 12.6 Group 5. CTX. 200 mg/kg IP 11.5 9.2* 9.5* 11.0' 12.1 12.0 ii. r 11.7' 11.6 75 Tahir 11. Number .'rguant females with one nr mure early deaths (lit)). Week 0 1 2 3 4 5 6 7 ft p < 0.001. V < o.ot. Group 1 (pooler! negative control) 0 1 ED 40 33 41 41 39 36 43 37 40 43 41 39 40 35 43 34 37 3b Group 2. VDC, 10 ppm (ft hr s 5) 0 a> 1 ED 14 16 IX 14 IX IX IX IA 17 19 15 19 14 19 15 18 13 15 Group 3, VIX . 30 ppm (6 hr > 5) 0 a 1 tit) 10 15 19 14 17 15 15 It It 16 It 15 15 17 15 13 13 13 Gioup 4. VIX . 50 ppm (6 hr >: 5) 0 1 HD 14 61 34 41 24 33 23 52 34 Group 5. C*TX. 200 mg'kg IP 0 * 1 ED 9X 0" 20 2" 23 r 21 12 14 12 17 12 13 10 9 15 5 Tahtc 12. Mean number of early deaths per pregnancy. Week 0 1 2 3 4 5 6 7 ft p < 0.001. Group 1 (pooled negative control) 0.39 0.72 0.69 0.76 0.79 0.7S 0.XX 0.65 0.X5 Group 2. VDC. 10 ppm (6 hr x 5) o.xo 0.72 0.75 0.6X 0.X3 0.AX 0.91 0.X5 0.75 Group 3. VDC. 30 ppm (6 hr x 5) 0.X4 0.61 0.56 0.46 1.00 0.73 0.94 0.57 0.X5 Group 4. VDC. 50 ppm (6 hr x 5) 2.00 0.29 0.71 O.XO 1.00 0.67 1.00 0.57 0.71 Table 13. Early deaths a.s a percentage nf (oral implants per pregnant female. Group 5, CTX. 200 mg/kg IP 1.06 4.0(V 4 04'* 2.32" 0 X5 0.X6 0.64 0.74 0.30 Week 0 1 2 3 4 3 6 7 X '/> < O.OOI. Group 1 (pooled negative control) 4.X 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 7.1 6.1 7.6 6.6 5.4 Gump 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 s- 5| 17.3 2.2 5.7 5J 10.3 5.6 7.5 4.3 5.X Group 5. CTX. 200 mg/kg IP 9.1 45.641.42I.X" 6.9 6.9 5.6 6.1 2.4 Fertility Successful Matin;' Frequency. The numbers of mules successfully muling ut cuch week ure 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 and the control at any week by using a chi-squared test. There was. how ever. a significant difference between the KMStreated 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 all weeks by comparison w ith 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. 1. and 7. This effect in the VDC highest exposure group was. however, probably due to infcitility of the males used. Pregnancy Frequency. The numbers of females in each group which became pregnant at each week of muling are show n in Tables 3 and 9. In the VC study, statistical differences between treated groups and the negative control group were found only in the HMS-treated group at weeks I and 76 Kuvirnnincntal Health Perspectives 2 by using a chi-square test. In the VI)C stiuly there were significant differences in the highest VDC exposure group at weeks 0-6 aiul the positive control group in weeks 0. I. and 7. Again this was probably due to infertility of the males. These results indicate that VC' at the three ex posure levels and VDC at least at exposures of 10 and 30 ppm did not cause a reduction in fertility. Any reduced fertility at 50 ppm VDC was tinproven. Total Implantations The mean total number of implants per pregnant female in each group is show n in Tables 4 ami 10. The 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 /-test. In the VC study statistically significant differences were evident in week I in the CTX-trcated group and weeks I and 2 in the EMS-lreated group. A significant difference (/) < 0.05) in week 4 was also found between the group exposed to the highest close of VC (Group 4t 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 from the negative control group in the pre-experimental nontreatment week. Thus there was no indication of a preimplantation loss of eggs in cither study except in week 4 after 30,000 ppm of VC. Early Deaths with CMS treatment*. In the VDC study only the Cl X positive control group was significantly dif ferent from the neealivc control ctoup in weeks I. 2. and 3 ( Table 12). Early Deaths as a Percentage of Total I inplants' per Pregnant Female. Again, it was necessary to stabilize the variance prior to analysis. In the VC study CTX and EMS treatment groups were significantly different from the negative con trol groups in weeks I and 2. whereas VC-treated groups were not ( fable 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). 'rims 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. Laic Deaths In each study late deaths were randomly distiibuled 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 (/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 be correlated with treatment. The data for early deaths have been presented in various ways. Snmber 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 was significant (chi-square) in weeks I and 2 for the CTX-trcated group and in week 2 for the EMStrealed group. No differences from the negative control group were seen in the VC-lreated group. Similarly in the VDC study there were only signiiicant differences in the CTX positive control group in weeks I. 2. and 3 (Table 11). The Mean Snmher of Early Deaths per Pregnancy. A large number of low or zero values were encountered, so it w<as necessary to stabilize the variance prior to analysis. There were no statis tically significant increases in early deaths after VC treatment liable 6). However, differences weie 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-implantalional foetal early deaths (/./). From the data for early deaths there was no evidence in either study of a mutagenic ef fect with VC oi VDC at the administered exposure levels. I his 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 Cl X and EMS weie used in our studies to obtain a highly significant positive result. However, the dominant lethal stiuly in our hands i/-/) 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 MO mg/kg body weight, which is computable to that reported previously (/5i for 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 t/6). 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 50 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, II). 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. It 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 to 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 1CI Pharmaceuticals Division, Alderley Park. REFERENCES 1. Maltoni. C., ct al. Vinyl chloride carcinogenesis--current results and perspectives. Med. Lasaro 65: 421 (1974). 2. Creech. J. I... 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 SelikolT, I. J. Vinyl chloride exposure and human chromosome aberrations. Mu tation Res. 31: 163 (1975). 4. Funes-Cravioto. F., et al. Chromosome aberrations in woikers exposed to vinyl chloride. Lancet I: 459 (1975). 5. Purchase, I. F. H,, Richardson. C. R.. and Anderson, D. Chromosomal effects in peripheral lymphocytes. Proc. Roy. Soc. Med. (London) 69: 290(1976). 6. S/cnlcsi. I., el al. High rate of chromosomal aberration in PVC workers. 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