Document gb6w6zDaqQnoY2eZgO4DGbB3e

Vinyl Chloride Cytogenetics Dante J. Picciano, Ph.D.; Ray E. Flake, M.D.; Peter C. Gay, M.D.; and D. Jack Kilian, M.D. This report presents cytogenetic findings from a group of 209 workers employed for up to 28 years in the manufacture of vinyl chloride monomer at the Texas Division of Dow Chemical U.S.A. Cytogenetic evaluation results from this group were compared to results found in examination of individuals being considered for employment. Statistical analyses were per formed on a group basis for chromatid aberrations, chromo some aberrations and proportion of abnormal cells; no statistical difference of significance was found between the two groups. Comparison of these results with reported studies suggests that the level of cytogenetic aberrations in vinyl chloride workers is probably related to the length and level of exposure, and that risk of adverse genetic effect can be avoided in cohtrolled, minimal-exposure environments. Vmyl chloride (VCI has been reported to be carcinogenic in animals1 1 and human beings' and mutagenic in bacterial test systems-4 '' h There have been reports of increased frequencies of chromosomal aberrations in workers exposed to vinyl chloride at polyvinyl chloride (PVC) facilities 7 8 , Similar increases were not found upon cytogenetic evaluation of German PVC workers10 nor in our own preliminary investigation of a group of American VC workers" One study based on interviews with male employees has indicated an increased rate of fetal wastage among wives of VC-exposed workmen involved in polymerization operations." We have recently completed cytogenetic evaluation of all currently-employed workers exposed to vinyl chloride at the Dow (Freeport) Texas Division Both vinyl chloride and vinylidene chloride are produced in this plant Vinylidene chloride has also been reported as mutagenic in bacterial test systems11 However, while these Dow workers are involved with the production of both compounds, the degree of contact with vinylidene chloride is far less than the exposure to vinyl chloride From Occupational Health and Medical Research Dow Chemical USA, (Drs Picctano and Kiliani and Department of Industrial Medicine Dow Chemical USA, Texas Division (Drs Flake and CavK Freeport TX 77541 Material from this report was presented at the Sth International Congress of Human Genetics Mexico City Octobei 10-1S l97f> Vinyl chloride monomer has been produced at the Texas Divi sion of Dow Chemical U.S.A. since 1948 There are no vinyl chloride polymerization operations. Average exposure levels, as is the case for almost all vinyl chloride monomer plants, have been generally lower than those reported for facilities involved in polymerization operations.14 Although the threshold limit values for vinyl chloride had been 500 parts per million (ppm) until 1974, Dow had established a goal of 50 ppm or below in 1959 11 The current Occupational Health and Safety Administration (OSHA) standard for vinyl chloride is one ppm as a time-weighted-average over an eight-hour workday. We have found that the TWA con centration of vinyl chloride for all VC-related |ob classifications was approximately 5 ppm from 1968 to 1973; in 1974, TWA con centrations were 1 to 2 ppm. Since 1975, average levels of expo sure have been lowered to less than 1 ppm. Estimates of average exposure levels for the years prior to 1968 are less reliable For the purposes of this study, estimates of exposure were calculated for specific job classifications, based on records of both personnel and area monitoring (Table 1). However, actual exposure may vary from individual to individual within the same |ob classification and accidental, short-term exposures of some workers to concentra tions in excess of the standard probably occur from time to time. Documentation of such incidents is difficult. Methods Our study group was composed of 209 workers who had worked in the vinyl chloride plant for periods ranging between one and 332 months (average, 48.3 months) at the time of this in vestigation As part of the medical surveillance program for this group, peripheral blood samples were obtained, and evaluation of lymphocyte chromosomes was performed. Lymphocytes were cultured using a modification of the Moorhead technique 16 Stan dard procedures, as previously described,17 were used for incuba tion, processing, and analysis. Findings were compared to cytogenetic data from a group of 295 "preemployment examinees" who had chromosome evalua tion done as part of routine preemployment examination and who, at that time, had no known exposure to chromosome-break- SL 041525 Reprinted from Journal of Occupational Medicine August. 1977, Volume 19. Mo 8 pp. 527-530 4 k V Table 1. -- Estimated Exposure to Vinyl Chloride (VC) tor Vinyl Table 2. -- Cytogenetic Study of 209 Workers Exposed Chloride-Related Job Classifications. to Vinyl Chloride- Job Classification Supervisor R&D Engineer Production Engineer Technical Specialist Development Lab (Lab Supv) Quality Control Lab Instrument Man Electrician CoMerers (Insulators) Painter Pipefitter Machinist Welder (Boilermaker) Maintenance (Utility Man) Material Handier Production Foreman Shift Foreman Control A Op. (V2 & Chloride) Control A Op. (E A Cl) Control A Op. (Vinyl) Control A Op (Chlorination) Control B Op (Oxy A Chloride) Control C Op. (Chloride & Vinyl) Control C Op.(Chlorination) Class 1 Op, Class 1 Op. (Parts) Class 3 Op. Production Clerk Unit Manager Estimated Exposure in Parts per Million, as Time-Wiifhted-Aventge 1973-1974 I960 1972 Before 1960 17 44 8.2 1 7 44 82 1,7 44 82 05 0,5 06 12 39 77 87 114 152 1.3 40 78 1.3 40 78 0.3 0.3 03 0.7 07 0.7 09 09 09 2.4 5.1 89 0.6 06 0.6 1 3 4.0 78 05 0,5 0.5 1.3 40 7,8 44 71 10.9 52 79 117 0.9 0.9 0.9 2.8 55 9.3 2.3 50 : 8.0 0,7 07 07 46 7,3 11 1 2 6 53 9.1 5.3 8,0 . 11.8 0,5 0.5 05 2 5 3 9-1 0.7 0 7 0.7 07 0.7 07 No of cultures No of celts Chromatid breaks Chromosome breaks Rings, dicentrics, and exchanges Abnormal cells Workers 209 10.483 2.4% 1.0% 04% 3 7% Controls 295 14.761 3 6% U% 0 2% 4 5% percent aberrations, and the vinyl chloride workers were separated Into those with estimated TWA exposure levels of lexxthan-1 ppm, from 1-to-5 ppm, and greater-than-1; ppm The estimated exposure levels were based on calculations for spectfir |ob classifications to which members of the vinyl chloride study group were assigned Three categories of aberration were chosen tor this analysis: chromatid breaks, chromosome breaks, and the proportion of abnormal cells As shown in Table 3, no significant differences were lound when these groupings were compared on the basis of chromatid aberrations. Results from those workers with estimated exposures of greater than 5 parts per million are almost identical to those ot the control group. Similar conclusions were reached upon evaluation of the data for chromosome breaks (Table 4) and the proportion of abnormal cells (Table 5). In all three cases, most of both groups were tound to have zero-to-five percent aberrations, and the vinyl chloride workers with the highest level of estimated exposure showed aberration rates not significantly different than those of the con trol group findings for both groups, workers and controls, are considered to be within the range of normal variation as seen in this laboratory mg agents The records selected for inclusion in the control group were matched to those of the study group, insofar as possible, for sex, number of cells analyzed, and time period during which the culture was initiated. Age variation between the two groups could not be completely eliminated1 the average age of the vinyl chloride workers was 39 5 years (range, 18 to 67 years), and the average age of the pre employment control group was 2S, 1 years (range 18 to SO years) Discussion The reports that have appeared, to date, concerning chromosome aberrations in vinyl chloride workers have not been in agreement. This conflict may be due either to the small number of workers studied or to differences in exposure levels, or both. The Swedish group studied by funes-Cravioto et al,B was com posed of seven PVC workers who had been exposed for nine to 29 years and who were found to have an increased frequency of chromosome breakage The level of exposure for this group was Results As shown in Table l, data from both groups -- vinyl chloride workers and preemployment controls -- were scored and com pared on the basis of chromatid breaks, chromosome breaks: rings, dicentrics, and exchange figures: and the proportion of ab normal cells The proportion of abnormal cells was calculated to show the overall frequency ot aberrant cells, that is, the ratio of cells with at least one aberration to the total number ol cells examined Results, expressed in terms of the mean percentage for these categories of aberration showed no ma|or different ex be tween the two groups tor anv of the classifications Using Chi-square analysis, it was decided to see if differences ir) various aberration rates could be detected within the two groups Accordingly, both groups were divided into those showing zeroto-five percent aberrations and those showing greater-than-tive Table 3. -- Distribution of Chromatid Aberrations Related to Vinyl Chloride (VC) Exposure. Eiposure to VC* < 1 ppm 1-5 ppm >5 ppm Controls No. in Group 209 70 98 41 295 % of Group with 05% Aberrations % of Group with > 5% Aberrations 90% 10% 11 23 80 20 75 25 X'O) .= 775 IP So 061 Exposure levels are estimates based on calculations for specific 10b classifications prior etpo sure for individuals may have been higher or lower exposure levels for individuals are known to vary within 106 classifications % s'- V Table 4. -- Distribution of Chromosome Aberrations Related Table 5. -- Distribution of Abnormal Cells Related to to Vinyl Chloride (VC) Exposure. Vinyl Chloride (VC) Exposure. Exposure to VC 1-5 ppm >5 ppm Controls No. In Group 209 70 98 41 295 % of Group with 05% Aberrations % of Group with > 5% Aberrations 96% 4% 94 6 95 5 94 6 JChsi = 0.355 (psoas) Exposure to VC < 1 ppm 1-5 ppm > 5 ppm Controls No. in Group 209 70 98 41 295 % of Group with 05% Aberrations % of Group with > 5% Aberrations 84% 16% 71 29 73 27 70 30 JC*(3I - 597 <P0 12) reported to have continuously decreased over a period of years until immediately prior to the study when VC concentrations in the polymerization department were estimated to be 20 to 30 ppm The U.S. group of 11 PVC workers surveyed by Ducatman et al" was also reported to show an increased rate of chromosome breakage following exposures estimated to have been in excess of 500 ppm at times. The group of ten German workers investigated by Fleig and Thiess20 worked with either VC or PVC or both for periods ranging between six and 34 years and with exposures esti mated to have decreased from greater than 500 ppm in 1945 to between 10 and 25 ppm in 1974. Evaluation of this group for in creased aberration rates was negative. The study of British PVC workers by Purchase et al.1' concluded that the frequency of cytogenetic aberrations was increased in 56 exposed workers as compared to 24 nonexposed individuals. Esti mates of the levels of exposure were not given in this report. The medical director of the surveyed group, however, has informed us that while further analyses of the data confirmed the findings of in creased aberrations in workers exposed to "higher" levels of vinyl chloride, no differences between worker and control groups were detected upon evaluation of the data for workers exposed to "lower" levels. An earlier report from our laboratory" concluded that there were no cytogenetic differences of statistical significance be tween a group of 121 workers exposed to vinyl chloride and a 75person control group. As in the work reported here, there was a discrepancy in the age composition of the two groups, because applicants seeking employment tend to be younger than those already settled into jobs. We do not believe, however, that this difference is a confounding factor in our comparison since the dif ference is not great and because it has been shown21 that the cytogenetic change most often associated with aging is chromosome loss, rather than chromosome breakage. On the basis of our negative findings and the conflicting find ings reported by others, we believe that the level of chromosome aberrations in workers exposed to vinyl chloride is probably re lated to the length and level of exposure and that the risk of ad verse cytogenetic effects can be avoided in controlled, minimalexposure environments Cytogenetic dose-response curves, similar to that suggested here, have been reported for x-irradiated ankylosing spondylitics,21 A-bomb survivors,21 radium dial paint ers,2,1 persons exposed to Thorotrast,27 and workers exposed to lead 28 It has also been noted that the groups exposed to radiation later demonstrated significant increases in neoplastic incidence. A-bomb survivors, for example, have shown increased .rates of leukemia and thyroid carcinoma;2' radium dial painters were found to be at increased risk of osteogenic sarcoma;10 and thorium dioxide-exposed persons have an increased rate of liver tumors 11 This relationship between chromosomal breakage and neoplasia strongly suggests that cytogenetic analyses may be a useful tool for detecting environmental situations which may be associated with increased cancer risks to the workers The present study is one of several involving Texas plant em ployees to investigate the possibility that increased rates of fetal loss or birth defects in offspring are experienced by wives of vinyl chloride workmen and to determine the morbidity-mortality ex perience of all past and present VC workers. A search has been made for cases of angiosarcoma of the liver; none have been found in our study group (533 individuals) which includes all past and present VC workers. Current workers are also being monitored as part of the continuing medical surveillance program. The authors acknowledge with appreciation the technical assistance ot Mrs A Linscombe and Mrs D Mensik, the suggestions of Mrs M Benge the editing of Ms T B Llovd, and the advice of Dr C B lacohson References 1 Viola PL. Bigotti A, and Caputo A Oncogenic response of rat skin, lungs, and bones to vinyl chloride. Cancer Res 31 516-519, 1971. 2 Maltoni C and Lefemine C Carcinogenicity to bioassays of vinyl chloride, I, Research plan and early results Environ Res 7*387-405, 1974. 3. Creech |l and lohnson MN- Angiosarcoma of the liver m the manufac ture of polyvinyl chloride. IOM 16:150-151. 1974. 4 Rannug U. lohansson A, Ramel C, and Wachtmeister CA: The mutagenicity of vinyl chloride after metabolic activation Ambio 3194-197, 1974 5 Malaveille C Bartsch H. Barton A et al* Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacetaldehyde and chloroethanol Biochem Biophys Res Commun 63:363-370, 1975 6 Bartsch H, Malaveille C. and Montesano R Human, rat and mouse liver-mediated mutagenicity of vinyl chloride in S lyphimunum strains Int / Cancer 15:429-437, 1975 7, funes-Cravioto f. Lambert B, lindsten I et al, Chromosome aberrations in workers exposed to vinyl chloride. Lancvt 1:459, 1975 8 Ducatman A, Hirschhom K, and Selikoff !)v Vinyl chloride exposure and human chromosome aberrations Mu at Res 31:163-168, 1975. 9 Purchase IFH, Richardson CR, and Anderson D Chromosomal and dominant lethal effects of vinyl chloride, Lancet 2 410-411, 1975 10 Fleig I and Thiess AM Chromosome analysis after vinyl chloride ex posure Arbeitsmed Sozialmed Praeventimcd 9:280-283. 1974 11 Kihan D). Picciano D), and lacobson CB: Industrial monitoring' A cytogenetic approach. Ann NY Acad Sc/ 269*4-11. 1975 12 Infante PF, Wagoner |K, MrMichael A|. Waxweiler R), and Falk Hl Genetic risks of vinyl chloride lancet 1,734-735. 1976 13 Bartsch H, Malaveille C, Montesano R, and Tomatis L1 Tissuemediated mutagenicity of vmylidene chloride and 2-chlorobutadiene m Salmonella typhimunum Nature 255 641-643, 1975 14 Occupational Safety and Health Administration: Exposure to vinyl chloride, Federal Register 39(194)' 35889-35898, October 4, 1974 15 Rowe VK Experience in industrial exposure control, Ann NY Acad Sci 246 306-310, 1975, 16 Moorhead PS, Nowell PC, Mellman W|, Battips DM, and Hungerford SL 041527 DA Chromosome preparations of leukocytes cultured irom human fJeriphera! blood fxp Coll Res 20 61 1-616, 1960 17 Kihan D| and Picciano D Cytogenetic surveillance of industrial popu lations In Chemical Mutagens Principles and Methods /or Then Deter von Vol 4, New York A Hollaender. ed Plenum Press, 1976, pp 121-119 10 Funes-Cravioto ct al. Reference 7, 19 Ducatman et al. Reference 8. 20 Fleig & Thiess, Reference 10 21 Purchase et al. Reference 9 22 Kihan et al. Reference 11 23 Court Brown WM: Human population cytogenetics In frontier* of Biology, Vol V, A Neuberger and EL Tatum, eds. North Holland Publishing, 1967, pp 1-31 24 Buckton KE, Jacobs PA, Court Brown WM, and Doll R A study of chromosome damage persisting after x*ray therapy for ankylosing spon dylitis, Lancet 2,676-682, 1962 25 Bloom AD, Nakagome Y. Awa AA. and Nerilshi 5 Chromosome aberrations and malignant disease among A-bomb survivors Amer / Pub filth 60 641-644, 1970 26 Vaughan | Bone disease induced bv radiation Int Re\ I \p Path 1 241-396, 1962 27 Fischer P, Colob E, Kunze-Mlihl [, Maim AB et al Chromosome aberrations in peripheral blood cells m man following chronic irradiation from internal deposits of Thorotrast Radiat Res 29 505-515, 1966 28, Garza Chapa R, Leah CH, Alvarez M, and Sanchez Fp Chromosome analysis in males occupationally exposed to lead (Abstract 3251 In AhMracts V Internationa} Congress of f Ionian CenetK s 5 Armendares and R Ltsker, eds Exccrpta Medica, Amsterdam, 1976 29 Sampson Rf. Key CR, Buncher CR, and li|ima A, Thyroid carcinoma in Hiroshima and Nagasaki I Prevalence of thyroid carcinoma at autopsy Hiroshima 1957-68, Nagasaki 1951-67 AffCC fee hmcaf Report 25-68, 1968 30. Muller |, David A, Rejskova M, and Brczikova D Chrome occupation al exposure to strontium-90 and radium-225 Uncer 2 129-131, 1961 31 Fischer P, Golob F. Kunze-Muhl E and Mullner T. Chromosomal aberrations in thorium dioxide patients Ann NY Arad 5c/ 145 769-766, 1967 SIj 041528 K Arch. Toxicol. 45, 1--7 (1980) TOXICOLOGY rQ Spnnua - Vci Liu I9NU Vinyl Chloride: An Example for Evaluating Mutagenic Effects in Mammals in vivo after Exposure to Inhalation Armin Basler and Gunter Rohrborn Institut fiir Humangenetik und Anthropologie der Universitat Universitatsstrasse 1, Gebaude 23.12, D-4000 Diisseldorfl, Federal Republic of Germany Abstract. As part of a programme of investigations on the mutagenic effects in mammals in vivo after inhalation of environmental chemicals, the effects of the in dustrial compound vinyl chloride (VC) was analysed. Chinese hamsters were exposed to 1.25%, 2.5%or5%(v/v) VC in air for 6,12 or 24 h. Bone marrow chromosomes were analysed for induced chromosome aberrations and sister-chromatid-exchanges (SCEs) 26 h after beginning of ex posure. The frequency of VC-induced chromosome aberrations and SCEs both de pend on dose and length of exposure. The highest measured effects were 33.25 SCEs/cell after an exposure to 2.5% VC for 24 h and 25.7% metaphases with aber rations, when exposed to 5% VC for 24 h. Key words; Vinyl chloride -- Inhalation -- Chinese hamsters -- Chromosome aberrations -- SCEs in vivo.' Zusammenfassung. Im Rahmen eines Programmes zur Untersuchung mutagener Effekte beim Sauger in vivo nach Inhalation von Umweltschadstoffen wurde Vinylchlorid (VC) untersucht. Chinesische Hamster wurden mit 1,25%, 2,5% oder 5%(v/v) VC in Luft fur 6, 12 oder 24 h begast. Chromosomen des Knochenmarks wurden 26 h nach Begasungsbeginn prapariert und auf induzierte Chromosomenaberrationen und Schwesterchromatid-Austausche (SCEs) hin untersucht. Die Haufigkeit VC-induzierter Chromosomenaberrationen und SCEs hangt sowohl von der Dosis ab, als auch von der Expositionszeit. Die hochsten ermittelten Werte waren 33,25 SCEs/Zelle nach einer 24stiindigen Begasung mit 2,5% VC und 25,7% Metaphasen mit Aberrationen nach 24stiindiger Begasung mit 5% VC. Schliisselworter; Vinylchlorid -- Inhalation -- Chinesische Hamster -- Chromo somenaberrationen -- SCEs in vivo. Offprint requests to: A. Basler at the above address 0340-5761/80/0045/0001/1 01.40 Si 4j52g 2 Introduction A. Busier and G. Rohrborn In man, most serious occupational diseases arising from environmental chemicals are caused by inhalation of these compounds. With the exception of gases, it might be pos sible to investigate biological effects of these substances also by oral, intraperitoneal, intravenous or subcutaneous application to experimental animals. However, it is preferable to expose the animals with the compounds in form of gas, aerosols or particles in order to estimate the potential mutagenic risk of chemicals inhalated by man. In the field of toxicology including cancer research inhalation toxicity has been per formed for decades (Gage, 1970). In mutation research especially with mammals, experiments with inhalation mutagenicity are rare and methodical experiments to analyse SCEs in animals exposed to gases are lacking. As part of investigations on the mutagenic effects in mammals in vivo after subacute inhalation of environmental chemicals, the applicability of the SCE test and the analysis of structural chromosome aberrations were investigated. The chosen test compound was Vinyl chloride, known to be mutagenic and cancerogenic in mammals and man (for review see Bartsch and Montesano, 1975). Material and Methods Animals. Adult (10--15 weeks old) Chinese hamsters(Cricetulusgriseus), weighing 30 g, were used. In all experiments the ratio offemale to male hamsters was 1 ; 1. During the exposure period the animals were al lowed access to food and water. Test Compound. Vinyl chloride (Chlorethylene, C2H,C1) was purchased from Merck-Schuchardt, Darm stadt, FRG. The gas was supplied via a two stage pressure regulator and a flow meter in order to measure the flow rate in 1/min. The gas was mixed in a mixing chamber with air in different amounts, which was me tered from the compressed air taps in the laboratory via a pressure reducing valve and an air flow meter. Experimental Design (Fig, 1). 1.25%, 2.5% or 5% (v/v) VC in air was conveyed through an inhalation chamber, made of Plexiglas with the size of 20 x 20 x 20 cm. The fresh gas inlet was on the bottom of the chamber, the outlet was at the lid of the box, diagonal opposite to the inlet. The continuing flow of the gas Experiment b; implantation of a 50 mg BrdU tablet Experiment a and b: group 1, II and III beginning of exposure i 1 End of exposure group I i Experiment a and b: group I, II and III 8 mg Colchicin/kg i End of End of Experiment a and b: exposure exposure group 1, 11 and HI group II group III preparation of chromosomes ii i 1 __l______ Oh +6h + 12 h + 24 h + 26 h Fig. 1. Experimental design. Analysis of Vinyl chloride induced structural chromosome aberrations (a) and SCEs (b) in bone marrow of Chinese hamsters SL 041530 Vinyl Chloride: Mutagenic Effects in Mammals 3 mixture was 41/min. Up to 4 hamsters (only one sex at a time) were housed in the inhalation chamber. Ani mals of the negative controls were kept under similar conditions. They were gased with a flow of air only. A nalysis ofStructural Chromosome Aberrratiorts. Chinese hamsters were exposed to 2.5% VC in air for 6, 12 or 24 h. Another group was gased with 5% VC for 24 h only. The hamsters received an intraperitoneal injection of 8 mg colchicine/kg body weight 24 h after the beginning of exposure. Bone marrow chromo somes were prepared 2 h later according to Boiler and Schmid (1970) and analysed for gaps, breaks, frag ments, deletions and exchanges. Metaphases with 5 and more aberrations were listed as multiple aberrations. Analysis of Induced SCEs. Chinese hamsters were exposed to VC for 6, 12 or 24 h. The VC con centrations were either 1.25% or 2.5%. The BrdU-tablet method (Allen et al., 1977) was used to obtain in vivo induced sister chromatid exchanges. Therefore a 50 mg BrdU-tablet (Boehringer, Mannheim, FRG) was implanted subcutaneously into the neck of 30 g weighing Chinese hamsters immediately before starting the VC exposure. Colchicine was injected 24 h later, bone marrow chromosomes were prepared another 2 h later. After an air drying of 2 to 3 days the chromosomes were stained with Bisbenzimide (Hoechst 33258) dye (Riedel de Haen, Seelze-Hannover, FRG) and Giemsa according to Epplen et al. (1975). Statistics. The data of the SCE test have been statistically analysed using the r-test. The significance of dif ferences in the yield of metaphases with aberrations was verified using the J^-test. Results The dose levels for VC were selected on the basis of toxicity studies. Groups of 4 ham sters were exposed for 24 h to VC in air at 1.25%, 2.5%, 5% and 7.5%. 5% VC was cho sen for the mutagenicity experiments as the highest exposure level as this was found to be the maximum tolerated dose. No mortality occurs in this group within the 24 hourly experiment, whereas higher doses were toxic in all cases. The number and percentage of metaphases with structural chromosome aber rations are shown in Table 1. In 1400 metaphases of 14 hamsters of the control group only 1 break (0.1%) and 8 gaps were found. The percentage of metaphases with Table 1. Chromosome aberrations in bone marrow cells of Chinese hamster Dose and time No. of of exposure animals No. of Metaphases with aberrations meta- ----- --............ ....... ....................... phases Gaps included Gaps excluded Metaphases with ................................................. G B+F D E m.a. No. % No. % Control 2.5% VC; 6 h 2.5% VC; 12 h 2.5% VC; 24 h 5.0% VC; 24 h 14 4 4 4 10 1400 400 400 400 1000 9 0.6 3 0.8 3 0.8 29 7.3" 257 25.7* 1 0.1 3 0.8* 3 0.8* 18 4,5* 225 22.5* 81 03 03 11 18 32 87 0 00 0 00 0 00 0 00 9 76 53 G gaps, B + F breaks a. fragments, D deletions, E exchanges, m.a. multiple aberrations ` p < 0.02 4 A. Basler and G. Rohrborn Table 2. In vivo induced SCEs in bone marrow cells of Chinese hamster Time of exposure No. of animals No. of meta phases 1.25% VC in air SCEs/cell + SD No. of animals No. of meta phases Control 6h 12 h 24 h 8 4 4 4 400 4.41 + 0.58 8 200 8.72" 4 1.36 4 200 17.51* 4 1.55 4 200 22.33* 0.67 4 400 200 200 200 * p < 0.02 2.5% VC in air SCEs/cell + SD 4.56 4 0.92 12.66* 4 3.72 19.80* 4 2.60 33.25* 3.26 1.25% Vinyl chloride in air Fig. 2. In vivo induced sister Itime of exposure Ih) 24 chromatid exchanges in bone marrow chromosomes of Chinese hamster aberrations was slightly increased in those hamsters, exposed to 2,5% VC for 6 h. Simi lar results were obtained following an exposure time of 24 h. In both groups the values for induced aberrations (only breaks and fragments) were 0.8%. A distinct increase was first noted following an inhalation time of 24 h. In 4.5% of the analysed meta phases breaks and fragments were induced. Gaps included, the percentage of affected metaphases increased from 0.6% in the controls to 7.3%. Even 22.5% aberrant metaphases (25.7% including gaps) were found if the dose of the 24 h exposure was in creased to 5%. The majority of aberrations were breaks and fragments, followed by ex changes, multiple aberrations, gaps and deletions. The number of in vivo induced SCEs (Table 2) depends -- as demonstrated for structural chromosome aberrations -- both on dose and length of exposure to VC. The 6 h lasting inhalation of 1.25% VC even doubled the SCE frequency. 4,41 SCEs per cell were counted in the controls, whereas 8.72 were detected in this experimental group. A lengthening of the inhalation time increased the number of SCEs. 17.51 SCEs per cell were found following 12 h lasting inhalation and 22.33 after an exposure time of 24 h. After application of 2.5% the SCE frequency increased in all exposure-timegroups as compared to the dose of 1.25%: 12.66(6 h of exposure), 19.80 (12 h) and at maximum 33.25 SCEs per cell (24 h) were analysed. The increase was nearly linear (Fig. 2) at the investigated dose range. 0^1532 Sb Vinyl Chloride: Mutagenic Effects in Mammals Discussion 5 Several studies on human lymphocytes of workers exposed to VC have been per formed to ascertain whether mutations were induced in somatic tissues: Kucerova et al. (1979) reported both, an increase of aberrations and SCEs in workers exposed for 10--27 years to mean annual doses of VC about 20-- 150 ppm of air. The SCE rates observed in this study (increase from 9.41 to 13.80 SCEs per cell), however, have to be considered with reservation. Four out of the examined persons were smokers and 8 of them had more or less drinking habits and both of these habits are supposed to be mutagenic. The average number of SCEs for example is 17.2 in heavy smokers, compared to 13.2 in nonsmokers (Lambert et al., 1978). The level ofchromosome aberrations was also positively correlated to the duration of employment, as demonstrated by Purchase et al. (1978). The different findings of Funes-Cravioto et al. (1975) may be the result ofother factors, such as job category. In a study of Purchase et al. (1978), for example, the autoclave workers who operated and cleaned the autoclaves, had the greatest number of aberrations of all plant workers. No increase was observed in workers who were not involved in the polymerisation process (Kilian et al., 1975). Correlations between the VC concentrations at work place and induced mutations were reported also by Hansteen et al. (1978). Cytogenetic studies were performed first in 1974 on workers who had been heavily exposed to VC for years. The study was re peated 2 years later with the same workers. Meanwhile the concentrations in plants were reduced and the workers had only a low exposure to VC (< 1 ppm). Chromosome aberrations increased compared to unexposed workers in the 1974 study, whereas in the follow-up study, the chromosome breakage frequency in these workers decreased to control level. The SCEs were tested in 1977 only, and no difference was found be tween workers and controls. In heavily exposed workers, resulting in symptoms of VC illness such as acroosteolysis, Raynaud syndrome, thrombocytopenia and liver disturbances, the rate of aberrations increased, compared to unexposed persons, as well as to VC workers without these syndroms (Fleig and Thiess, 1978). Comparing the history ofexposure, it becomes evident that the yield of VC-induced chromosome aberrations in lymphocytes of workers depend on different factors: the category of workplace correlated to the VC concentration in the PVC plant and the length of exposure. Besides the induction of chromosome aberrations in somatic tissues mutational events in germ cells and the transmission to the following generations were discussed by Infante et al. (1976). They confirmed a significant excess of fetal mortality among wives of workers, who were occupationally exposed to VC. The data however, have been criticised (Paddle, 1976) because of inaccuracies in statistical methods. Experimental investigations with mammals did not support the findings in man. Anderson et al. (1976) exposed mice to VC by inhalation up to 30000 ppm for 6 h a day for 5 days. No mutagenic effects on any maturation stage ofspermatogenesis in treated males were detected as measured by the dominant lethal test. Negative results were ob tained also in drosophila when tests on dominant lethals and translocations were carried out with VC at 30000 ppm for 2 days. It was mutagenic only in the recessive le thal test (Verburgt and Vogel, 1977). The conclusion that chromosome breakage is not *1533 6 A. Basler and G. Rohrborn a reliable measure of mutagenic activity of VC (Verburgt and Vogel, 197 7) might be rel evant for the test animal drosophila, not however for mammals, as shown by Fleig and Thiess (1978a, b). Induced aberrations were provable in Chinese hamster bone marrow chromo somes when treated by inhalation up to 5000 ppm VC for 4 h a day for 5 days (Fleig, 1977; Fleig and Thiess 1978b). In contrast to our finding with higher doses no doseeffect-relationships were obtained. In the present experiments a distinct increase was measured following an exposure to 2.5% VC. Considering the average concentrations of VC at the work place of PVC plants in the years 1950-- 1954 were 2000 ppm (Hansteen et al., 1978), the chosen dose levels in our experiments were approximately 60 to 250 times higher. We have to state, however, that chromosome aberrations were induced following an exposure to in halation of only 6 to 24 h. The SCE test in vivo is increasingly used in the field of mutagenicity testing because of the high sensitivity. Performing the primary method (Vogel and Bauknecht, 1976; Allen and Latt, 1976) BrdU had to be injected every hour for 6 to 8 h. Today using the BrdU tablet method (Allen et al., 1977), BrdU is subcutaneously implanted in form of a 50 mg tablet into the neck ofChinese hamster and chromosomes are prepared 26 h lat er. This technique enables to expose test animals by inhalation immediately after the tablet implantation without interruption up to 24 h when colchicine is injected. The ap plicability of this method is demonstrated in the experiments presented here. The SCE frequency was even doubled following an exposure to 1.25% VC for only 6 h and in creased with increasing doses of VC and duration of exposure. As mentioned above, the VC concentration at the work place was reduced within the last years. Clues, whether the present technical guiding concentration of 5 ppm (Deutsche Forschungsgemeinschaft, 1979) is a tolerable dose, cannot be drawn from these experiments. As demonstrated, the exposure time plays a considerable part in the yeald ofinduced mutations. A long term exposure for years, the situation for man at the work place, cannot be reproduced experimentally. The establishment of maximum tolerable values, however, was not the aim of this study. We intended to demonstrate that both cytogenetic methods in vivo are applicable to evaluate a possible mutagenic risk of environmental agents in gaseous state. For this reason, as usually in the field of mutagenicity testing, the animals were acutely exposed to the test compound with dose levels selected on the basis oftoxicity studies. On the basis of this experimental design, the SCE test as well as the analysis of chromosome aberrations lead to clear cut dose response relations. This study was supported by the Bundesministerium fiir Forschung und Technologie. Contract No. CMT16. References Allen, J. W,, Latt, S. A.: Analysis of sister chromatid exchange formation in vivo in mouse spermatogonia as a new test system for environmental mutagens. Nature 260, 449--451 (1976) Allen, J. W., Shuler, C. F., Mendes, R. W., Latt, S. A.; A simplified technique for in vivo analysis of sister chromatid exchanges using 5-bromodeoxyuridine tablets. Cytogenet. Cell Genet. 18, 231--237 (1977) SL 041534 * Vinyl Chloride: Mutagenic Effects in Mammals 7 Anderson, D., Hodge, M. C. E., Purchase, I. F. H.: Vinyl chloride: Dominant lethal studies in male CD-I mice. Mutat. Res. 40, 359--370 (1976) Bartsch, H., Montesano, R.: Mutagenic and carcinogenic effects of vinyl chloride. Mutat. Res. 32,93-- 114 (1975) Boiler, K,, Schmid, W.; Chemische Mutagenese beim Sauger. Humangenetik 11, 35--54 (1970) Deutsche Forschungsgemeinschaft: Maximum concentrations at the workplace 1979. Commission for in vestigation of health hazards of chemical compounds in the work area. Report No. XV. Boppard: Harald Boldt Verlag 1979 Epplen, J. T., Siebers, J. W., Vogel, W,; DNA replication patterns of human chromosomes from fibroblasts and amniotic fluid cells revealed by a Giemsa staining technique. Cytogenet. Cell Genet. 15, 177-185 (1975) Fleig, I.: Zur Mutagenitat von Vinylchlorid. Untersuchungen an Sauger und Mensch. Dissertation, Ruprecht-Karl-Universitat, Heidelberg, 1977 Fleig, I., Thiess, A. M.: External chromosome studies undertaken on persons and animals with VC illness. Mutat. Res. 53, 187 (1978a) Fleig, I., Thiess, A. M.: Mutagenicity of vinyl chloride. J. Occup. Med. 20, 557--561 (1978b) Funes-Cravioto, F., Lambert, B., Lindsten, J., Ehrenberg, L., Natarajan, A. T., Osterman-Golkar, S.: Chromosome aberrations in workers exposed to vinyl chloride. Lancet 1, 459 (1975) Gage, J. C.: The subacute inhalation toxicity of 109 industrial chemicals. Br. J. Ind. Med. 27, 1 -- 18 (1970) Hansteen, I.-L., Hillestad, L., Thiis-Evensen, E., Storetvedt Heldaas, S.: Effects ofvinyl chloride in man. A cytogenetic follow-up study. Mutat. Res. 51, 271--278 (1978) Infante, P. F., Wagoner, J. K., Waxweiler, R. J.: Carcinogenic, mutagenic and teratogenic risks associated with vinyl chloride, Mutat. Res. 41, 131--142 (1976) Kilian, D. J., Picciano, D. J., Jacobson, C. B.: Industrial monitoring: A cytogenetic approach. Ann. NY Acad. Sci. 269, 4-11 (1975) Kucerova, M., Polivkova, Z., Batora, J.: Comparative evaluation of the frequency of chromosomal aberrations and the SCE numbers in peripheral lymphocytes of workers occupationally exposed to vi nyl chloride monomer. Mutat. Res. 67, 97--100 (1979) Lambert, B., Lindblad, A., Nordenskyjold, M., Werelius, B.: Increased frequency of sister chromatid ex changes in cigarette smokers. Hereditas 88, 147--149 (1978) Paddle, G. M.: Genetic risks of vinyl chloride. Lancet 1, 1079 (1976) Purchase, I. F. H., Richardson, C. R., Anderson, D., Paddle, G. M., Adams, W. G. F.: Chromosomal analyses in vinyl chloride-exposed workers. Mutat. Res. 57, 325--334 (1978) Verburgt, F. G., Vogel, E,: Vinyl chloride mutagenesis in Drosophila Melanogaster. Mutat. Res. 48, 327-336 (1977) Vogel, W., Bauknecht, T.: Differential chromatid staining by in vivo treatment as a mutagenicity test sys tem. Nature 260, 448--449 (1976) Received December 21. 1979