I.F.H. Purchase

Chromosomal analysis in vinyl chloride exposed workers: comparison of the standard technique with the sister-chromatid exchange technique.

A group of 21 workers occupationally exposed to vinyl chloride and 6 controls were examined for the presence of chromosomal aberrations or sister-chromatid exchanges in their peripheral lymphocytes. These people comprised a second sampling from a group of exposed workers and controls first examined 18 months earlier. The vinyl chloride exposed workers showed levels of chromosomal aberrations elevated above those of the controls, but there was only a slight increase in sister-chromatid exchanges (per cell or per chromosome) and this increase was not statistically significant. Sister-chromatid exchanges (SCEs) were also examined from in vitro cultures of lymphocytes exposed in G0/early G1 and late G1/early S phase to vinyl chloride, both with and without metabolic activation. There was no increase in SCEs in vitro without metabolic activation but there was a marked increase with metabolic activation and this increase was shown to be independent of cell-cycle phase. It thus was apparent that the small increases of SCEs in workers were not due to the inability of vinyl chloride to induce SCEs in human lymphocytes but were probably because of low exposures and SCE levels could have returned to normal relatively quickly after exposure. The present study suggested that the analysis of longer-living conventional chromosomal aberrations appeared to be a more sensitive monitor of exposure to vinyl chloride in exposed workers than the estimation of SCEs; however, it should be noted that in a 3rd sampling taken 24 months later the exposed workers had chromosomal aberration levels similar to the controls.

Chromosomal analyses in vinyl chloride exposed workers. Results from analysis 18 and 42 months after an initial sampling.

In a previous study (Purchase et al., Mutation Res., 57 (1978) 325-334) it was reported that 57 workers occupationally exposed to vinyl chloride had an increase in the incidence of chromosomal abnormalities in their lymphocytes by comparison with 24 control workers. Since that time (July 1974) threshold limit values for vinyl chloride and plant exposure levels have been reduced. In the present study, 2 further samples from the same population of workers have been analysed for chromosomal aberrations 18 and 42 months after the initial sampling. At 18 months, 21 VC workers and 6 on-site controls were investigated as were 23 workers and 8 on-site controls at 42 months. In the second sampling there was a tendency to an increase in chromosomal abnormalities of VC-exposed workers except in those people who had changed occupation. By the third sampling, however, there was a decrease by comparison with previous samplings and the levels of abnormalities had returned to values similar to those of the controls. Thus, reduction in exposure to vinyl chloride is accompanied by a reduction in the chromosomal abnormalities to levels indistinguishabe from those of controls.

Vinyl chloride: Dominant lethal studies in male CD-1 mice

The mutagenic activity of vinyl chloride (VC) at three exposure levels was assessed in fertile male CD-1 mice with the dominant lethal test. Male mice were exposured by inhalation to VC at 3000, 10,000 and 30,000 ppm for 6 h a day for 5 days. By comparison with control males exposed to air, no mutagenic effects on any maturation stage of spermatogeneisis in treated males were detected. There was no significant increase in the number of post-implantational early foetal deaths as shown by the number of females with one or more early deaths or number of early deaths/pregnancy or the number of early deaths/total 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 lack of effect was not due to the insensitivity of the system used since a dominant lethal effect was clearly demonstated in male mice dosed i.p. with cyclophosphamide (CTX) at 200 mg/kg body weight and ethyl methanesulphonate (EMS) orally at 200 mg/kg body weight once a day for 5 days. During dosing these animals were housed under dimilar exposure conditions to those animals exposed to the test substances but with a flow of air through the exposure chambers. Thus vinyl chloride is not mutagenic in the mouse at the stated exposure levels as measured by the dominant lethal test.

Dominant-lethal test results with known mutagens in two laboratories.

Abstract During investigations of the potential dominant lethal activity of various chemicals, concurrent controls were run to check the sensitivity and the reproducibility of the test systems. These experiments were carried out over an 18-month period in two laboratories using similar protocols. Negative control substances used were maize oil, dispersol, tween 80 and water. Positive control substance used were cyclophosphamide, ethyl methane-sulphonate and mechlorethamine hydrochloride (nitrogen mustard). The substances were administered either intraperitoneally or by gavage. The results were analysed using, principally, a hierarchical analysis of variance of the total implants per pregnancy and the transformed early deaths per pregnancy data. Pregnancy frequency generally did not fall below 80%. The total number of implantations per pregnancy was usually about 11.8 in negative controls and was variably reduced by the mutagens used. With cyclophosphamide or ethyl methanesulphonate (EMS), there were some quantitative variations in the results obtained but qualitative agreement was good in the results both between experiments in the same laboratory and between the two laboratories. It was demonstrated that EMS is a useful positive control substance in experiments with orally administered materials. Sensitivity of the system was indicated by positive effects with a relatively low dose of EMS and consistent positive results with mechlorethamine for which a dominant lethal effect has not been demonstrated previously. It is concluded that the dominant lethal test gives reproducible data and it is, thus, possible to have confidence in the results and to compare findings in different laboratories using similar protocols.

Dominant lethal studies with paraquat and diquat in male CD-1 mice

The herbicides paraquat and diquat were tested for dominant lethal activity in male CD-1 mice. No mutagenic effects were detected on any stage of spermatogenesis in treated males if these compounds were administered orally up to 4 mg paraquat/kg/body weight/day of 10 mg diquat/kg/body weight/day for 5 days. There was no increase in the number of post-implantation losses as shown by the number of females with one or more early deaths or number of early deaths/pregnancy or the number of early deaths/total implants/pragnancy. There was no evidence of pre-implantation losses as indicated by the total implants/pregnant female. There was no anti-fertility effect on the treated males as measured by the pregnancy frequency or successful mating frequency. The lack of dominant lethal effect was not due to particular insensitivity of the animals used since such effects were amply demonstrated in mice doses orally on 5 days with ethyl methanesulphonate at 100 mg/kg/body weight/day, or intraperitoneally on one day with cyclophosphamide at 200 mg/kg/body weight.

Reflections on the declining ability of the Salmonella assay to detect rodent carcinogens as positive

It is suggested that urgent attempts should be made to define and gain general agreement for the existence of two classes of animal carcinogen, those which are genotoxic and those which are not. In the absence of such a step, attempts to validate in vivo genotoxicity assays, and to derive a meaningful structure-activity database for chemical carcinogenesis, will be frustrated. These suggestions are supported by the preliminary findings of a detailed analysis of the carcinogen database accrued by the United States National Toxicology Program. The possibility that many non-genotoxic carcinogens should be regarded as tumour-promoting agents is considered.

GENETIC TOXICOLOGY APPLIED TO ASSESSMENT OF MUTAGENIC, CARCINOGENIC AND TERATOGENIC ACTION OF PESTICIDES AND RELATED COMPOUNDS

Abstract More is known about the molecular mechanisms by which chemicals induce mutations than is known about carcinogenesis or teratogenesis. There is a large body of knowledge about genetics which, combined with our understanding of the role of DNA in hereditary processes, provides a basis for genetic toxicology. Apart from recognising that there are two classes of carcinogens (genotoxic and non-genotoxic) and the circumstantial evidence of the involvement of DNA, little is known of the molecular mechanisms of carcinogenesis. Knowledge of the processes of teratogenesis is equally rudimentary. The techniques for detecting chemicals which produce these toxic phenomena, reflect this underlying knowledge. Mutagenicity tests are well understood, sophisticated and varied although there are no clear examples of chemically induced human mutations. Those for carcinogenicity rely on life-time studies in laboratory animals which mimic the human disease. Screening tests based on mutagenicity are finding increasing use in detecting genotoxic carcinogens, leading to support for the somatic mutation theory of cancer. For teratogenesis relatively obvious in vivo tests are currently the only ones available and even these show a high degree of species specificity. In vitro tests are not developed to the stage where they find general applicability and the limited evidence available does not support the use of mutagenicity tests for screening teratogens.