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Antimony trioxide (Sb2O3, CAS 1309-64-4) has been examined in a range of in vitro and in vivo genotoxicity assays. Negative results were obtained with the Salmonella/microsome assay and the L5178Y mutation assay, but a positive response was observed in the in vitro cytogenetic assay using isolated human peripheral lymphocytes. However, in vivo, antimony trioxide was non-clastogenic in the mouse bone marrow micronucleus assay, following oral gavage administration for 1, 7, 14 or 21 days at dose levels of up to 5000 mg/kg (single dose) or 1000 mg/kg (repeat dose). A negative result was also obtained in the in vivo rat liver DNA repair (unscheduled DNA synthesis) assay following a single oral gavage administration of doses up to 5000 mg/kg. These data show no genotoxicity for antimony trioxide in vivo and do not confirm a previous report of clastogenicity in the mouse on repeated dosing. It is concluded that antimony trioxide is not genotoxic in vivo and does not present a genotoxic hazard to humans.

While sodium hypochlorite is widely used as a disinfectant for municipal sewage effluents and power station cooling waters discharged into coastal environments, there is limited information on the potential in vivo genotoxicity of such disinfection procedures to marine organisms. Using a recently developed test system based on the marine polychaete Platynereis dumerilii, we have evaluated impacts based on embryo-larval development, cytotoxicity and genotoxicity following exposure to disinfected settled (primary) effluent from a municipal sewage treatment works (STW). Sewage samples were collected from Newton Abbot STW, Devon, UK and then disinfected with sodium hypochlorite based on standard operational procedures. Exposure of polychaetes to dilutions of disinfected sewage in seawater (20 +/- 1 degree C) led to a marked reduction in normal embryo-larval development (7 h EC50 from 0.57-1.88% (v/v), n = 4), with a simultaneous increase in cytotoxicity. Following the calculation of the Maximum Tolerated Dose (MTD), based on developmental and cytotoxic effects, the organisms were also analysed for the induction of chromosomal aberrations. This investigation demonstrated the absence of genotoxicity in polychaetes exposed in vivo to sewage disinfected with sodium hypochlorite. These observations extend our previously published studies in which polychaetes exposed to non-disinfected sewage, while showing developmental toxicity and cytotoxicity, did not exhibit any evidence of cytogenetic damage.

Aneuploidy plays a significant role in adverse human health conditions including birth defects, pregnancy wastage and cancer. Although there is clear evidence of chemically induced aneuploidy in experimental systems, to date there are insufficient data to determine with certainty if chemically induced aneuploidy contributes to human disease. However, since there is no reason to assume that chemically induced aneuploidy will not occur in human beings, it is prudent to address the aneugenic potential of chemicals in the safety assessment process. A wide range of methods has been described for the detection of chemically induced aneuploidy including subcellular systems, tests with fungi, plants and Drosophila as well as in vitro mammalian systems and in vivo mammalian somatic and germ cell assays. However, none of these methods is sufficiently validated or widely used in routine screening. Underlying the efforts to develop aneuploidy-specific assays is the presumption that current genetic toxicology tests do not detected chemicals that have aneuploidy-inducing potential. To address this, we have critically evaluated data from standard genetic toxicology assays for 16 known or suspected aneugens. The conclusions from the review are listed below. 1. At present there are only nine chemicals that can be classified as definitive aneugens, as determined by positive results in in vivo rodent assays. 2. As expected, the majority of definitive and suspected aneugens are negative in the bacterial mutation assay. 3. The majority of definitive aneugens evaluated induce polyploidy in vitro. With few exception, they also induced structural chromosome aberrations in vitro. 4. All of the definitive aneugens that have been sufficiently tested induce micronuclei in rodent bone marrow cells in vivo. A number of these chemicals also induced structural chromosome aberrations in vivo. 5. There is no evidence for a unique germ cell aneugen, that is a chemical that induces aneuploidy in germ cells and not in somatic cells. Furthermore, an analysis of several databases indicates the proportion of chemicals which induce polyploidy and not chromosome aberrations in vitro is low. Based on these conclusions, the following recommendations are made: for screening purposes, a standard genotoxicity test battery (including an in vitro cytogenetic assay with an assessment of polyploidy and clastogenicity at the same harvest time) should be performed; in the absence of polyploidy induction in vitro no further evaluation of aneuploidy-inducing potential is needed; if polyploidy is observed, in vitro follow-up testing to investigate further the aneuploidy-inducing potential should be conducted; such follow-up testing will generally start with the conduct of a standard in vivo somatic cell micronucleus assay; if the in vivo somatic cell micronucleus assay is negative, with adequate evidence of exposure of the bone marrow to the test compound, no further testing of aneuploidy-inducing potential is needed; if the in vivo somatic cell micronucleus assay is positive, further information on mechanisms of micronucleus induction can be obtained by using kinetochore/centromeric staining in vitro and/or in vivo; an assessment of potential germ cell aneuploidy activity may then be considered; aneuploidy induction which does not involve the direct interaction of a chemical or its metabolite(s) with DNA is expected to have a threshold. This must be considered in the risk assessment of such chemicals; this is not addressed by current risk assessment guidelines.

Samples of settled (primary) effluent were collected from a municipal sewage treatment works at Newton Abbot, Devon, UK, a site which discharges primary effluent via long sea pipeline into the English Channel (minimum of 200-fold initial dilution). Sewage samples were collected during the period February-April 1995 and were analysed for standard physico-chemical parameters (ammonia, chemical oxygen demand, conductivity, non-purgeable organic carbon and settled solids). Samples were also tested for cytotoxicity, genotoxicity, and for developmental effects in the embryo-larval stages of the marine worm, Platynereis dumerilii. Exposure to sewage concentrations of > or = 10% (v/v) in seawater at 20 +/- 1 degrees C led to a marked reduction in normal embryo-larval development (7 h EC50 values from 10% to 18% v/v, n = 5). There was also evidence of a simultaneous delay in the cell cycle progression (as determined by sister chromatid differential staining) following embryo-larval exposures to sewage concentrations of > or = 10% (v/v). Following the calculation of the Maximum Tolerated Dose (MTD), based on cytotoxic and developmental effects, cells from the same embryo-larvae were analysed for chromosomal aberrations (CAs). Results were consistent for all samples tested, demonstrating the absence of cytogenetic damage following the in vivo exposure of polychaete embryo-larvae to settled sewage.

An in vivo genotoxicity test system has been developed using the embryo-larval stages of the marine annelid, Platynereis dumerilii (Polychaeta: Nereidae). This species is representative of an ecologically important group of marine invertebrates, it is amenable to laboratory culture and has a well defined and stable karyotype (2n=28) which is suitable for the analysis of a range of cytogenetic endpoints, including chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs). An evaluation of the cell cycle kinetics using the embryo-larval stages allowed selection of exposure times for cytogenetic work. Subsequently, 12-h-old embryos were exposed to reference mutagens, dissolved in sea water, in the presence of 5-bromodeoxyuridine (BrdU) for 12 h (SCE analysis) or 8 h (CA analysis) at 15 +/- 1 degree C, by which time they had reached the first larval stage (20-24h). Dose response-relationships for cytotoxicity, SCEs and CAs were observed for both direct acting mutagens (methyl methanesulfonate, mitomycin C) and mutagens which require metabolic activation (cyclophosphamide, benzo[a]pyrene). The sensitivity of the embryo-larval stages of P. dumerilii to both direct and indirect acting mutagens, their suitability for laboratory culture, together with the presence of a good karyotype and chromosome morphology for cytogenetic analyses, makes this species a potentially valuable in vivo model for marine genotoxicity testing.

Sister-chromatid exchange and micronucleus frequencies are reported for lymphocytes cultured in vitro from 15 patients receiving sulphasalazine therapy for ulcerative colitis and 15 controls matched for age and sex. While the patients did not differ from matched controls for micronucleus frequency there was a substantial rise in their sister-chromatid exchange frequency. This evidence of DNA damage is discussed in relation to the known cancer risk that ulcerative colitis carries and the possibility of an increased mutation rate in the germ cells of those receiving sulphasalazine therapy.

Two different endpoints, sister-chromatid exchange and micronucleus induction, were measured in human peripheral blood lymphocytes stimulated to divide in short-term in vitro cultures. The cultures were exposed to sulphasalazine and 6 of its metabolites for 72 h in the absence of any exogenous metabolic activation system. Analysis of the sister-chromatid exchange and micronuclei frequencies clearly indicates that sulphasalazine itself is capable of inducing both sister-chromatid exchange and micronuclei while sulphapyridine and its acetylated metabolites only induce sister-chromatid exchange. 5-Aminosalicylic acid, the therapeutic moiety of sulphasalazine, and its acetylated metabolite did not induce either sister-chromatid exchange or micronuclei at the concentrations tested. The data from these in vitro experiments are discussed in relation to the previously reported elevations in sister-chromatid exchange and micronucleus frequencies in inflammatory bowel disease patients receiving sulphasalazine therapy.