An assessment of the genetic toxicology of antimony trioxide.

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.

Aneuploidy: a report of an ECETOC task force.

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.

CI solvent yellow 14 shows activity in the bone marrow micronucleus assay in both the rat and mouse.

CI Solvent Yellow 14 has been reported to be carcinogenic in the rat, inducing neoplastic liver nodules, but non-carcinogenic in the mouse. The present experiments have extended previously reported investigations on the activity of CI Solvent Yellow 14 in in vivo genotoxicity assays. CI Solvent Yellow 14 has been examined for genotoxicity in vivo in the bone marrow micronucleus assay in both the rat and the mouse, and in the rat liver unscheduled DNA synthesis (DNA repair) assay, to limit doses of 5000 and 2000 mg/kg respectively, by oral gavage. CI Solvent Yellow 14 showed evidence of clastogenic activity in both the rat and mouse bone marrow (clear effect in the rat; weak effect in the mouse), but no evidence of DNA repair in the rat liver. In view of the latter finding, the contribution, if any, of the genotoxicity expressed by the micronucleus assay to the formation of liver nodules on chronic administration of the compound, is unclear.

Evaluation of phenobarbital/beta-naphthoflavone as an alternative S9-induction regime to Aroclor 1254 in the rat for use in in vitro genotoxicity assays.

A series of bacterial mutation, mammalian cell (L5178Y) gene mutation and in vitro cytogenetic assays were performed to compare the efficacy of using S9 fractions prepared from rats induced with a combination of phenobarbital (PB) and beta-naphthoflavone (beta NF), with S9 fractions from rats treated with the general enzyme inducer Aroclor 1254. Although some quantitative differences in the magnitudes of the mutagenic/clastogenic effects were observed between the two induction regimes, no qualitative differences were observed. The use of a combined PB/beta NF induction regime using oral dosing is therefore considered to be a suitable substitute for Aroclor 1254.

Trichloroacetic acid: investigation into the mechanism of chromosomal damage in the in vitro human lymphocyte cytogenetic assay and the mouse bone marrow micronucleus test.

Trichloroacetic acid (TCA) was tested for its ability to induce chromosomal damage in cultured human peripheral blood lymphocytes and in bone marrow cells of male and female C57BL/6JfBL10/Alpk mice. Two in vitro cytogenetic assays were conducted with TCA. In the first TCA, as free acid, was added to whole blood cultures at final concentrations of 500, 2000 and 3500 micrograms/ml in the presence and absence of an auxiliary metabolic activation system (rat liver S9-mix). Statistically significant increases in the percentage of aberrant cells compared with solvent control values were observed in cultures treated with TCA at 2000 and 5000 mu/ml. Investigation into the effects of TCA on the pH of the culture medium revealed significant reductions in pH at both these TCA concentrations. Neutralized TCA was then tested at concentrations of 500, 2,000 and 5000 micrograms/ml, also in the presence and absence of S9-mix. No statistically or biologically significant increases in the percentage of aberrant cells were observed in any of these cultures. In the mouse micronucleus test, neutralized TCA was administered in two equal intraperitoneal doses 24 h apart to C57BL/6JfBL10/Alpk mice (337, 675 and 1080 mg/kg in males; 405, 810 and 1300mg/kg in females). These dose levels represent 25%, 50% and 80% of the median lethal dose (MLD) in this strain of mouse. Bone marrow samples were taken 6 and 24 h after the second dose and the chromosomal damage assessed by analysis of the bone marrow for micronuclei. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes compared with the solvent control dosed animals were observed in either sex at the 6 h sampling time or in the females at the 24 h sampling time. A small but statistically significant increase in micronucleated polychromatic erythrocytes was observed in male mice 24 h after a dose of 675 mg/kg (50% MLD). Since no increases were noted at the 25 or 80% MLD, and the levels recorded are within the range of the concurrent solvent control values, the small increase observed in the males at the 50% MLD is considered not to be biologically significant. Flow cytometric studies on suspensions of isolated liver cell nuclei revealed that changes in FITC binding (indicating altered chromatin conformation) were induced by pH changes alone and were not caused by neutralized TCA.(ABSTRACT TRUNCATED AT 400 WORDS)

Procedures for the detection of chemically induced aneuploidy: recommendations of a UK Environmental Mutagen Society working group.

The development of assays to detect numerical chromosome aberrations has not kept pace with that for assays used to detect other genotoxicity endpoints such as gene mutations and structural chromosome aberrations, even though the importance of aneuploidy in relation to heritable defects in germ cells and to carcinogenesis in somatic cells is acknowledged. Regulatory bodies at present have no formal requirements concerning aneuploidy detection and decisions are made on a case-by-case basis. The aim of this review is to indicate which assays are available for the detection of chemically induced aneuploidy and what aspects should be taken into account when testing for chemically induced aneuploidy using in vitro, in vivo somatic and in vivo germ cell assays without dictating exact protocols. Our recommendations concentrate on systems that, to date, have been most extensively used and we indicate where future developments may lie. It is important that the currently available and future tests for chemically induced aneuploidy should be adequately validated before being implemented into screening strategies or regulatory guidelines. This requirement has not yet been met and is confounded by the lack of a well defined reference database of animal and human chemical aneugens.

Evaluation of the genetic toxicity of the peroxisome proliferator and carcinogen methyl clofenapate, including assays using Muta Mouse and Big Blue transgenic mice.

The rodent liver carcinogen and hepatic peroxisome proliferator methylclofenapate (MCP) has been evaluated for genetic toxicity in a range of in vitro and rodent genotoxicity assays. It gave a negative response in each of the following assays: mutagenicity to S. typhimurium and E. coli (+/- S9 mix, plate and pre-incubation assays), clastogenicity to cultured human lymphocytes and CHO cells (+/- S9 mix), a mouse bone marrow micronucleus assay (24h and 48h sampling), a rat liver assay for UDS in vivo (12h sampling), assays for lac I (Big Blue) and lac Z (Muta Mouse) mutations in the liver of transgenic mice, and an assay of the ability of MCP to modify the mutagenicity to the liver of dimethylnitrosamine in both transgenic mutation assays. The micronucleus and UDS assays were conducted using a single administration of MCP at its maximum tolerated dose, while the transgenic assays were conducted using nine daily administrations of MCP at its cancer bioassay dose level. These nine daily administrations were shown to double the weight of the liver of non-transgenic, Big Blue and Muta Mice, as well as leading to a dramatic proliferation of peroxisomes (electron microscopy) in the livers of each strain. These changed parameters had returned to control levels when the mutation analyses were conducted (10 days after the final dose of MCP). Despite the liver enlargement observed following MCP administration, no evidence of mitotic activity was observed in treated livers, although an increased number of cells were undergoing replicative DNA synthesis during the final 3 days of the 9 days of administration (BUdR assessment of S-phase). Liver biochemistry parameters (ALT, AST, AP, CK, GGT and albumin) were unaffected by the chronic (9 day) administration of MCP indicating an absence of hepatic toxicity. These combined observations favour a non-genotoxic mechanism of action for the hepatic carcinogenicity of MCP. The clastogenicity in vitro of the perixisome proliferator Wyeth 14,643 has been confirmed in CHO cells, but it is noted that this chemical is more soluble than is MCP. In particular, at the highest dose level at which MCP could be tested, Wy 14,643 was also non-clastogenic.

Failure of N-acetylcysteine to protect the mouse bone marrow against the clastogenicity of 7,12-dimethylbenzanthracene.

De Flora et al. (1991a) have demonstrated a marked protective effect afforded by N-acetylcysteine (NAC) to the liver and lung of rats exposed to benzo[a]pyrene (BP) by intratracheal injection. Due to the protocol used by De Flora et al., BP was inactive in the bone marrow micronucleus assay and, consequently, the possible protective effect of NAC in this tissue could not be assessed. In the present study, three daily administrations of 7,12-dimethylbenzanthracene (DMBA; 15 or 25 mg/kg/day via oral gavage) resulted in the expected increased in micronucleated polychromatic erythrocytes (MPE) in the bone marrow of male C57BL/6 mice 24 h after the final dose. Pretreatment of similar groups of mice with NAC (1 g/kg/day via oral gavage) 5 h before each administration of DMBA had no effect on MPE frequencies. It is concluded that NAC does not have a protective effect on the mouse bone marrow.

Lung cancer: political measures.

In countries where prolonged smoking of manufactured cigarettes is a widely established habit, it is responsible for about 90% of lung cancer. As lung cancer is usually incurable, even with expensive technology, the key to its control lies in prevention. World experience has shown the crucial need for government commitment, funding and action in controlling the epidemic of tobacco-related disease. It is recommended that each country establish a national council of 'tobacco or health' to coordinate a comprehensive tobacco control programme. This programme should incorporate data collection, including evaluation of specific anti-tobacco measures; legislative measures, including strong, rotating health warnings, limits on harmful substances, establishment of smoke-free areas, bans on any new forms of tobacco use, and a total ban on all direct or indirect promotion of tobacco products; health education campaigns; and taxation and price policies. The support and involvement of the medical profession is vital. Obstacles to success include the effect of advertising revenue in silencing the media, the inertia of governments and the medical profession, but most importantly the tobacco industry--the largest, wealthiest, most determined and strongest opposition to tobacco control worldwide.

Tobacco control--action and obstacles.

World experience has shown the crucial need for government commitment, funding, and action in reducing the tobacco epidemic. It is recommended that each country establish a national council on tobacco or health to coordinate a comprehensive tobacco control program. This program should incorporate data collection including evaluation of specific anti-tobacco measures; legislative measures including strong, rotating health warnings, limits on harmful substances, establishment of smoke-free areas, bans on any new forms of tobacco use, and a total ban on all promotion of tobacco products; health education campaigns; and taxation and price policies. The support and involvement of the medical profession are vital. Obstacles to success include cultural factors, the intertia of governments and the medical profession, but most importantly the tobacco industry, the largest, wealthiest, most determined, and strongest opposition to tobacco control worldwide.

Effects of strong government measures against tobacco in Hong Kong.

The government of Hong Kong grasped the political nettle of control of tobacco in the early 1980s, since when a comprehensive policy of legislation, education, and publicity, together with large increases in taxation on tobacco products, has been introduced. This has led to almost all of the population of Hong Kong having knowledge of the harmful effects of tobacco and of antismoking measures taken by the government. From 1982 to 1984 the number of people who smoked daily fell appreciably from 888 300 to 744 500, a reduction of 16%, while the number of teenage smokers was halved (from 22 600 to 11 200). Government commitment is crucial in programmes against tobacco in developing countries; without it antismoking efforts are unlikely to be successful.

Increased sister-chromatid exchange frequency in patients receiving sulphasalazine therapy for ulcerative colitis.

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.

Co-cultivation of human cell lines with synovial fluids of patients with rheumatoid arthritis.

We have interpreted the above findings as implying that the changes seen may have arisen as a result of co-cultivation, albeit accidental, of Chang cells and cells derived from RA synovial tissue or fluid. A further obvious approach to this problem is to attempt deliberately to repeat the co-cultivation of Chang cells with RA synovial fluids.