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.

Chromosomal aberrations in vitro induced by aneugens.

Various aneugens were reported to induce structural chromosomal aberrations beside their influence on cell division and their aneugenic potential To asses, whether a relationship between disturbance of cell division and clastogenic potential exists, CHO cells were treated with the well-known aneugens colcemid, colchicine and vincristine and investigated for the induction of structural chromosomal aberrations, polyploid cells and alterations in mitotic index. At low and intermediate concentration, all compounds induced polyploidy and an increase in mitotic index, but no structural aberrations at all. However, at high concentrations, colcemid and colchicine both induced numerous structural chromosomal aberrations in diploid cells. Colchicine was also clastogenic in tetraploid cells. Vincristine did not induce structural chromosomal aberrations in diploid cells, but in tetraploid cells. The clastogenic effects showed a clear-cut threshold with all three compounds. Furthermore, it was found that the tetraploid condition in CHO cells is generally accompanied by an increase in structural chromosomal aberrations, in vehicle controls as well as in cultures treated with the aneugens. Nevertheless, this study demonstrates that for the three aneugenic compounds tested, no direct relationship between compound induced disturbance of cell cycle and compound induced structural chromosomal aberration incidence exists.

Assessment of the potential germ cell mutagenicity of industrial and plant protection chemicals as part of an integrated study of genotoxicity in vitro and in vivo.

An approach is described that enables the germ cell mutagenicity of chemicals to be assessed as part of an integrated assessment of genotoxic potential. It is recommended, first, that the genotoxicity of a chemical be defined by appropriate studies in vitro. This should involve use of the Salmonella mutation assay and an assay for the induction of chromosomal aberrations, but supplementary assays may be indicated in specific instances. If negative results are obtained from these 2 tests there is no need for the conduct of additional tests. Agents considered to be genotoxic in vitro should then be assessed for genotoxicity to rodents. This will usually involve the conduct of a bone marrow cytogenetic assay, and in the case of negative results, a genotoxicity test in an independent tissue. Agents found to be non-genotoxic in vivo are regarded as having no potential for germ cell mutagenicity. Agents found to be genotoxic in vivo may either be assumed to have potential as germ cell mutagens, or their status in this respect may be defined by appropriate germ cell mutagenicity studies. The basis of the approach, which is supported by the available experimental data, is that germ cell mutagens will be evident as somatic cell genotoxins in vivo, and that these will be detected as genotoxins in vitro given appropriate experimentation. The conduct of appropriate and adequate studies is suggested to be of more value than the conduct of a rigid set of prescribed tests.

Automated modification of the Ames test with COBAS Bact.

The automatic analyser for microbiology "COBAS Bact" from Roche, supplemented by a Hewlett-Packard 9816S is used to automate the Salmonella mutagenicity test, developed by Ames. More than 30 compounds were tested in this system and the results were shown to be in good agreement with those obtained with the standard Ames test.

Criteria for the standardization of Salmonella mutagenicity tests: results of a collaborative study. II. Studies to investigate the effect of bacterial liquid culture preparation conditions on Salmonella mutagenicity test results.

The influence of various parameters and growth conditions in the "overnight culture" of Salmonella typhimurium strains on mutagenicity test results was investigated. A number of factors were first suspected to be of some importance for the quantitative outcome of the mutagenicity test. None of them, however, was found to influence the results to such a marked extent as to be a major source of variability. Only the brand of nutrient broth used for the propagation of the bacteria proved finally to have a certain effect on the number of (spontaneous and induced) revertant colonies, although no precise and quantitative statements can be made with regard to a possible standardization of this experimental segment in the Salmonella mutagenicity test. The occurrence of such unpredictable but noticeable influences is, however, evidence for the importance of an intralaboratory optimization and standardization of all parts of the test procedure.

The emission of corrosive vapours by wood. Sweet-chestnut (Castanea stiva) and wych-elm (Ulmusglabrau) O-acetyl-4-O-methylglucuronoxylans extracted with dimethyl sulphoxide.

1. O-Acetylated 4-O-methylglucuronoxylans were isolated from sweet chestnut and wych elm, either green or incubated at 48 degrees and 100% relative humidity for 36 weeks. 2. The chlorine-ethanolamine method of delignification resulted in a 50% loss of O-acetyl groups from green wych elm compared with an 18% loss from green sweet chestnut. 3. The acid-chlorite method gave an acceptable loss of O-acetyl groups in three cases, but incubated sweet chestnut showed a 44.6% loss. However, it is believed that this is due to the loss of simple O-acetylated xylose sugars resulting from glycosidic hydrolysis, rather than removal of O-acetyl groups by direct hydrolysis. Assuming that this occurs in a random manner, it is unlikely to have much structural significance. 4. Dimethyl sulphoxide extraction of chestnut holocellulose and elm holocellulose, green and incubated, yielded O-acetyl glucuronoxylans containing 10.2, 3.8, 13.1 and 7.7% O-acetyl groups respectively. 5. The location of these O-acetyl groups was determined by Bouveng's method in which phenyl isocyanate is used as a blocking group.

The emision of corrosive vapours by wood. Hot-water-extracted O-acetylated hemicelluloses from sweet chestnut (Castnaea sativa) and wych elm (Ulmus glabrau) and a discussion of O-acetyl-group changes occurring in these woods during incubation at 48 degrees and 100 per cent relative humidity.

1. O-Acetylated polysaccharides were obtained from green wood of both sweet chestnut and wych elm by treatment of the residue remaining after dimethyl sulphoxide extraction with water at 98 degrees . This gives a mixture of polysaccharides containing xylose, galactose, glucose and uronic acids. Analysis of these and their fractionated products suggest that only xylans in green sweet chestnut and green wych elm are O-acetylated. 2. The isolated O-acetylated xylans are not representative of the total O-acetylated xylans occurring in sweet chestnut and wych elm. 3. Application of the method developed by Bouveng for the location of O-acetyl groups to all four O-acetylated xylans obtained in this series of investigations by dimethyl sulphoxide extraction showed that those from sweet chestnut and wych elm, under the same conditions of incubation, lost: 74.2 and 43.4% of acetyl groups respectively, at C-2; 58.0 and 28.5% of acetyl groups respectively at C-3; 41.8 and 82.2% of acetyl groups respectively at C-2 and C-3. 4. A consideration of electronic and steric factors indicates that there does not appear to be a purely chemical reason for the difference in loss of O-acetyl groups between sweet chestnut and wych elm. It is suggested that the location of O-acetylated xylans in the wood cell walls and the presence of extractive may play some part in this difference.