The current state of mutagenicity testing 213 reproducible response. Simpler 'spot' tests measuring the degree of differential killing in repair-deficient microbial strains by comparison with wild type strains (64, 65) provide an indirect answer to the problem of mutation testing, since they detect damage to DNA but do not measure gene mutation directly. Such tests at the present time are not con- sidered particularly sensitive, however. MAMMALIAN CELL SYSTEMS Cell 'transformation '/mutation assay In this laboratory, concurrent with the Ames' test the 'cell transformation' assay system of Styles is used (8, 66). It is based on the ability of mammalian cells transformed by a carcinogen to form colonies in soft agar and this is only one of the accepted criteria for cell transformation. A recent paper (67) suggests that the ratio between transformation and mutagenesis for ouabain resistance in normal diploid cells is about 20: 1 (after treatment with benzo(a)pyrene and its 7,8-dihydrodiol), which suggests that any one of 20 genes may be involved in transformation as opposed to 1 for mutation. This ratio is also apparently substantiated for normal hamster embryo cells treated with benzo(a)- pyrene. If transformation is, therefore, a genetically based system, it may be useful for preliminary genetic monitoring. We have found it to be equally predictive for carcino- genicity as the Ames' test, and by inference, therefore, it is equally predictive for mutas genicity. Baby hamster kidney (BHK 21/C12) and either human diploid lung fibroblast- (WI 38) or human liver cells (Chang) were treated as described (47). Five doses of com- pound were used with the S-9 mix of the Ames' test. Survival was assessed independently and a transformation frequency calculated. The test is not as rapid as the Ames' test, but in our 'blind' study for 120 compounds with these two tests in combination the tests only missed detecting as carcinogens/mutagens diethylstilboestrol and vinyl chloride. This latter compound was later detected when tested in the gaseous phase. This therefore seems a very promising system for a preliminary mutation screen. The limitations of using these two test systems for a simple screening programme have been discussed elsewhere (8). Lymphoma Cells in Culture Whilst human cells or normal diploid cells are obviously desirable for mutation assays in culture, they are more difficult to handle from a screening viewpoint than malignant cells in culture due to their low plating efficiencies and lack of perpetual proliferation. Lymphoma cells, which grow in suspension, are even easier to handle than cells which grow as a monolayer. They do not require trypsinisation, are very easily subcultured and they are not subject to metabolic co-operation which can cause a loss of mutants. Also they can be used in a host-mediated type assay (68) or in a fluctuation test (69). Lymphoma cells in culture can readily detect direct-acting mutagens and carcinogens (70-78) and may also be used in combination with S-9 mix to detect indirect acting muta- gens and carcinogens (Anderson, unpublished). The lymphoma cell mutation system is manageable in that induced mutation frequencies are readily detectable and vast numbers of plates do not have to be used to detect a spontaneous frequency (e.g. 105 cells per petri dish in soft (0.3 •o) agar and selective medium gives a background frequency of about 2-20 colonies in P388F cells with selective media containing 5-iodo-2-deoxyuridine and excess thymidine respectively). Induced frequencies can increase from 10- to 100-fold above these

214 Diana Anderson levels. Absolute increases in induced colony numbers are observed over some of the dose range above control values- which is not always the case for human cell systems, where mutants may be very sensitive to the inducing agent and are being killed more rapidly than non-mutant cells (79, 70). Bone marrow in Mammals This is a useful system in that no auxiliary metabolising system is required, animals can be dosed with compounds directly and bone marrow cells do not need stimulation to divide since they have a high proliferative cell activity (80-83). Rats are suitable species to use but others will do. With rodents, groups of 5-8 animals, 8-10 weeks of age, should be used per concentration of the test substance and positive control substance. A larger number of animals should be used in the negative control group to provide a better data base for comparison. A minimum of 50 cells, preferably 100, from each animal should be analysed from coded slides to avoid observer bias when scoring chromosome damage. The higher the background frequency, the higher the number of cells needed to be examined in order to detect a statistically significant effect (Buffier, personal com- munication). A maximum tolerated dose of compound should be used, together with a more realistic dose related to human exposure and an intermediate value. In a preliminary screen only the maximum tolerated dose need be used - or even a micronucleus test may be used (84, 85). Animals can be sacrificed at various time intervals after exposure. From our own studies and those of others (86, 81, 60) 6 h seems to be suitable time after multiple chemi- cal exposures and 24 h after single exposures. However, these times may not be suitable for all chemicals. The most relevant route of administration of the compound should be used. If man is exposed to the chemical by inhalation, then an inhalation route should be used. If he is likely to ingest a pesticide sprayed on crops, then an oral route is recommended either by gavage or in the diet. Rarely does man receive intraperitoneal or intravenous injections of compound except in the case of some medicines. Positive control animals should be housed under identical conditions to those dosed with the test compound of animals even if identical routes of administration of test com- pound and positive control substances cannot be achieved. Cells should be scored for all type of chromosome aberrations including chromosome gaps. We have shown with several positive control mutagens such as ethyl methane- sulphonate, mitomycin C, benzene and vinyl chloride that gaps are at least as sensitive an indicator of damage as other types of damage: they may indicate a toxic event as opposed to or in addition to a genetic event (Anderson and Richardson unpublished). Dominant Lethal Mutation Assay This is one of the whole mammal tests on germ cells (87). The tests should be carried out on random-bred rodents of 8-10 weeks of age. The comments which apply to dosing regimes in bone marrow cells also apply to dominant lethal tests, i.e. a maximum tolerated dose should be used together with a dose related to human exposure levels and an inter- mediate value. Again, the most relevant route of administration should be used, and animals treated with the positive control substance sould be housed under identical conditions to those of animals exposed to the test compound.