The current state of mutagenicity testing 215 Each test group should consist of at least 10-20 males if males are exposed. (Domi- nant lethal effects in females are difficult to separate from systemic effects.) A pre-treat- ment fertility test should be undertaken to check fertility of the animals used and to determine a background dominant lethal frequency. Each male is bred with 2-4 females once a week, for 8 weeks in the case of mice or 10 weeks in rats, to sample all stages of the spermatogenic cycle. Negative control animal numbers should be larger, again to provide a better data base for comparison. (The size of the negative control group can be determined by the square root of the number of treatment groups, i.e. if there are 9 treatment groups each of 10 animals, then there should be 30 in the control group (88).) The statistics used in the dominant lethal test are complicated various statistical methods can be used, each with its own bias. The greater the number of statistical methods used, the greater the chance of producing a false positive result (Anderson, unpublished). Different evaluation methods are used for dead implant data. The US Food and Drug Administration has analysed all data on dead implants in two ways (both on dead implants/total implants/female basis and on dead implants/female) and infer that equally significant data are provided by both (89). If late deaths are eliminated from the analysis of the above parameters a more sensitive index of dominant lethality is obtained. Preimplantation losses can be indicated by comparing values of total implants in females mated with treated males and those mated with control males, as suggested by Epstein (90) instead of counting corpora lutea. Decreases in total implants which re- present increases in preimplantation losses without a corresponding increase in early deaths may not represent a mutagenic event. Other than genetic factors can explain a preimplantation egg loss. Fertility effects can also be determined in the dominant lethal test if fertile animals only are used for the study. The dominant lethal test is best con- ducted with at least three concentrations of test compound in order to try to obtain a dose-response relationship from which extrapolation of risk might be made. Clear-cut genetic effects are best claimed when a dose dependent increase in post-implantational foetal deaths is evident. Other methods such as the heritable translocation test detect transmitted damage and are useful is this respect. However, such a test is extremely expensive, as is the specific locus gene mutation test, requiring very large numbers of animals, extensive housing and maintenance. A negative result in any one of the systems so far discussed may mean that the wrong species of animals or microsomes has been used, or a result may be negative for any one of the reasons discussed earlier, or it may be a true negative. Human Peripheral Lymphocytes from Exposed Workers This sort of study is usually carried out retrospectively after a compound has been identified as a hazard, witness the many publications on workers exposed to vinyl chloride (91). Before such a study can be undertaken. many ethical problems have to be taken into consideration, e.g. if the exposed work-force need to be told the results of the study and what the results mean in the light of current knowledge. Such a study and decisions relating to it involve negotiations with medical officers, workers, unions, etc. Negotiation may be more difficult on a prospective basis where compounds present an unknown hazard. If a positive response is obtained, while it is possible to show a cor- relation between exposure and level of abnormal cells on a groups basis, the range of

216 Diana Anderson individual values within groups of exposed people is wide and overlaps the ranges of adjacent or control groups. In retrospective studies where exposure has occurred for up to 20 or 30 years, it is often difficult to know exact exposure levels and exposure can often be only estimated roughly from occupation. We are investigating whether such a study is useful in trying to determine safe exposure levels. If the exposed population is not significantly different in terms of chromo- some damage from the control population, then it might be assumed that safe exposure levels have been achieved, because a negative result suggests that the chemical concerned is not a mutagen. However, there are limitations to this approach, since the exposure level may be too low to produce the chromosome damaging effect but may still cause sister chromatid exchanges or gene mutations which are not detected by conventional chromo- somal analysis. Controls should be taken from both on and off site where possible, and should be age and sex matched. Cells are generally cultured for 48 and 72 h and we have found no significant differences in data at these two times after vinyl chloride exposure (91), although after irradiation this is not the case (92) and 48 h cultures are desirable. At least 100 cells per individual should be analysed from slides coded to avoid observer bias. Whilst there is a correlation bteween carcinogenesis and mutagenesis (earlier references), a review paper by Hamden (93) puts the correlation for clastogenic and carcinogenic effects into perspective, as does the book edited by German (94). There appears to be a fairly good but non-quantitative correlation. Other techniques such as sister chromatid exchange may be useful on exposed workers, but effects are much more short-lived (95, 96). and may to some extent have disappeared before culturing is possible. Once it is established that a chemical is clastogenic, regular population monitoring of the work force may be initiated, in which the workers are monitored both pre-exposure and during employment The results of the monitoring will be useful for checking plant hygiene, and an increase in absormal cells in an individual could be used as an indication that the worker should not continue to be exposed to that chemical. If an individual is found to have a chromosomal abnormality linked to a certain disease he can be advised through appropriate medical channels of the risk of inheritance of the disease in any children he may have. If all workers in chemical plants were monitored as part of a routine medical surveil- lance service, then many of the difficulties involved in initiating prospective studies would disappear. Computerised microscopic techniques are available which can reduce the time spent by a technician by about 40•o. At present, however, metaphase spreads are merely located and the amount of damage still has to be assessed visually. Initial cost of purchas- ing a computer microscope is very high. Other techniques for direct application to man are available, such as the use of urine or blood plasma from exposed workers in combination with a microbial assay (97), electrophoretic monitoring of enzymatic markers in man (98), detection of variants in haemoglobulin molecules (99-100), investigations of sperm morphology (101) and an increase in the presence of YY bodies (102). INTERPRETATION OF DATA Assuming that experiments are reproducible, in well-conducted experiments using