Document 91VVVYwdom01brEyR5aRRr5xp

Warure V0l 261 'lay 20 1926 m review article Short term screening tests for carcinogens Bryn A. Bridges* There are now short term tests with a high predictive valuefor mammalian carcinogens. Many of them are based on the ability to detect damage to DNA in bacteria or mammalian cells after metabolic activation by microsomal enzymes. Their introduction will enable provisional safety assessments to be made for the many thousands of industrial and environmental chemicals for which long-term animal testing cannot at present be considered. It has been estimated' that if one could totally abolish human cancer tt would add a mere two yean to the average lifespan. Most cancer tufferen art past retiring age so that industrial production would be little affected by the abolition of cancer. The fight against cancer must instead be justified in terms of the cost of hospital services and of basic humanity; treatment of cancer, even when it is successful, is a miserable process. When it fails as it so often docs, one feels guilty of a double offence, not only the loss of the patient, hut the imposition of heroic measures that them selves may cause conviderable physical and mental suffering. The International Agency for Research on Cancer holds it as a rule of thumb that around S0% of cancer baa an environmental cause' ': others would give a higher figure'. The evidence is indirect, being based on differences is tumour incidence between genetically similar populations in different environments1 *'. Even if this estimate is only approximately correct it leads ineluctably to the conclusion that a substantial proportion of cancers, possibly s majority, are in principle preventable. In past decades those responsible for the disbursement of cancer research funds have tended either to look for s breakthrough in the area of curative treatments or to make a long term investment in basic biology in en attempt to understand the daease (or more properly diseases since "cancer" is but a general term for hundreds of different malignant conduces). Recently, however, these two essential approaches hero been aimple mented by a third, the search for the specific environmental factors involved in caronofsaeris. The nature of these environmental factors is net known in detail, but it seems likely that many of them are man made or natural chemicals. Even factors such es diet or stress may act indirectly by altering the metabolism of chemicals in the gut or in the body uacif. Of course, identification of environmenial carcinogens does not necessarily lead to their removal but it does open the way to control so that the risk that they prrisni is no more than is necessary when weighed against any nm they may give. The most direct method of identifying environmental carcinogens for man is based on population studies, but unfortunately it is expensive and seems to have tether low resolving power. Only a handful of chemicals are known to be carcinogenic to man and moat of these have been detected following the study of watfcsn occupationally aSSTiTM**?sux u""'UM,,nh, *%mu- exposed to chemicals capable of giving rise to specific and rather rare neoplasms. The classic case is soot which has been known for 200 yean to produce scrotal cancer in young chimney sweeps'. More recent examples are 2-naphthylsmine. vinyl chloride and asbestos which produce, respectively, rare cancers of the bladder, angiosarcomas of the liver, and mesotheliomas of the lung cavity. The problems involved in identifying two populations differing only in their exposure to one chemical are formidable and are further compounded if the chemical gives rise not to specific and otherwise me tumours, but to a variety of common cancers. Population studies are thus likely to be of limited value in identifying environmental (as distinct from occupational) carcinogens but they will be indispens able in providing the basis for risk evaluation, particularly where dose-response data can be obtained. The alternative is to screen chemicals to which man is exposed. The generally accepted method of doing this is to cany out long term carcinogenicity tests with laboratory mammals. Not only are those tests very demanding of resources but any extension of loans] testing on tucb s wide scale would be vigorously nppamd by a number of animal welfare lobbies, la practice, it is mcoocetvaW* that rtsourom could be mods icallable (sitter men, money or mice) on the necessary scale to screen all the tens of thousands of sutetsnees to whieh humans are exposed. Of necessity, therefore, testing with whole mammals will be restricted to certain groups of suspect substances, for example ttaee suspect but already in use en s large teak, or those substances which it is proposed to administer on a large scale, as food additives or ceaetks. for instance. If one is to screen for carcinogenic cternseals, therefore, one must use short term tern with a high piadhaive value. I propose to review a number of possible systems which have been aiuaiiiil in recent yean. A* wiB become apparent, many of them are in fact systems for the detccrioa of agents causing damage to DNA. Damage to DNA leading to heritable changm may te important to mao not only became of camnogasucity but herante it may cams hereditary disease'"'. Moreover, DNA damage may eonccivabty te involved is xpeasg and diaeaim associated with ageing'. I take it m self-evident that any agent likely to damage the DNA of man. whether in somatic or germ ceils, is potentially temrdoua. Screening system The induction of cancer is but one aspect of long term loxtaiy and for the evaluation of such haxards a thrtc-tier I f I \ r tt r/! ucc 007935 ^3 BFG61958 m approach has been proposed" " The first tier would consist of simple short term sub-mammalian tests with u high predictive value for the human effect ultimately jf interest. As many substances as possible should be screened with these tests. Second-tier tests would he both short and long term, on mammals. Only selected, high priority sutwcances would be screened by these tests in addition to the hrst-ner tests Tests in the third tier are designed not to detect toxic agents but to evaluate as quantitatively as possible the hazards to man from agents shown to be potentially tOXIC. Only substances whose use or presence seems inescapable would be subiect to the third tier of evaluation, the obtect of which would be to make a risk-benefit assessment and institute appropriate regulatory action. Tests in successive tiers show in principle increasing relevance to man but this is often accompanied by decreasing sensitivity and practicability. Not all of the sub-mammalian tests depend upon the postulated electrophilic nature of the active forms of carcinogens and in particular on their ability to react with DNA. Williams and Rabin", for example, have proposed that substances might be screened using a test based on membrane-polysome association. They found that a number of carcinogens caused degranulation of rough endoplasmic reticulum (microsomal membranes) from male rat liver. This test has been further developed by Purchase and Lefevre" who have measured the loss of radioactive RNA from rough endoplasmic reticulum. Preliminary results (D. Anderson tt a!., unpublished) with a large number of carcinogens and non-carcinogens indicate that the method predicts the activity of arylamtnes rather well fS5% correct) although tt is lest successful with polycyclic hydrocarbons and direct acting alkylating agents. The necessity for metabolic activation of many carcino gens hy microsomal enzymes prompted the suggestion of McPherson et ul." that the specific in vitro enhancement of biphenyl 2-hydroxylation activity in rat liver microtome preparation might be used as a screening test They found that of eight known carcinogens, all caused an increase of around 100% in such activity, four compounds whose carcinogenicity is in doubt gave lower but significant increases, and eleven non-carcinogenic compounds gave no significant increase. This test system, like that of degranulation of ribosomes, is obviously promising and in need of a much more exhaustive validation on a scale similar to that used with some ocher systems. Metabolic activation There is s widespread belief among cancer workers that DNA damage is involved in the induction of cancer. That is the basis for tha supposition that carcinogens might be detected by the consequences of DNA damage in simple systems. Two recent developments have enabled this possibility to be realised. First, it has become dear that many carcinogens arc the products of metabolism of inactive chemicals by mixed function niirtssei in the animal", and that preparations of liver mietoaomes can be used in vitro to carry out this metabolic activation1-". Second, ultra sensitive bacterial systems, usually involving strains deficient in DNA repair, have been developed for the detection and characterisation of agents musing damage to DNA'--". The Ant published work in which the mutagenic activity of metabolites was detected sfter metabolic activation of carcinogens was by Mailing". The methodology of his quantitative liquid assay system has been recently described". Later. Ames tt ol.m showed that tnicroaomcs could be added to the semi-solid agar overlay in a plate test, a procedure that is in some way* rather better for routine screening although it fails with a few compounds, for example, dimcthylnitrosamme. possibly because the agar interferes with the diffusion of short-lived active metabolites. Bacteria deficient in repair of DNA arc killed more Santrt Vot. .'0/ Way 20 .y-'j tisily bv DN A-damagtng agents than are wild type bacteria, and this is the basis for several simple tests. Bacteria deficient in excision repair have been used" but these are sensitive only to certain types of DNA damage. Much more useful have been bacteria lacking DNA polymerase I or deficient in genetic recombination iRec')" These tests are usually conducted on the surface of agar plates but are also amenable to rather more quantitative procedures with liquid-pnase treatment" " Another way of revealing the existence of DN A damage is to look for the repair chat it usually mutates and this ts the basis of a very useful test developed by Stich and his colleagues. It depends on estimating the amount of DN A synthesis involved in repair by measuring autoradiographically the uptake of trmated thymine during the period immediately following exposure to the test chemical. The method has the advantage that it can be used with cultured human skin fibroblasts. To prevent normal DNA synthesis the cells are kept in an arginine deficient medium for 3 d before exposure. In a report on 6* substances tested. Han and Stich" found that all directly acting carcinogens elicited unsche duled DNA synthesis whereas no repair synthesis was observed after treatment with 16 non-carcinogens. Most carcinogens known to need metabolic activation gave nega tive results although a few were active after prolonged exposure to high concentrations. More recent results41 indicate that metabolic activation systems can be incorpor ated in this assay and make possible the detection of procarcinogens. Mutation induction Perhaps the most sensitive assay for DNA damage is the induction of mutations in bacteria, particularly ir the bacterial strain carries a mutation rendering it unable to excise damage from DNA (Uvr'). Excision-proficient strains should always be included in any assay, however, because certain agents able to cross-link DNA are only mutagenic in such strains"; presumably the mutational event occurs as an error during excision-initiated repair. Reversion to prototrophy is generally regarded as the most sensitive type of asssy and the methodology has recently been reviewed"'". Etchrrichia coii WF1 is a tryptophanrequiring strain that responds to mutagens causing base-pair substitution mutations at both adenine; thymine and guanine: cytosine sites. A more complete set of tester strains has been developed in SabnontOa lypMmumim by Ames and collaborators" Individual strains respond to base-pair substitution mutagens or to compounds causing various types of frameshift. The permeability of these Sotmontfl* strains to some chemicals has been increased by the incorporation of a cell wall mutation ("deep rough")*1. From recent data one can calculate that these strains an capable of detecting muta genic activity of between 61%" and 90% (D. Anderson, unpublished) of known carcinogens. In an attempt to detect the ''false negatives" obtained with the deep rough strains. Ames's group developed a fourth generation set of strains containing the drag resisiance plasmid pKMIOl". as suggested by MacFhee certain plasmids confer a mutator activity on their host ceil which becomes more sensitive to many mutagens and carcinogens". The ability of these plasmid-contauung strains to detect carcinogens as mutagens is impressive (sec below). A word of caution is in order, however, since although the mechanism by which the plasmids act is still unknown, it is dear that they convert into mutations damage which would not hr muugenic in a normal cell. They may even act as amplifying systems and product mutations at rites where no damage exists. The value of such strains lies in the correlation they show with carcinogenicity but there is at least a theoretical possibility of real "false positives'' ucc 007936 BFG61959 25013108 }1 apprnimal carcinogenieit* and bacterial mutagenicity wiin and of prr A up of compounds Carcinogens detected as bacterial mutagens . amines etc. ..aiidcs. etc /Cyclic aromatics epostdes. caroamam. etc. _ Nitro aromatics anu neteroeycles F Miscellaneous organics O Nirrosammes H Fungal toxins anl antibiotics l Mi%turesteiir*tte*mo*econdemaie> J Miscellaneous nctenxydes K Miscellaneous nitrogen compounds L AzoUyesJftUUut/o compounds M Common laboratory bnichcmicals Tout 23/25 t?'I0 :/27 13-18 :s:s 16 :o/2t d/9 l/l 1(4 719 l I/I i - 137/17* Sature Vo! HI .Vfey 20 lift metabolic activation 'from McCann tt oi *i Non-carcinogens not mutagenic to bacteria Compounds of uncertain carcsnogentcjty UetecteC as mutagens 10/12 1.3 :;9 S/9 1/4 13/13 :j2 5-5 - 7/7 2-4 2/3 46.46 101/11 T 5.7 U ;i O'! 0.2 0't li - - - 3-3 - 11/17 non is only as good as the confidence one has in the reliability of both parameters. Whereas positive and negative mutagenicity results can be both unambiguous and repro ducible. the same is not true of carcinogenicity remits where, as will be discussed below, there are several factors which could result in a failure to detect relatively weak carcinogens. As discussed by McCann and Ames**, there is good reason to believe that many of the "false" positive chemicals will eventually be shown to be carcinogenic. This has already happened with the food additive (uryl futamide which had been used for many yean in Japan and had given negative results in two carcinogenicity trials'*. After positive results had been obtained in Bacillus subtiiis end . eoli systems for detecting DNA damage, it waj re examined and shown to produce a low hut significant yield for tumours when given to foetal and young mice**. There is also the real possibility that some of the "false" negatives are genuine, that metabolism m vivo is different from that with isolated microsome preparations. Only further studies in depth can resolve this. It is worth analysing the data of McCann er of., further to see whether there is any particular type of mutational event (as detected hy the Salmonella) that is correlated with carcinogenicity. It has been postulated*' that carcinogenicity is associated with the ability to produce specific types of frameshift mutation. This hypothesis docs not hold up in any general application. As can be seen from Table 2. whereas most members of some groups of carcinogens (for example, amoratic amines, polycyclic aromatics and nitroaromatics) gave nsc to bods frameshifts and base-pair sub stitutions. others (for exampie. esters, epoxides and carbamates, mtrosamuses, (nacelleneous nitrogen com pounds) gave rise exclusively to bass pah substitutions. There was no group that gave tin exclusively to frameshirts. Taken together. 43.2% of mutagenic eardnogena gave rise solely to ban pair fliuiloni. I4.g% soiciy to frameshifts, and 40% prm rim to both. Roienkrartx (cited in ref. 42) usntf a PoC strain, of E. soil together with the Salmtmail* .set without plasmids. has obtained results as encouraging *s those of McCann et of. with the plasmid-containing satmoneflax. Of about 100 com pounds tested. 151 of the known carcinogens were detected (91% of direct acting carcinogens. 72% of proeamnogens). The proportion of now-cxrcinogtni delected as positive was rather high. 30%. but the figure it not comparable with the lower value derived from the data of McCann tt of." since it did not include the 44 common laboratory biochemicals tested by the latter workers, none of which was positive. A comparison of the efficiency of various microbial systems for detecting DNA damaging agents has been carried out by Shirasu tt of ". They found that the hyper sensitivity of repair-deficient bacteria (Rec* B. submit) eras the most sensitive. Of 164 pesticides studied. 23 were pas ture in the Rec-essay (carried out without microsomal activation). Of the 143 negatives, none proved to be positive when tested with . coil or Salmonella reverse mutation systems. Of the 2J positives 9 were positive in reverse muta tion systems, and of these 9, I was not detected by the . eoli strains and 1 by the Salmonella strains. As far as base-pair substitution mutations are concerned, the non plasmid . eoli strains were found to be preferable to the non-ptaamid Salmonella strains at least with some groups such as nttrofurans. With other groups such as the organic phosphates a similar small proportion of mutagens was missed hy both S. typhimurium and . eoli strains". The only study in which a single laboratory has compared a number of different tests for predicting carcinogenicity appears to have been carried out by the Central Toxicology Laboratory of ICI (D. Anderson et of., unpublished). The preliminary results with 120 chemicals point io the value of the bacterial .mutation tesa when metabolic activation is incorporated. The carcinogenicity, or non carcinogenicity was accurately predicted for 90% of the chemicals by this test. Ceil transformation in vim came dose with S3% accuracy. Rather less accurate was d(granulation of endoplasmic reticulum. 72%, and morphological changes following subcutaneous implanta tion. 70% correctly predicted. Sehtceous gland suppression" was good for polycyclic hydrocarbons (90%) but little better than random for other substances (32-42%). Tetmolium reduction in mouse skin was eiso poor (42% overall). The authors conclude that soma of these rapid tesa ire capable of distinguishing between carcinogens and non-caranogens with sufficient accuracy to enable them to be used for selecting potential carcinogens. They also make the point that figures for successful prediction must be treated with TaMe 2 Number of caianogms diwetsd as beoeriai mutagens (with or without metabobc scuvanoni dauified as to type of mutation induced. 1m*mr Fwneshifti Both baae*oir tuOetmioora only only substitution and franashifi muiaoons A c* D E P o H 1 J K L M Tool 1 14 7 13 3 1 19 0 0 0 7 1 0 70 9 2 J 0 0 0 0 3 1 1 0 2 0 23 13 1 14 0 23 0 0 5 0 0 0 6 0 42 * Oau nor available (or one member. Key (or minwsI poups as for TaMe I. (From McCaiui tt el.") ucc 007937 GuTGTCTIo BFG61960 Sature Voi. 26t \toy 20 1976 It u possible to improve sensitivity by altering the methodology, and a modified fluctuation test has been proposed which achieves between ten and one hundredfold greater sensitivity rhan the conventional assay without the need for plasmid-containing strains" There is also the russibiiitv of developing a single tester strain that can be used to detect many different types of mutational events" Although hnererul screening systems have proved very useful, there is soil scope for further improvement. As well as the bacterial tests that have now been ettensiveiv studied, a large number of other techniques can be used to detect DNa damaging activity and may perform a useful supplementary role, probing ambiguous or suspect results and characterising more fully the nature of the genetic damage. One may. for example, study the induction of mutations tn cultured mammalian cells'*"'. Mammalian cells may also be used for cytogenetic study of visible chromosome aberrations". Recently developed staining techniques for demonstrating sister-chromatid exchanges show a greatly enhanced sensitivity" and their role is screening has been recently discussed**. Sister-chromatid exchanges may now be detected in spermatogonia" and bone marrow cells" following exposure of the whole animal to carcinogens. It is already clear, however, that although the induction of sister-chromatid exchanges is a very sensitive response to some carcinogens it occurs hardly at alt with others". Other eukaryotic mutation systems include fungi, yeasts and insects. All of these have their own advantages and disadvantages. Malignant transformation in cultured ceils Rather than develop a model system depending on mutation or DNA repair, others have worked towards a screening method by which transformation to the malignant condition could be brought about and detected in ceil culture. The only really valid criterion for malignant transformation is the ability of a cell to produce a tumour when inoculated into an appropriate host. There are. nevertheless, several secondary criteria (discussed by Freeman and Huebner**. of which the most commonly used is the ability of cells to grew into clones in soft agar or. in the case of fibroblast cultures, to produce clones of piled-up cells when growing on a solid surface. Most human cancers arc carcinomas which arc derivad from epithelial cells. Relatively little work has. however, been done on the transformation in rim of epithelial ceils. Such cells art usually obtained from tat liver end are not easy to retain in culture in the differentiated state. Never theless they have been succeratoRy transformed hr 4-nicraquinaline-l-oaide". aflatoain &. V-hydrexy-2acetylaminofluorcne. and 7.H-dimsthylbenx(e) anthracene", dimcthylnitrosamine and iV-methyl-N'-nltro-N-nitrosofuanv cells transformed in vitro uwaQy show no altered morph ology although they may grow fat soft igv. Work on ftbrebiares is considerably more advanced. Fibro blasts. when transformed, give rise to sarcomas. responsible for a minority of human malignant disease. They show, nevertheless, great premiss as the basis of a potential screening system for chemical carcinogens. As with bacterial mutation systems. H has often been foond necrerery to supplement the fibreblast's relatively poor ahffity to metabolise carcinogens into their active form. This has been achieved, either by recultivation with other cells capable of carrying out metabolic activation" or by ooiaring the cells (rent hamster embryos after treatment of the pregnant mother". Much of the work on transformation has been carried out with treatment of mass cultures often for long periods of time and has been subject to some criHeiim Trans formation has. however, been reported with then treat ments followed by cloning" and this would seem to be a irr better approach to adopt in future. A noteworthy feature of much work with both fibroblast and epithelial systems has been the high spontaneous rates of transformation, sometimes considerably higher than one would expect for a gene mutation. Many workers regard this as the result of the artificial environment in which the cells are cultured, and have looked (often successfully) for conditions in which the spontaneous rate ts lower. Nevertheless it is likely that the rate of transformation in vivo is higher than has been thought and that the oody is normally able to deal effectively with the aberrant cells. If this were true the role of ONA-damaging carcinogens might be seen as increasing the already high spontaneous rate and thus overloading the ability of the natural defences of the body to cooe with malignant cells. Most of the work on transformation in vitro has con centrated on the development of systems that can be used as models for the study of carcinogenesis (for reviews see refs 55 and 561. To the uninvolved observer, a certain amount of contradiction and inconsistency is apparent. There is. notwithstanding, an impression that this tech nique 'may soon be a valued constituent of the battery of techniques for detecting carcinogenic and DNA-damaging substances. Two recent studies have shown successful pre diction of carcinogenicity almost as good as that of the bacterial mutagenicity tests"1*. Validation Some of the test procedures described above ere very sensitive, but how good are they as predictors of carcino genicity? Recent reports have presented" and discussed" carcinogenicity and mutagenicity data obtained for more than 300 chemicals. All die results come from the use of Sdmotitila strains developed by Ames. Where negative mutation results hid beta obtained with now-obsolete strains, the test was repeated with the latest plasmidcontaining strains. Testing was dene both without metabolic activation and (generally) with microtomes embedded together with the bacteria in soft agar. With some nitresamines incubation was carried out with microtomes before plating. A summary of the results it given in Table 1. Since publication, one of the non-carcinogens. 5-hydroxy 2-acctytamino-fluortna, has been shown to be mutagenic because of the presence of an impurity and has now been rertastifled as aon-mutagensc. It can be seen that of 179 compounds whose carcinogenic effect on animals is wed documented. 1S7 (or 17.7%) were detected as potitive in the bacterial iol This level of confirmsoon was obtained with encmially ail types of compound tad was sire evident for the small group of compounds for which evidence exists for carcinogenicity in man. The proportion of rrunpounds believed to be noncaroaofease vrbieh gave negative results in the muta genicity teats was also high: 101 out of 117 (or M.3%). Tham included 46 common biochemscah all of which srarr negative. Sivaneaen compounds ware tested for which careinognicity data an uncertain: of then 11 were positive in the mutagenicity ml The apparent "false" pusilha* and negatives have bean dttcMMd elaewhere". It is apparent that many of the latter damage DNA or cause mutations in other systems, or have mutagenic metsbnlhai Furthermore three is a very obvious limitation to the use of liver msercaoenes for activation: tome chemicals may need reductive activation, or may be metabolised by the got flora, by orpins other than the liver, or by call components ocher than mirromima Indeed it is surprising that liver aucrocomes ate as effective as they seem to bo; certainly the method is capable of further improvement. When one ennmrtm the "fsbe" positives. that is the tupposed non-earemotens that register as mutagenic with bacteria, certain difficulties become apparent. Any correla- ucc 007938 BFG61961 Sature Vql. 261 May 20 IV76 some caution since they can be manipulated within wide limits by the choice of substances tested. As their substances include'a large number of non-carunogenic chemicals closely related to known carcinogens they feel that their results give a reasonably good indication of the likely value of ihe tests in practice. D NA damage and human cancer The correlation between mutagenicity and carcinogenicity is tativfwng io those who believe m the somatic mutation theory of cancer*4 and distressing to those who do not** 1 think the correlation can be more correctly described as being between UNA damaging ability and carcinogenicity. Gene mutation is hut one consequence of DNA damage; others such as chromosomal structural rearrangements, virus integration and excision, and changes in gene expres sion. may well be important in the carcinogenic process. Non-genetic effects are also probably involved. One could argue that detecting ONA damage is merely a very sensitive way of detecting electrophilic reagents, and that the actual targetts) may svell be in other molecules as well as or instead of DNA. This is quite possible: but there is other evidence strongly implicating DNA damage as the rate-limiting step in many carcinogenic processes. In man. for example, mutations in live complementation groups are known to reduce or abolish the ability of ceils to remove ultraviolet photoproducts from their DNA". In all cases they enormously increase sensitivity to the car cinogenic effect of sunlight (resulting in the hereditary disease xeroderma pigmentosum). A further mutation causing the same symptoms has been shown to be associ ated with a deficiency in another DNA repair pathway active on newly synthesised DNA". Another human muta tion responsible for the disease ataxia telangiectasia has been shown to Mock repair of ionising radiation damage'* and also results in proneness to develop malignant disease". Thus, the human data strengthen our confidence in the reality of the observed correlation between DNA damaging ability and carcinogenicity. It must he emphasised, however, that even an empirical "litmus paper test**, with no known theoretical basis, which gave an SO to 90% prtdictrveness for carcinogenicity would be a powerful tool in the screen ing of chemicals for human toxicity. The place of tests with mammals No single test is adequate for a first-tier (sub-mammalian) screen: most authorities agree that a battery of tews anas be used as false negatives may occur with any oae tea. The results of these teem would be used to asMgn priorities for further testing using nasmsHu rymaeu. At one extreme, a substance wish no apparent effect on submammalisn systems might be given a priority so low that no further team would be conaidared ealea a large (man population exposure were to occur or be contemplated. At the other extreme a wrongly active tuhmama might well he regarded as hanrdwu* without tetter tasting if tte population exposed ware small. If it warn. say. an industrial chemical, then production workers and users outfit to treat it as if it were a known toxic agent or carcinogen, at least until such time as it became powiblt to carry out full scale animal tests. The greatest problem in testing ter carcinogenicity or mutagenicity with mammals it tte bmsnsitmty of mom of the tests. This has led. for example, to difleuttim in vali dating microbial carcinogenicity greening systems tines many of the "false'' positive* obtained with these are based on animal experiments that may be madeqnatc**. There have been, and still are. too many carcinogenicity tests with 20 or 30 animals per group. Provided that the number of animals in caeh group it kept small, even a large increase in the frequency of ae 199 plasms can fail to be statistically significant and enable a conclusion of "non-carcinogenic" ;o be drawn tsee for example a recent study on the carcinogenicity of hair dyes *). As long ago as 1954. Barnes and Denr' pointed out that to detect with a probability of 0.01 an effect occurring in l . of the animals, one would need a group of at least 455 animals. If the effect also occurred spontaneously then the number of animals per group would have to be increased manyfold. Today, notwithstanding, carcinogenicity experi ments with lOO animals per group are often regarded as "good" and those with 200 animals per group are extremely rare. But although "kilomouse" experiments are theoreti cally attractive there may he little to be gained from (hem in practice. Logistical problems dictate that such experi ments he phased over many weeks and involve slightly varying conditions. "Spontaneous" rates of tumour occur rence unfortunately often vary in time and place, perhaps reflecting slight differences in diet, and it is often difficult to run an adequate control group. Errors in handling are also more likely in very large experiments. Whereas a significant reproducible positive result in a mammalian test may be taken as indicating the existence of a potential hazard for man. a negative result taken should not necessarily he taken to indicate the absence of hazard, particularly if the human population to be exporod is very large, and the number of animals in the test small. We may take some comfort where the disparity in dose between the animal and human exposure is great. This is not always so. Anaesthetic gases, for example, ms given to an appreciable fraction of the population hi Western society at concen trations which are not far from the lethal level. One might well feel that a negative result in s screening test with a few dozen mice would be of little value. It can he seen that mammalian tests are not wholly appro- ` prate for the validation of sub-mamma!tan tests, and it is perhaps remarkable that they should show such good agree ment. Validation of one type of test against another must not Mind one to the real objective, which is to predict long term toxic effects in man. There are few proven human carcinogens and most of these can be detected by both mammalian and sub-mammalian tests. In man. carcinogens tend to be recognised only when the tumour is of a rare type and there is a sufficient cluster of cases to enable association with a particular occupation to be seen by an alert clinician. Genetic effects in man are even harder to detect raiwepaetivcly. There are several examples of somatic chromosome damage in lymphocyte* of persons capeasd to known mutagenic and caremogtnic substances (for example, vinyl chloride", oaooc", benzene", toluene", cadmium"-" and methyl mercury**. Recent evidence for the powtailiry of dominant lethal damage in man by vinyl chloride fP. Infanta, unpublished) and anaesthetic gases" is ominous but neads closer examination. Ultimately quantitative risk amessments must be attempted, for we must face the unpalatable fact that man will almost certainly have to be exposed to some carcino gens and mutagens whose benefits cannot be dispensed with and often which it is impracticable to eliminate from the environment. Risk evaluations at tte present time almost always require information which is not availsMe. such as the narure of tte item tffret rmpoose at low Seers. Never theless. approach** must be found that wffl lend eventually to risk-benefit evaluation baaed leas on guestwuik and men upon knowledge In tte meantime tte oae of short term tests would enable potentially carcinogenic substances to be identified among the many thousands for which long term animal testing cannot at present be contemplated. This in turn would open tte door to provisional regulatory action to minimtie human expoaure. Taken seriously and on a large enough sale, then it good reason to believe that this approach would ultimately result in a reduction in the incidence of chemically induced cancer. I I t f f* i con UCC 007939 BFG61962 ZM I thank Drs D Anderson. E. J. Ashby. P A. Lefivre. E. Lcngstaff. I. F. H. Purchase. J. A. Styles and F. R. Westwood of ICI Central Toxicology Laboratory, and Dr Bruce Ames for allowing me to see their data before oublication. 1 liinw. P. M.. fotrtmie WfgfiwUi Gomottt 4#*naarA f# Afrf <Maima! ano Technical. Lancaster. 1774). 1 J.. C-n. Comoro Camf.. dO-75 fPerfaBMfl. Otforl. 1741). J Htfpnaon. J.. ano Muir. C- S.. n Cm*** omd Dototttmm. I (in tha Carrm. J.. Sti. Aaee.. 133. te*! W>. 1 Oort. A.. Acr-rren of Comoro--Potmtorr fm CmtAomtmimfy (Wimafnan Pro*. LanOan. I74T) Pat. P . m C--/mu> of of Cmtmmmmo Cm***. NCI. Miimpmii Nn. 10 (1*43). Lefaior. M. $.. a. Moo. kfo4.. 13. 41M21 (1972V Cmotomm. HlrM Pr-*#erf . 4 <wnnA iMI) I17T3). Drug. J. W . ot iowmoe. 107, J03-JU (1775). '* ftrMifn. 0. 4h fit-tea# hum Mommott.. 4, 22t-Cf 0*77). ` Bnaaaa. . A . m kwwm Tetri < CMomoemi CmimnwUifaBH. hy Mmmnm. R Barm*. H.. #n0 Tmuiv L.I flABC. Cyan. 17741. I Wiliams. O J . and Ia*m. B. Narary. 132. lOJ-lOS0471). ' Purenam. I. P M . mb Uftufi. P. a.. Ctmmo Imo. IB. 419-414 fifty). miPwim. P . BnOpea. J. w.. wB Pit. 0. V . team. 132. 400-07 f97V > * Crwaar. J W . 44*llr. i. A_ aaO M*Uv. L C-/. te. Cte.. 233. UJ-400 (1740). ' SUtar. 8. L. Ai iwi. M. Ow MB >ifinn M. ft. Cmkm In.. 31. 770*73 077. <* MaMm* H. V.. Warar. Xn. U 423*2* (1971V " Cmr. ft. C- Millar. I. C. mB MiOv. i. ft Cmm In. 32.3090-2044 (19T2V >* IlOtaBltii. 31.. "B~o1ptem. ft. Ai AsBmaaO-OBmft M. J. MB Saao. ft. ft 1mm. Baa.. . lt-30 0979V * Bnif ft Aw-------------- if ft. Pw ______ M. AiM Gte M. 9C. U Cte-te. ....... .. ft T7-0B (I9T2V 11 ABM. ft. M.. in Ctaaaraf WaNfiaf' PmafM*4-_ t <40*. ay Haltmaim. A). 247-2S3 (PM M* Yam. ttl). ** ABM. ft. Nw LA P. Ow mb Dwa W. ft. PMC. ON. Bte. 3*. C/-ftft 71. Tt2-7BflT3V SI MacMB. J.. 3><NBan>. M. ft. KBBart. A MB am. ft. W, Pmc. com. Bte. id. UHw . *74*09 fllTJl. PfMtA C M.. MB Momma M. V, *HM. JUt. 31. 34^900 flm ss ABM. ft. N.. Don. L. YI--IIH LOOU.P. O. Ppm m Boa* tf-J.Bw 7ft 2211-2213 flYTlL Hunbtt.ifvt. ft. uiinii, M. D.MBIbbm* M.ft.Cbnpb.3t.HMD >* KaBo. T. laBaia Y.. aaB TbMbm ft. INiia Baa. tft t4J-mfl9TZ). ftnOBW ft Aw Mini........... ft. P, Om 9C K. U mB Of*.. 1. H, Mm. te. 11. 2fft-)B3 llfll). ____ Cm. M. H. L.. m0 Mb*. W. Jw AftMt. tew 2ft J3UJJ4/lfTJ>. * Ha*, ft. H. Cw aaB SiH*. H. P- tar. /. Cmhp. 1ft te-2l (tWV Smml H. P.. Lm. P.. Lb. L. w.. ftaraaBMaft D. J. mB Sba ft. K. C. Cm. / rart. CM 17.471^02(1479). Wa*M. I, mBOmbb. Hw teM. ftatw Ift llT-tt4(t0tD. ___ J ABMA ft N.. McCMaX mb Yiaiwli, ft. tear. te. 31. 9B7-M4 (1173). > (tea. M. H. Lw te Manat. W. Jw tear. te. 3ft VllMOTf). is MrCaaa. J.. CM. ft. YhmM L. te abb. ft Mw tar. m Bte. JM. C/.ftBw 71. 9I1V9I34 (1973V * Mactea, o. O. Ami. ^vi nr 2ft IDM !> (I97JV j' CfMk M. M. Lw MteL w. J,, te BrteA ft Aw ABM. In. 3ft ))-Bt (19744. MaPa. Cw llliiMtp. Jw te MaOaaBM. D. Mm. te. 2ft II7-I44(I974V Naturt Vot. 241 Vfav 20 /tte * Ota. ft H. Y.. to Ckomicai W*ravm* taatr/n aaB Wr*rwi >* r*#r 0r*rrrraa- j (adit, tv HaiiaaaOar. A.t. *i i im (PNum. s von. 47i > * Amtt. C F . Twrtiaall. O . Harwaort. S a. LrfimaiM. a. R.. an C^Niia. C. N . Mmtm Mrs.. 33. :H-i7| nt7). 1 Ci*.O.F!*nn.W C- MO Ftmrw. J. 1 * CMomfui WuMreei Pi<h rr<r VrraoOjrarer Doiert<nm. } iMu. or Hoitaraorr. A > *9-101 iPmum. Nn. Torfc. tf7jy s Lilly- L. J . Bafmar. ft. 0 Maaa. P ^avarr. Ut. < 1-412 f 177)) * Parry. P.. and Cvam. H. i . vararr. 191. >21-129 M47)> Sauaoa. J ft < - vmrr. 134. >03-104 ft'?!) ` Allan. 1. W.. ana Lett. S A., vaiwmm ma oraul. * Vofat. W . ana Bawanacnc. T.. <m mg ami. * Fraaman. a ft. ana Hwaoaar. ft. 1.. >. wr. C>*mr /<.. 90. .'03-304 (H7j) * ramaoa. T.. TaRaoAa. T . iLatMia. M.. M . ana V*io J.. J<* j. re#. WrO.. 42. 377.314(177^. * Wihiama. t. M . wgimriif. ft 10^.11211771), ana tMataavrcar. J. M . Frrf rrf/ Art.. 47. * Mantaaana. ft.. Ina-Vagau. L.. ana Tamaiia. L.. 8* J C^mro*. 29. 219-220 (177J), * ramaeveta. H..te Waatatam. I. ft. tar. 4*.<. Ci*rr* Art.. 19. 74<i7t4) < 1 HCwraamar.mCa.AH. f.t.aannaoOStaPcatoai.aL. .j..taa r..Cmumnr.r4%Anrdt.. Sn 31. V a * . 54. t > 23-1 i^nanni lOtS-maa i7t)> * OiMararco. Cw Graanaiait. M.. Trawiftan. T. Saftar. a. ana C<artano. ft . prar. mtm. AraO. fri. U.S.4.. 70. ufit7 t HaPHaia. C- A. Ar* Or*m.. 44. ta.i 2i (t779) * Caaca. ft C.. and OtPaaia. J. A., tar mW k*#.a . 14. i^7nay)v * PtaaiA ft J.. oaaaad 4t StM rr af.*. * MaCaaa. 3.. ano ARM. ft N.. Pmr. "mm. ate. .fr< L'. 9 4 fm t*a araan * T.. TaAaAa J. on. Mo4- 103. 331-347. 30l-]00(l77i) * Naaan. T.. Narva. 13ft 410-411 (1479). t abm ft N.. Camay, ft G.. MiUar. J. A. aao Bamclt. M.. tair. nara. tr*. in. C/.3^4w 44. 3123-9132 (I4T2V s BraaAaa. P. aao Oa Ssrraa. P J- AOarar. Art. <ia tnt trtnl s Hina T . Many*. M.. lUao. ft PvMaata. Aw aao AaOa. T.. .Warn Art.. M. 14-JO (1474V * Mote. P. J.. te Ovor. ft Pw !Hmm. Mot.. 2ft 40VB20 (1479). t Book. P. O.. te Maaft ft. Coarar Art.!. U7-J7) (1490V * ABM. ft Nw Setmto. 141. 201-149 (IVHi. 7 |M. H.. 9mm 141. 241 (1474V * Knmm. ft Mw 0a WwrO Kmpwn. ft A. Mte J. M.. KatMr. 3. P . Barvac R.A. taM. B. Aw te Bawari. O.. tear. Prw 33. 377-740(1479). * LitMOa*. A. ft m* OaB. ft Aitao.C. P.. Paaanaa. M. C.. LohatM. P. M. M.. 0* Watesftoante. ft Aw te OaataoM. O.. Pmr. aara. Acni Sot. U.S.A.. 7ft 214-223 (1479). * Taytar. a. M. ft Morte*. O. O- AOblC. F. Maraaart. 3. A. LaBaiaaa. A. ft tuii-- ft te OnOte ft A. Woraar. 20.4r*B24 (1475). Hamit. O. 0~ ia CMaaaaan te Caarr faOa. 4y Cw. J.V 414-434 (TMor. Naa Yam. I47v l Ote A. ft Oaa. C Ww te KaaMMMa. Cw J. Tanratf. aa. IMA. 1.433-te (1474V S lam J. Mwte Dm. P. ft Pte*. Am., ft 141 (I4MV * DtMA ft Mtetem. ft te MRlC I. Jw tev Art^ 31.143-<M(I4T9V Mam. T. item. M. ft Karr. K. D.. te ftaO*. T. i.. 1mm. Am. 31.2*0-902 0479V 9 Parai. a. Mw CooaaOtaC ft Ptetaa. ft te Vioaa. ft Cw Bte cm. Mrft 13. Nftlll (19TIV T TaaMV L Mwte Cm jraaa, W. M. tern. 1.400 0449). * Owm. Y . te Vote. T. H.. Prar. Job. Ate.. O. 240-191 0472). * 0m.TMw I iaMW Jw te Waif. C. P. lama. Art.. 7.107-179 0479). s 3VarPitap, ft. Motaa. ft. Mmba C.. LaaOnt. J- te Bymoa. N.. n-*raa. Aar. 7.19-70 (1974V s Cam*, ft M.. tea*, ft W.. Bm O. t. CimarM. M. P..Cataart.T M . Jana* T. W.. te W4IIM*.C ft/. Ba^ Aa. AaOft 1^1-1144 (1479). articles Isolation and N-terminal amino acid sequence of membrane-bonnd human HLA-A and HLA-B antigens John Bridgea Lahoraiory of MotaBi Mepr. Ml loi CHAridpi CS2 2QH. UK David Saary A Mkhad J. Cmaiptoa* Nenoael la----foumart P--iep. M Hg. ImUb-WWTUA.UK Coiia Banstabk. Peter GoodfeDow A Walter F. Bodmer Genedei Ubwmn. Depniwi of WNiy. Uammy of Oxford. Oxford 0X1 JQU.UK MtmbrmtAowtd HLA-A mi HLA-M mttifmu Itor* btm txmunHy porifiti in fooi yitii. 7V j-f-owm of dm ftttrmmot It amimo adit kmt btm Atttrmimi tsm? Am / nmol of protm tbati from poiymoyiamhk got after tkanpkonsti in sodom dodteyijolphmt. Tm major hi--ocompodbifity region of man (HLA) occtiprcs at laaat 1 to 2 i-combrnanew units on chrornoeotna 6 and pro* babfy eeoeaina t larpa oumbar of gma invoKod m divana immuna and poMibty orhar funeboai1. Tho pna product! that l to M. J. r..n ucc 2 007940 C-3 BFG61963