Environmental and Molecular Mutagenesis Volume 14, Supplement 16:51-59 (1 989)

Origins of Current Uncertainties in CarcinogedMutagen Screening

John Ashby IC1 Central Toxicology Laboratory, Alderley Park, Cheshire, England

The origins of current uncertainties regarding how best to screen for agents likely to pose a

rnutagenic/carcinogenic hazard to humans are discussed, and a solution is proposed.

Key words: assays for carcinogens/mutagens, toxicology, genotoxicity

At the foundation of the Environmental Mutagen Society (EMS) in the United States, in January, 1969, five objec- tives for the Society were agreed, as follows: to encourage interest in mutagens in the environment, to publish a mono- graph on mutagenicity testing, to publish a newsletter, to form a registry of chemical mutagens, and to act as a con- sulting resource to government and industry on such mat- ters. The first official meeting of officers and councilors took place a month later, and in the minutes of that gath- ering it was suggested that the newsletter should also be circulated to people interested in carcinogenicity, but car- cinogenicity figured little in the early meetings of the soci- ety.

In July, 1970, at Munich, Dr. Hollaender gave the open- ing address at the foundation of the European EMS. He restated the Society goal of “dealing with the factors from our environment which could have a genetic effect. ” Only two references were made to carcinogenicity, the most per- tinent being by Lars Ehrenberg, who suggested that, in countries with only limited resources, research in carcino- genesis and mutagenesis should be coupled. Likewise, in 1973 the long-range planning committee of the U.S. EMS presented a report that identified nine areas of possible fu- ture endeavours for the Society, but the specific study of carcinogenicity and the evaluation of mutagenicity assays for its prediction were not mentioned.

In 1973, Ames and his colleagues published a paper that demonstrated selected carcinogens to be mutagenic to Sal- monella ryphimurium in vitro [Ames et al., 19731. Within the next few years, the results of several cooperative studies were presented, and these demonstrated that -90% of ro- dent carcinogens were mutagenic to Salmonella, whereas only -10% of the noncarcinogens evaluated were muta- genic. This caught the interest of both the scientific and governmental communities, for it appeared that, by com- plementing the Salmonella assay with one or two others, all animal and human carcinogens could be detected as muta- gens in vitro. The apparently compelling nature of the ev-

idence supporting this proposition essentially silenced those without contrary data, and only when it was called into question by papers such as that of Tennant et al. [ 19871 did the need for a reappraisal become apparent.

It is suggested that two fundamental problems beset this field, and that only when they are separated will either be open to resolution. The first is that a significant number of animal carcinogens are not mutagenic or reactive to DNA but rather increase cancer incidences by specific chronic toxicities or by modulating the overall homeostatic balance of animals. The second is that a general failure to accept this first proposition has led to the multliplication and abuse of mutagenicity assays in a vain attempt to detect all carcino- gens as mutagens. This has eventually led to instances of poor science. An attempt to separate these two problems is made below under the broad headings of experimental and conceptual factors leading to uncertainty. Primary reliance has been placed herein on discussion of the -250 chemicals studied for animal carcinogenicity by the U.S. National Toxicology Program (NTP), as discussed by Ashby and Tennant [1988] and Ashby et al. [ 19891. This is because the biological properties of these agents have given rise to much of the current uncertainty.

EXPERIMENTAL FACTORS LEADING TO UNCERTAINTY

The Need to Confirm Observations Nearly all the founding ‘ ‘validation” studies were based

on single observations. The problem is still with us in the literature and has spread into in vivo genotoxicity assays.

Received December 5 , 1988; revised and accepted February 21, 1989.

Address reprint requests to John Ashby, ICI Central Toxicology Labora- tory, Alderley Park, Cheshire, England.

0 1989 Alan R. Liss, Inc.

52 Ashby

For example, the vast majority of published rodent bone marrow assays represent single observations. The need at hand is not so much to repeat an experiment exactly but rather to probe for extra information relating to the reality of the initial observation. It is of interest that the U.S. NTP do not repeat their cancer bioassays, and the incidence of stud- ies classified as equivocal is relatively high (32 of 264 stud- ies reviewed by Ashby et al. [1989]). One can be fairly confident that a proportion of the NTP carcinogens could not be confirmed in a repeat bioassay, for example, agents such as piperonyl sulfoxide in Class D of Table 1 in Ashby and Tennant [1988]. The real danger is when two uncon- f m e d observations are correlated with each other, leading to an unsound tertiary conclusion, for example, when an unconfirmed positive response observed in vitro for a weak and unconfirmed carcinogen is used to justify the use of that mutagenicity assay in general screening. The probability that this happened in the study reported by Tennant et al. [ 19871 was responsible for the subsequent discussion [see Parry and Venitt, 1988; Brockman and DeMarini, 19881, and the same problem besets the approach to testing out- lined by Lave et al. [ 19881.

A basic need of this field, therefore, is that, in the ab- sence of compelling evidence to the contrary, journals should unite in rejecting unconfirmed observations. The publication of qualitative results in the absence of support- ing quantitative data should cease.

Presentation of Adequate Test Data and Statement of Response Criteria

It seems redundant to suggest that investigators should furnish a sufficient amount of data to support the conclu- sions they draw, but this is often not done. This concern is supported by the rejection rate of papers considered in re- views conducted by the U.S. Environmental Protection Agency (EPA) Genetox Committees. Thus Mitchell et al. [ 19831 rejected 69% of the 244 papers considered for in- clusion in their review of unscheduled DNA synthesis (UDS) assays, and Kier et al. [ 19831, 40% of the 501 Sal- monella assay papers; in most of these cases, missing or inadequate data were the cause of rejection. This matter can really be resolved only by the peer review/editorial policies of scientific journals. To illustrate this point, Environmental Mutagen Information Centre (EMIC) has now collected qualitative genotoxicity results from publications on over 20,000 chemicals, but data supporting these results are available for only about 10,000 of the agents (E. Von Halle, personal communication). This matter assumes particular importance when the qualitative test responses on chemicals of unknown structure, and for which the primary test data are not available, are used to justify a particular testing strategy (see the debate between Garner and Kirkland [ 19861 and Ashby [ 19861). An ancilliary concern is that the

criteria by which positive or negative test responses are judged are either insecure or absent from papers. For ex- ample, Salmonella data are assessed for activity by a range of methods, some investigators still using the arbitrary two times rule, irrespective of the nature and reproducibility of any mutagenic activity this might conceal. Likewise, the present debate about the true status of the mouse lymphoma L5178Y Tk+’- mutation assay is predicated mainly on the absence of agreed test response criteria [Arlett and Cole, 19881. To be specific, the test protocol and response criteria used by Caspary [Tennant et al., 19871 do not enjoy uni- versal approval. Such matters require urgent resolution.

The design of experiments

Inadequate or inappropriate test protocols can prejudice the outcome of experiments, a possibility that is at its great- est when testing “confidential” chemicals under code. For example, a series of papers were published in the late 1970s indicating that volatile chemicals should be evaluated for mutagenicity in a sealed container. This advice is usually ignored, but the issue was raised again recently by Proctor et al. [ 19861, by Forster [ 19861, and by Parry and Venitt [ 19881 and, finally, by Ashby [ 19891. During the interven- ing 10 years, many inadequate data on volatiles have been published, and this illustrates how important findings and advice can be lost in the rush to test more chemicals in more assays. Two personal instances can be cited in which lack of sufficient thought contributed to an element of experimental confusion. First, volume 100 of Mutation Research is de- voted to the first U.K. EMS collaborative study [Parry, 19821. In that study, 4-chloromethylbiphenyl (4CMB) was assayed using a wide range of in vitro and in vivo genotox- icity tests. The confusing database that resulted was ex- plained towards the end of the study, when it was realized that 4CMB was probably hydrolytically unstable; in fact, its half-life in buffer at 37°C was a mere 10 min. That fact could and should have been anticipated. Similarly, Me- shram and Rao [ 19881 recently questioned negative Salmo- nella data on methylisocyanate (cf. Bhopal) for the same reason-by reducing the assay temperature, they estab- lished an increase in hydrolytic half-life that resulted in the demonstration of a mutagenic response. Chemical hydrol- ysis can usually be anticipated and can be determined with ease.

The second example relates to chemical purity and is derived from the second U.K. EMS collaborative study [Parry and Arlett, 19851. In that study, 4-cyanodimethylan- iline (CDA) was provided with a purity of >99.5%. Ex- periments conducted during the course of the collaborative study led to the realization that the mutagenicity of CDA to Salmonella was due to an impurity present at < O . 1%. It is, in fact, unusual for chemical purity to be stated in papers, and this provides ample scope for confusion.

Uncertainties in Carcinogen/Mutagen Screening 53

TABLE 1. Review of the Literature Citations of the Ten Most Frequently Tested Genotoxins*

Mutagenicity to Standard chemical on chemical in EMIC files Salmonella

(+S9 mix) abbreviation

No. of papers (%) with data

(total of 62,500 papers)

Selection of dose levels

Some agents are mutagenic in vitro only within a narrow, subtoxic dose range. This suggests the need to use a suffi- cient number of dose levels and to pursue signs of activity in repeat experiments. Failure in this regard probably ac- counts for a significant number of false-negative results. At the other extreme is the testing of what are probably bio- logically irrelevant doses of an apparently nonmutagenic carcinogen in the hope that it will elicit a mutagenic re- sponse in vitro (discussed in detail by Brusick [1987] and recently encountered again in a paper by Schiestl [ 19891). Concern in this general area led to a recent editorial state- ment by Parry and Venitt [ 19881, and one can anticipate similar problems occurring with in vivo genotoxicity assays unless attention is given to this matter.

Summary Conclusions of Experimental Sources of Uncertainty

A proportion of current problems would be solved, or at least be brought into sharper focus, by the requirement that test data be accepted for publication only if papers provide sufficient experimental details and data to establish that a real response was observed, using an appropriate test pro- tocol and with appropriate dose selection, test criteria, and positive and negative control observations.

CONCEPTUAL FACTORS LEADING TO UNCERTAINTY

In comparison with the experimental factors discussed above, consideration of the conceptual ones is more com- plex; here one enters the realm of dogma, often unsupported by facts.

Selection of Chemicals to Evaluate Test Batteries

The outcome of a “validation study” of a new short-term test for carcinogens can now be designed to the extent that an accuracy of 100% or 0% can be arranged by the appro- priate choice of chemicals [see Ashby, 19881. A limited form of this ability has always been available with reference mutagens, whose activity in virtually any assay can be guar- anteed in advance. The data shown in Table I illustrate the extent to which mutagenicity/carcinogenicity literature has been dominated by the activity of a few reference bacterial mutagens. The fact that these agents are mutagenic to Sal- monella should have encouraged their future avoidance in seeking to justify the use of complementary or supplemen- tary assays, but in fact these same reference agents have generally been used for this purpose. The real justification for additional screening assays should be their ability to detect as positive mutagenic rodent carcinogens such as procarbazine and acrylamide, agents that are nonmutagenic to Salmonella.

EMS M ” G BP MMS MNU DMN CP ENU 4NQo 2AAF

4,455 (7) 4,470 2,555 2,249 1,942 (3) 1,523 1,417 1,321 (2) 1,252 1.191

+ (-S9) + (-S9) + (+S9) + (-S9) + (-S9) + (+S9) + (+S9) + (-S9) + (-S9) + (+S9)

~~~ ~ ~

*Activity is usually assured for each of these in any genotoxicity assay. Thus, beyond use as positive controls, they should be avoided when de- termining the individual attributes and sensitivity of an assay. Data pro- vided by the Environmental Mutagen Information Centre (EMIC). NB: mitomycin C (1,787 papers) was eliminated; it is diagnostic for uvrE- in Salmonella. Caffeine (1,362 papers) also was eliminated; this chemical is a source of separate study and is of uncertain carcinogenicitylmutagenicity .

False-Positive In Vitro Responses

Pharmaceutical houses accept that an in vitro antibacte- rial screen, for example, will detect many new antibacterial agents, only a proportion of which will be active in vivo. That is what a screen is for, so that the fact -30% of the NTP noncarcinogens are mutagenic to Salmonella should be accepted as inevitable; they are probably genuine mutagens that are not active in vivo. That does not devalue the assay, it merely confirms it as a screen rather than as an animal carcinogenicity bioassay. Substantial effort is currently be- ing expended to confirm the proposition that short-term ro- dent genotoxicity assays will detect as positive those in vitro mutagens that will be carcinogenic while finding negative those in vitro mutagens that are non-carcinogenic. This ap- proach to testing requires that newly defined in vitro geno- toxins be evaluated further in rodents if refined predictions of carcinogenicity/mutagenicity for man are to be derived. The point here is that it is unlikely that any in vitro assay can anticipate that a genotoxin will prove inactive to ani- mals because of its nonabsorption, detoxification, or non- activation in vivo, yet this “attribute” is enforced on in vitro assays by the requirement that the perfect test is one with a specificity for noncarcinogens of 100%. No such in vitro assay does or could exist.

A subsidiary question is as follows: How low can the specificity of an in vitro assay be before its value is abol- ished? There is no simple answer to this quesiton; rather, it lies with the in vitro assay in question-how genetically valid is the change being monitored, how stable is the cells’ karyotype, to what extent is the end point of the test subject to modulation by toxicity, etc.? Even with well established

54 Ashby

tests such as the Succhromyces assay, it is necessary to retain interest in its performance characteristics; sometimes a positive test response does not define unequivocally a mutagenic response [see, e.g., Albertini and Gocke, 1988; Ashby et al., 1988al.

The Carcinogen/Noncarcinogen Pair Concept

The main principle of the first International Study [de Serres and Ashby, 19811 was that the 42 test chemicals be structurally related carcinogenlnoncarcinogen pairs, e.g., benzo[a]pyrenelpyrene, 2AAF/4AAF, etc. When a short- term test found the putative noncarcinogen positive, it was penalized by a reduction in its accuracy score. This approach was troubled by two problems. First, the non-carcinogens had all been inadequately evaluated for carcinogenicity. Second, the resolution of carcinogen/ noncar-cinogen pairs is probably an inappropriate goal for in vitro assays (see above). As it happened, a range of in vivo short-term tests was included in that first study, and they provided evidence of the ability to resolve these pairs. This fact led to the IPCS study of in vivo assays [Ashby et al., 1988bl. Furthermore, although insecure “noncarcino- gens” such as pyrene and 4-acetylaminofluorene (4AAF) were employed in the several international studies, the fact of a significant proportion of noncarcinogens being genotoxic in vitro has been confirmed using the relatively secure noncarcinogens defined by the U.S. NTP [see, for example, Tennant et al., 1987; Ashby and Tennant, 19881.

Invalid Short-Term Tests

The fact that >200 different genotoxicity assays have been described makes it inevitable that the majority will have been inadequately studied. Most assays are the product of parallel evolution, and most are therefore repetitive of other assays. Purchase [ 19821 has listed objective criteria by which a new assay should be judged before being accepted as suitable for routine use; but these are rarely achieved before an assay enters routine use.

An early example of a problematic test was the baby hamster kidney (BHK) cell transformation assay. As an in-house assay with the cells we used, this test performed as claimed. However, when it was evaluated by others, par- ticularly as part of the international study [de Serres and Ashby, 19811, it was found deficient, and its use was even- tually discontinued. Subsequently, McGregor used the last set of the 42 test chemicals from that international study to evaluate the more established C3H 10T 1/2 transformation assay; he was experienced in its use, but the study outcome led him to discontinue the assay for routine testing (personal communication). That decision should not reflect badly on data generated with this assay in research laboratories; it merely endorses that the use of an assay for research pur-

poses is often different from its employment with a fixed test protocol for routine testing. There are many similar instances. For example, the nonreproducibility of the mac- rophage transformation and liver single-strand break assays in the second IPCS study [Ashby et al., 1988b], the dem- onstrated insensitivity of the 5’. pombe forward mutation assay in the second U.K. EMS study [Parry and Arlett, 19851, the lack of carcinogednoncarcinogen resolution by Salmonellalurine assays [McGregor, 19881, etc. It is essen- tial that the new generation of molecular assays should be assessed for their practical utility and true role before they are pronounced a panacea-a matter discussed by Brock- man and DeMarini [I9881 in relation to transgenic mice mutation assays.

Conditions of Exposure In Vitro and In Vivo

It is only recently that consideration has been given to the question of whether the conditions under which a test re- sponse is observed should influence its extrapolation, ulti- mately to humans. The minimum effective dose level for positive responses, and the maximum dose tested for a neg- ative response now figure widely in data assessment, as exemplified in the histograms of Waters et al. [1988] and the ICPEMC initiative [Brusick et al., 1989; Mendelson et al., 19891. An extension of this is the concern that positive effects observed in vitro at elevated dose levels (e.g., >M-’) may be of no predictive significance for mammals [Brusick, 19871.

A subtle point that probably accounts for some instances of contradictory reports of activity for the same chemical, in the same in vitro assay conducted in different laboratories, concerns the meaning of “tested to the limit of solubility.” In some regulatory guidelines, this phrase is unqualified, and as a result it is interpreted in different ways. Some laboratories take it to mean the use of a saturated stock solution of the test agent in, for example dimethyl sulfoxide (DMSO). Others take it to mean that dose level in DMSO that when added to the assay medium will not lead to an observable precipitate. A few laboratories take it to mean a saturated solution of the test agent in the assay medium. Orders of magnitude of difference in delivered dose can separate these three interpretations, with only the second seeming to be legitimate.

Similar concerns apply to in vivo genotoxicity data. For example, the unresolved matter of whether the intraperito- neal (ip) injection route of exposure is legitimate in present circumstances (the assessment of genotoxicity, not the seek- ing of it) remains to be resolved. However, the value of the thoughtful use of routes of exposures is illustrated by the demonstration by Doolittle et al. (19841 that inhalation of 500 ppm of dimethylnitrosamine leads to UDS in rat nasal epithelium, trachea, and liver; only the liver being affected by the oral or ip routes of exposure.

Uncertainties in Carcinogen/Mutagen Screening 55

activity of direct-acting mutagens such as nitrogen mustard in all assay systems supports this suggestion. Similarly, Crosby et al. [I9881 have demonstrated the induction of a total range of genetic changes for formaldehyde, dependent on the dose level tested and the cell type and culture con- ditions employed. Most chemical mutagens require meta- bolic conversion (using S9 mix in vitro) to an electrophilic/ radical species, and differences in S9 mix are probably the major determinant of differential mutagenicity of a chemi- cal between different in vitro assay systems. Thus conclu- sions of genetic specificity of mutagenic action for a chem- ical should be drawn only when the possible role of competing metabolic variables has been eliminated. Fur- thermore, although gene amplification, insertion mutations, hypomethylation, and recombination events may in future be required to refine an efficient prescreen for the detection of carcinogens, the need for such assays should not be as- sumed in the absence of data. Perhaps of greatest impor- tance, many animal carcinogens are probably devoid of genotoxicity and will not therefore be detected by genetic screening tests, as is discussed below.

Role of Statistics in Assay Design and Interpretation

All statisticians emphasize the importance of designing appropriate experiments, but they are usually appealled to for the rescuing of inadequate ones. Except in a few legit- imate cases, such as the mouse heritable translocation assay and the mouse spot test, the use of statistics is of secondary importance to the investigators’ conclusions. The long- standing debate about biological significance vs. statistical significance usually recedes if one but repeats the experi- ment.

Premature Testing Strategies: Need for an Expanded Database

Although EMIC has mutagenicity “information” on -20,000 chemicals, the number of chemicals for which a basic set of adequate assay data exists is small. Thus, among the several hundred genotoxicity profiles derived by Waters et al. [ 19881, only 1 13 chemicals currently have a database comprising three in vitro assay results and two in vivo assay results (after Genetox editing; see Brusick et al. [1989]). Mindful of this, the early promise shown by in vivo assays [e.g., Ashby, 19861 is being pursued by several laboratories by the acquisition of further data.

Bone Marrow-Germ Cell Relationship

In 1980, Brusick observed that rodent germ cell muta- gens were also active as clastogens to the rodent bone mar- row. This observation was elaborated and confirmed by Holden in 1982 and by Adler and Ashby in 1989. The mouse bone marrow micronucleus assay forms a common component of most strategies for the detection of potential genotoxic carcinogens. Thus inactivity in this assay appears to preclude mutagenic activity for the test chemical in the germ cells. Nonetheless, this is not yet a common assump- tion of all regulatory authorities.

Chemical Structure as an Aid to Testing

The Millers’ electrophilic theory of chemical carcinogen- esis [Miller and Miller, 19771 remains a useful aid to pre- dicting possible human mutagens and carcinogens [Shelby, 1988; Ashby and Tennant, 19881. It fails to explain the selective carcinogenicity of some rodent carcinogens, but that is because not all rodent carcinogens are reactive to DNA. as is discussed below.

Genetic Specificity of Action

Most of the regulatory guidelines and requirements in existence, worldwide, were based on the assumption that some chemicals would show selective and specific genetic toxicities. Experience has not borne this out. The uniform

Genotoxic/Nongenotoxic Carcinogen Debate

Weisburger and Williams [for review, see Weisburger and Williams, 19811 were among the first to respond to the problem posed by the rodent liver carcinogenicity of agents such as dieldrin and chloroform. These agents do not bind covalently to hepatic DNA, but they do induce changes in liver morphology and homeostasis, and this exemplifies the ‘‘epigenetic” (nongenotoxic) theory of carcinogenesis. Such chemical disturbances of normal body homeostasis could be highly specific in terms of the species, strain, sex, or tissues affected [Malling and Chu, 19741. Clayson [1987, 19891 has attempted to define some of the mecha- nistic factors responsible for the existence of these pre- sumed nongenotoxic carcinogens. He and others have con- cluded that toxic properties of a chemical other than mutagenicity may play a critical role in the expression of tumors in its bioassay for carcinogenicity, and, further, that these toxicities may of themselves lead to selective carci- nogenic responses for certain nonmutagens in some species or strains of animal.

An enduring problem when discussing “nongenotoxic” carcinogens is that there is no general agreement on how to test for genotoxicity. At this point, three examples are given to illustrate how individual authors could accelerate under- standing in this area by emphasizing observations contrary to their own. First, sodium saccharin (NaS) is probably the best studied and most credible of nongenotoxic carcino- gens. It has also given rise to an extensive literature on the potential for artefactual ‘‘genotoxic” responses when test- ing elevated dose levels of it and other salts in vitro (10-20 mg/ml). It was therefore interesting when Suzuki and Su-

56 Ashby

Fig. 1. Schematic summary of data for 264 agents tested for Salmonella mutagenicity and rodent carcinogenicity by the U.S. NCVNTP as reviewed in Ashby and Tennant [I9881 and Ashby et al. [1989]. a: The perfect assay; mutagens ( M + ) are carcinogens (C+), nonmutagens (M-) are noncarcinogens (C-), b: Noncarcinogens that are Salmonella mutagens, c: Rodent carcinogens that are nonmutagenic to Salmonella, d: Agents scored as structurally alerting superimposed over Figure Ic in pink. This confirms that most bacterial mutagens can be explained in terms of their

structure irrespective of their carcinogenic status, e: Positive Salmonella mutagenicity (red) and structural alerts (pink) for carcinogens to the 16 tissues listed in the figure [see Ashby et al., 1989; for tissue codes], f: As in Figure le, except for the 13 different tissues shown, illustrated here for the 22 mouse liver (ML)-specific carcinogens. For these carcinogens, the Salmonella assay shows a similar proportion of positive responses as for the 91 noncarcinogens, g: Figure Id, with three fields of chemical selec- tion identified (see text).

Uncertainties in CarcinogedMutagen Screening 57

mammalian cells, and probably most effectively in vivo. Ultimately, DNA controls tumor growth, but the difference between a chemical inducing new mutations, as opposed to selecting for preexisting ones, could prove critical in human risk assessment; such differentiations should soon be exper- imentally practical. At present, these two mechanisms are fused, and are reflected as low performance figures for mutagenicity assays, as typified by the recent NTP studies.

An updated summary of the U.S. NTP rodent carcinoge- nicity database [Ashby and Tennant, 1988; Ashby et al., 19891 now incorporates 264 chemicals; they provide an op- timal set of chemicals for future study (Fig. 1) . The three points of relevance to this discussion are, first Salmonella mutagenicity and structural alerts to DNA reactivity are closely correlated in predicting carcinogenicity in 16 tissues of the rodent (Fig. ld,e). Second, when these two param- eters are negative, carcinogenicity can still be recorded in one or more of the 13 different tissues listed in Figure 1 (illustrated by data for the 22 mouse liver-specific carcino- gens [ML]). The prediction of the latter agents is a present challenge to the field. Third, the most important aspect of the paper by Tennant et al. [1987] is suggested to be that the authors forced the study of complementary in vitro assays from established bacterial mutagens (area 1 in Fig. lg) to apparently nongenotoxic carcinogens (area 2 in Fig. lg) and, further, into the area of noncarcinogens (area 3 in Fig. lg). It was only then that the supplementary assays studied were found to perform less well than had hitherto been assumed [Tennant et al., 19871. The difference in the car- cinogenicity of agents in areas 1 and 2 of Figure lg is

zuki [1988] announced the mutagenicity in vitro of NaS to human cells (10-20 mg/ml) but much more so when viewed against the extensive literature on the nonmutagenicity of NaS, which was barely discussed. Second, the potentially seminal paper by Reynolds et al. [1987] concerning muta- tion of the H-ras gene in mouse liver tumors was slightly confused by reference to furfuraldehyde as a nongenotoxin based on a single negative Salmonella response; in fact, other published data define this chemical as genotoxic, in- cluding to Salmonella. Finally, two papers on the induction of sister chromatid exchange (SCE) recently appeared in the same issue of this Journal. The first of these [Best and McKenzie, 19881 demonstrated that by lowering the dose of an alkylating agent its induction of SCE in human lympho- cytes in vitro could be reduced with a retention of statistical significance. The authors concluded that ‘‘increases in SCE less than twice background can be reliable indicators of genotoxic exposure.” In the second paper, Ghosh and Ghosh [1988] concluded that such increases were also a reliable indicator of human pregnancy. Thus, in these two papers, the heart of the debate about the true meaning of the term “genotoxic” was captured. Within this context, cau- tion is required when assessing the significance of the fact that elevated dose levels (up to 60 mg/ml) of several non- genotoxic carcinogens induce intrachromosomal recombi- nation in yeast [Schiestl, 19891.

SUGGESTED DIRECTIONS OF RESEARCH A new generation of genetic assays will soon be avail-

able. These will measure mutation at the molecular level, in

Fig. 2. Carcinogenicity data for cupferon and chlorinated paraffin, as published by the NTP and abstracted by Ashby and Tennant [ 19881. The tissue codes are CS, circulatory system; HG, Harderian gland; L, liver; S, stomach; ZG, Zymbal’s gland; HS, hemopoietic system. The first corn- pound, cupferon, was classified as positive (P) in all four test groups. The second compound was classified using current levels of evidence and gave

clear evidence (CE) of carcinogenicity only as a leukemogen in the male mouse. These two chemicals are therefore representative of putative geno- toxic and nongenotoxic carcinogens. The acquisition of DNA adduct data (postlabeling or I4C binding) for such agents, coupled to acute histopatho- logical assessment of the major tissues of the treated rodents, would aid clear classifications of carcinogenic hazard.

58 Ashby

usually dramatic, as is shown in Figure 2 (see legend for further discussion).

In summary, it is suggested that two major initiatives are required to clarify and to resolve current problems associ- ated with the prediction of possible new human carcinogens and mutagens. “Nongenotoxic’ ’ carcinogens should be studied to define their mechanisms of action. Much progress has already been made in this area. [Clayson, 19891, and future studies should lead to the development of (nongenetic) screening tests and appropriate risk assessment models [Moolgavkar and Knudson, 1981; Park and Snee, 1983; Moolgavkar, 1988; Watanabe et al., 19881. Once the concept of nongenotoxic carcinogenesis is accepted, atten- tion can be focused on refining protocols for the detection and assessment of genotoxins, which current evidence in- dicates pose the greatest hazard to man [Shelby, 19881. If genotoxicity assays are relieved of the need to detect non- genotoxins, the conducting and development of such tests will return to the established principles of good science, and order may return to this field.

ACKNOWLEDGMENTS

Liz Von Halle, Rosie Elspuru, Mike Shelby, Helen Tin- well, and Jane Steele gave invaluable help with the writing and editing of this paper.

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